Distinct Biological Functions Research Articles

Gene Regulatory Strategies that Decode the Duration of NFκB Dynamics Contribute to LPS- versus TNF-Specific Gene Expression.

Pathogen-derived lipopolysaccharide (LPS) and cytokine tumor necrosis factor (TNF) activate NFκB with distinct duration dynamics, but how immune response genes decode NFκB duration to produce stimulus-specific expression remains unclear. Here, detailed transcriptomic profiling of combinatorial and temporal control mutants identified 81 genes that depend on stimulus-specific NFκB duration for their stimulus-specificity. Combining quantitative experimentation with mathematical modeling, we found that for some genes a long mRNA half-life allowed effective decoding, but for many genes this was insufficient to account for the data; instead, we found that chromatin mechanisms, such as a slow transition rate between inactive and RelA-bound enhancer states, could also decode NFκB dynamics. Chromatin-mediated decoding is favored by genes acting as immune effectors (e.g., tissue remodelers and Tcell recruiters) rather than immune regulators (e.g., signaling proteins and monocyte recruiters). Overall, our results delineate two gene regulatory strategies that decode stimulus-specific NFκB dynamics and determine distinct biological functions.

Evolved resistance to partial GAPDH inhibition results in loss of the Warburg effect and in a different state of glycolysis

Aerobic glycolysis or the Warburg effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although the WE is ubiquitous, its biological role remains controversial, and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, here we evolved resistance to koningic acid (KA), a natural product that specifically inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme, during the WE. We found that KA-resistant cells lose the WE but continue to conduct glycolysis and surprisingly remain dependent on glucose as a carbon source and also on central carbon metabolism. Consequently, this altered state of glycolysis led to differential metabolic activity and requirements, including emergent activities in and dependences on fatty acid metabolism. These findings reveal that aerobic glycolysis is a process functionally distinct from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

Emerging Functions of Human IFIT Proteins in Cancer.

Interferon-induced protein with tetratricopeptide repeats (IFIT) genes are prominent interferon-stimulated genes (ISGs). The human IFIT gene family consists of four genes named IFIT1, IFIT2, IFIT3, and IFIT5. The expression of IFIT genes is very low in most cell types, whereas their expression is greatly enhanced by interferon treatment, viral infection, and pathogen-associated molecular patterns (PAMPs). The proteins encoded by IFIT genes have multiple tetratricopeptide repeat (TPR) motifs. IFIT proteins do not have any known enzymatic roles. However, they execute a variety of cellular functions by mediating protein-protein interactions and forming multiprotein complexes with cellular and viral proteins through their multiple TPR motifs. The versatile tertiary structure of TPR motifs in IFIT proteins enables them to be involved in distinct biological functions, including host innate immunity, antiviral immune response, virus-induced translation initiation, replication, double-stranded RNA signaling, and PAMP recognition. The current understanding of the IFIT proteins and their role in cellular signaling mechanisms is limited to the antiviral immune response and innate immunity. However, recent studies on IFIT protein functions and their involvement in various molecular signaling mechanisms have implicated them in cancer progression and metastasis. In this article, we focused on critical molecular, biological and oncogenic functions of human IFIT proteins by reviewing their prognostic significance in health and cancer. Research suggests that IFIT proteins could be novel therapeutic targets for cancer therapy.

Specification of BMP Signaling.

Bone Morphogenetic Proteins (BMPs) together with the Growth and Differentiation Factors (GDFs) form the largest subgroup of the Transforming Growth Factor (TGF)β family and represent secreted growth factors, which play an essential role in many aspects of cell communication in higher organisms. As morphogens they exert crucial functions during embryonal development, but are also involved in tissue homeostasis and regeneration in the adult organism. Their involvement in maintenance and repair processes of various tissues and organs made these growth factors highly interesting targets for novel pharmaceutical applications in regenerative medicine. A hallmark of the TGFβ protein family is that all of the more than 30 growth factors identified to date signal by binding and hetero-oligomerization of a very limited set of transmembrane serine-threonine kinase receptors, which can be classified into two subgroups termed type I and type II. Only seven type I and five type II receptors exist for all 30plus TGFβ members suggesting a pronounced ligand-receptor promiscuity. Indeed, many TGFβ ligands can bind the same type I or type II receptor and a particular receptor of either subtype can usually interact with and bind various TGFβ ligands. The possible consequence of this ligand-receptor promiscuity is further aggravated by the finding that canonical TGFβ signaling of all family members seemingly results in the activation of just two distinct signaling pathways, that is either SMAD2/3 or SMAD1/5/8 activation. While this would implicate that different ligands can assemble seemingly identical receptor complexes that activate just either one of two distinct pathways, in vitro and in vivo analyses show that the different TGFβ members exert quite distinct biological functions with high specificity. This discrepancy indicates that our current view of TGFβ signaling initiation just by hetero-oligomerization of two receptor subtypes and transduction via two main pathways in an on-off switch manner is too simplified. Hence, the signals generated by the various TGFβ members are either quantitatively interpreted using the subtle differences in their receptