The mutational landscape of a US Midwestern breast cancer cohort reveals subtype-specific cancer drivers and prognostic markers.

BACKGROUND: Female breast cancer remains the second leading cause of cancer-related death in the USA. The heterogeneity in the tumor morphology across the cohort and within patients can lead to unpredictable therapy resistance, metastasis, and clinical outcome. Hence, supplementing classic pathological markers with intrinsic tumor molecular markers can help identify novel molecular subtypes and the discovery of actionable biomarkers. METHODS: We conducted a large multi-institutional genomic analysis of paired normal and tumor samples from breast cancer patients to profile the complex genomic architecture of breast tumors. Long-term patient follow-up, therapeutic regimens, and treatment response for this cohort are documented using the Breast Cancer Collaborative Registry. The majority of the patients in this study were at tumor stage 1 (51.4%) and stage 2 (36.3%) at the time of diagnosis. Whole-exome sequencing data from 554 patients were used for mutational profiling and identifying cancer drivers. RESULTS: We identified 54 tumors having at least 1000 mutations and 185 tumors with less than 100 mutations. Tumor mutational burden varied across the classified subtypes, and the top ten mutated genes include MUC4, MUC16, PIK3CA, TTN, TP53, NBPF10, NBPF1, CDC27, AHNAK2, and MUC2. Patients were classified based on seven biological and tumor-specific parameters, including grade, stage, hormone receptor status, histological subtype, Ki67 expression, lymph node status, race, and mutational profiles compared across different subtypes. Mutual exclusion of mutations in PIK3CA and TP53 was pronounced across different tumor grades. Cancer drivers specific to each subtype include TP53, PIK3CA, CDC27, CDH1, STK39, CBFB, MAP3K1, and GATA3, and mutations associated with patient survival were identified in our cohort. CONCLUSIONS: This extensive study has revealed tumor burden, driver genes, co-occurrence, mutual exclusivity, and survival effects of mutations on a US Midwestern breast cancer cohort, paving the way for developing personalized therapeutic strategies.

Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling.

STUDY QUESTION: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? SUMMARY ANSWER: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. WHAT IS KNOWN ALREADY: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. MAIN RESULTS AND THE ROLE OF CHANCE: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. LARGE SCALE DATA: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

Splice-altering variant in COL11A1 as a cause of nonsyndromic hearing loss DFNA37.

PURPOSE: The aim of this study was to determine the genetic cause of autosomal dominant nonsyndromic hearing loss segregating in a multigenerational family. METHODS: Clinical examination, genome-wide linkage analysis, and exome sequencing were carried out on the family. RESULTS: Affected individuals presented with early-onset progressive mild hearing impairment with a fairly flat, gently downsloping or U-shaped audiogram configuration. Detailed clinical examination excluded any additional symptoms. Linkage analysis detected an interval on chromosome 1p21 with a logarithm of the odds (LOD) score of 8.29: designated locus DFNA37. Exome sequencing identified a novel canonical acceptor splice-site variant c.652-2A>C in the COL11A1 gene within the DFNA37 locus. Genotyping of all 48 family members confirmed segregation of this variant with the deafness phenotype in the extended family. The c.652-2A>C variant is novel, highly conserved, and confirmed in vitro to alter RNA splicing. CONCLUSION: We have identified COL11A1 as the gene responsible for deafness at the DFNA37 locus. Previously, COL11A1 was solely associated with Marshall and Stickler syndromes. This study expands its phenotypic spectrum to include nonsyndromic deafness. The implications of this discovery are valuable in the clinical diagnosis, prognosis, and treatment of patients with COL11A1 pathogenic variants.

Genetic Variants in Circadian Rhythm Genes and Self-Reported Sleep Quality in Women with Breast Cancer.

INTRODUCTION: Women diagnosed with breast cancer (BC) are at increased risk of sleep deficiency. Approximately 30-60% of these women report poor sleep during and following surgery, chemotherapy, radiation therapy, and anti-estrogen therapy. The purpose of this study was to examine the relationship between genetic variation in circadian rhythm genes and self-reported sleep quality in women with BC. METHODS: This cross-sectional study recruited women with a first diagnosis of breast cancer at five sites in Nebraska and South Dakota. Sixty women were included in the study. Twenty-six circadian genes were selected for exome sequencing using the Nextera Rapid Capture Expanded Exome kit. 414 variants had a minor allele frequency of ≥5% and were included in the exploratory analysis. The association between Pittsburgh Sleep Quality Index (PSQI) score and genetic variants was determined by two-sample t-test or ANOVA. RESULTS: Twenty-five variants were associated with the PSQI score at p < 0.10, of which 19 were significant at p<0.05, although the associations did not reach statistical significance after adjustment for multiple comparisons. Variants associated with PSQI were from genes CSNK1D & E, SKP1, BHLHE40 & 41, NPAS2, ARNTL, MYRIP, KLHL30, TIMELESS, FBXL3, CUL1, PER1&2, RORB. Two genetic variants were synonymous or missense variants in the BHLHE40 and TIMELESS genes, respectively. CONCLUSIONS: These exploratory results demonstrate an association of genetic variants in circadian rhythm pathways with self-reported sleep in women with BC. Testing this association is warranted in a larger replication population.

Targeted myocardial gene expression in failing hearts by RNA sequencing.

BACKGROUND: Myocardial recovery with left ventricular assist device (LVAD) therapy is highly variable and difficult to predict. Next generation ribonucleic acid (RNA) sequencing is an innovative, rapid, and quantitative approach to gene expression profiling in small amounts of tissue. Our primary goal was to identify baseline transcriptional profiles in non-ischemic cardiomyopathies that predict myocardial recovery in response to LVAD therapy. We also sought to verify transcriptional differences between failing and non-failing human hearts. METHODS: RNA was isolated from failing (n = 16) and non-failing (n = 8) human hearts. RNA from each patient was reverse transcribed and quantitatively sequenced on the personal genome machine (PGM) sequencer (Ion torrent) for 95 heart failure candidate genes. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite™. Differential expression analyses were conducted by empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes between failing and non-failing groups, and between responder and non-responder groups respectively. Targeted cardiac gene messenger RNA (mRNA) expression was analyzed in proportion to the total number of reads. Gene expression profiles from the PGM sequencer were validated by performing RNA sequencing (RNAseq) with the Illumina Hiseq2500 sequencing system. RESULTS: The failing sample population was 75% male with an average age of 50 and a left ventricular ejection fraction (LVEF) of 16%. Myosin light chain kinase (MYLK) and interleukin (IL)-6 genes expression were significantly higher in LVAD responders compared to non-responders. Thirty-six cardiac genes were expressed differentially between failing and non-failing hearts (23 decreased, 13 elevated). MYLK, Beta-1 adrenergic receptor (ADRB1) and myosin heavy chain (MYH)-6 expression were among those significantly decreased in failing hearts compared to non-failing hearts. Natriuretic peptide B (NPPB) and IL-6 were significantly elevated. Targeted gene expression profiles obtained from the Ion torrent PGM sequencer were consistent with those obtained from Illumina HiSeq2500 sequencing system. CONCLUSIONS: Heart failure is associated with a network of transcriptional changes involving contractile proteins, metabolism, adrenergic receptors, protein phosphorylation, and signaling factors. Myocardial MYLK and IL-6 expression are positively correlated with ejection fraction (EF) response to LVAD placement. Targeted RNA sequencing of myocardial gene expression can be utilized to predict responders to LVAD therapy and to better characterize transcriptional changes in human heart failure.

Genome-wide mutation avalanches induced in diploid yeast cells by a base analog or an APOBEC deaminase.

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.

Mammalian alteration/deficiency in activation 3 (Ada3) is essential for embryonic development and cell cycle progression.

Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3(FL/FL) mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G(1) to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G(2)/M to G(1) transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex.

Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters.

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay-Bovine Gene Chip-was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons.

Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate.

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes.

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/ß subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHß subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHß results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHß subunit in pituitaries of women.

Morphine suppresses peripheral responses and transforms brain myeloid gene expression to favor neuropathogenesis in SIV infection.

The twin pandemics of opioid abuse and HIV infection can have devastating effects on physiological systems, including on the brain. Our previous work found that morphine increased the viral reservoir in the brains of treated SIV-infected macaques. In this study, we investigated the interaction of morphine and SIV to identify novel host-specific targets using a multimodal approach. We probed systemic parameters and performed single-cell examination of the targets for infection in the brain, microglia and macrophages. Morphine treatment created an immunosuppressive environment, blunting initial responses to infection, which persisted during antiretroviral treatment. Antiretroviral drug concentrations and penetration into the cerebrospinal fluid and brain were unchanged by morphine treatment. Interestingly, the transcriptional signature of both microglia and brain macrophages was transformed to one of a neurodegenerative phenotype. Notably, the expression of osteopontin, a pleiotropic cytokine, was significantly elevated in microglia. This was especially notable in the white matter, which is also dually affected by HIV and opioids. Increased osteopontin expression was linked to numerous HIV neuropathogenic mechanisms, including those that can maintain a viral reservoir. The opioid morphine is detrimental to SIV/HIV infection, especially in the brain.

Microarray analysis of Paramecium bursaria chlorella virus 1 transcription.

Paramecium bursaria chlorella virus 1 (PBCV-1), a member of the family Phycodnaviridae, is a large double-stranded DNA, plaque-forming virus that infects the unicellular green alga Chlorella sp. strain NC64A. The 330-kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To monitor global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDSs) was constructed. Competitive hybridization experiments were conducted by using cDNAs from poly(A)-containing RNAs obtained from cells at seven time points after virus infection. The results led to the following conclusions: (i) the PBCV-1 replication cycle is temporally programmed and regulated; (ii) 360 (99%) of the arrayed PBCV-1 CDSs were expressed at some time in the virus life cycle in the laboratory; (iii) 227 (62%) of the CDSs were expressed before virus DNA synthesis begins; (iv) these 227 CDSs were grouped into two classes: 127 transcripts disappeared prior to initiation of virus DNA synthesis (considered early), and 100 transcripts were still detected after virus DNA synthesis begins (considered early/late); (v) 133 (36%) of the CDSs were expressed after virus DNA synthesis begins (considered late); and (vi) expression of most late CDSs is inhibited by adding the DNA replication inhibitor, aphidicolin, prior to virus infection. This study provides the first comprehensive evaluation of virus gene expression during the PBCV-1 life cycle.

Induced pluripotent stem cells generate both retinal ganglion cells and photoreceptors: therapeutic implications in degenerative changes in glaucoma and age-related macular degeneration.

The direct reprogramming of somatic cells to a pluripotent state holds significant implications for treating intractable degenerative diseases by ex vivo cell therapy. In addition, the reprogrammed cells can serve as a model for diseases and the discovery of drugs and genes. Here, we demonstrate that mouse fibroblast induced pluripotent stem cells (iPSCs) represent a renewable and robust source of retinal progenitors, capable of generating a wide range of retinal cell types that includes retinal ganglion cells (RGCs), cone, and rod photoreceptors. They respond to simulated microenvironment of early and late retinal histogenesis by differentiating into stage-specific retinal cell types through the recruitment of normal mechanisms. The depth of the retinal potential of iPSCs suggests that they may be used to formulate stem cell approaches to understand and treat a wide range of retinal degenerative diseases from glaucoma to age-related macular degeneration (AMD).

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells.

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H(2)O(2)) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H(2)O(2). These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.

Identification of anti-inflammatory vesicle-like nanoparticles in honey.

Honey has been used as a nutrient, an ointment, and a medicine worldwide for many centuries. Modern research has demonstrated that honey has many medicinal properties, reflected in its anti-microbial, anti-oxidant, and anti-inflammatory bioactivities. Honey is composed of sugars, water and a myriad of minor components, including minerals, vitamins, proteins and polyphenols. Here, we report a new bioactive component‒vesicle-like nanoparticles‒in honey (H-VLNs). These H-VLNs are membrane-bound nano-scale particles that contain lipids, proteins and small-sized RNAs. The presence of plant-originated plasma transmembrane proteins and plasma membrane-associated proteins suggests the potential vesicle-like nature of these particles. H-VLNs impede the formation and activation of the nucleotide-binding domain and leucine-rich repeat related (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome, which is a crucial inflammatory signalling platform in the innate immune system. Intraperitoneal administration of H-VLNs in mice alleviates inflammation and liver damage in the experimentally induced acute liver injury. miR-4057 in H-VLNs was identified in inhibiting NLRP3 inflammasome activation. Together, our studies have identified anti-inflammatory VLNs as a new bioactive agent in honey.

High-throughput analysis of lung immune cells in a combined murine model of agriculture dust-triggered airway inflammation with rheumatoid arthritis.

Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden.

CD38 regulation in activated astrocytes: implications for neuroinflammation and HIV-1 brain infection.

Reactive astrogliosis is a key pathological aspect of neuroinflammatory disorders including human immunodeficiency virus type 1 (HIV-1)-associated neurological disease. On the basis of previous data that showedastrocytes activated with interleukin (IL)-1beta induce neuronal injury, we analyzed global gene changes in IL-1beta-activated human astrocytes by gene microarray. Among the up-regulated genes, CD38, a 45-kDa type II single chain transmembrane glycoprotein, was a top candidate, with a 17.24-fold change that was validated by real-time polymerase chain reaction. Key functions of CD38 include enzymatic activities and involvement in adhesion and cell signaling. Importantly, CD38(+)CD8(+) T-cell expression is a clinical correlate for progression of HIV-1 infection and biological marker for immune activation. Thus, CD38 expression in HIV-1 and/or IL-1beta-stimulated human astrocytes and human brain tissues was analyzed. IL-1beta and HIV-1 activation of astrocytes enhanced CD38 mRNA levels. Both CD38 immunoreactivity and adenosine 5'-diphosphate (ADP)-ribosyl cyclase activity were up-regulated in IL-1beta-activated astrocytes. CD38 knockdown using specific siRNAs significantly reduced astrocyte proinflammatory cytokine and chemokine production. However, CD38 mRNA levels were unchanged in IL-1beta knockdown conditions, suggesting that IL-1beta autocrine loop is not implicated in this process. Quantitative immunohistochemical analysis of HIV-seropositive without encephalitis and HIV-1 encephalitis brain tissues showed significant up-regulation of CD38, which colocalized with glial fibrillary acidic protein-positive cells in areas of inflammation. These results suggest an important role of CD38 in the regulation of astrocyte dysfunction during the neuroinflammatory processes involved in neurodegenerative/neuroinflammatory disorders such as HIV-1 encephalitis.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex.

BACKGROUND: The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. RESULTS: Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuitytrade mark Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. CONCLUSION: Inactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.

The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling.

The canonical Wnt pathway is known to influence multiple developmental events such as patterning, cell proliferation and cell specification. Recent studies have provided evidence of the involvement of the canonical Wnt pathway in the emergence and development of the optic neuroepithelium and its derivatives, particularly the retina. However, the mechanism of its action during retinal development remains rather obscure. Here, we demonstrate that (in agreement with observations in the blood, intestine, and skin) the canonical Wnt pathway influences retinal development by maintaining stem cells/progenitors. For example, the activation of this pathway keeps the early retinal stem cells/progenitors proliferating and uncommitted, while its attenuation facilitates their differentiation into retinal ganglion cells in vitro and in vivo. In addition, we demonstrate that Wnt signaling acts in concert with Notch signaling during retinal histogenesis, where the latter calibrates the influence of the former on the differentiation status of retinal stem cells/progenitors by regulating Lef1 and sFRP2.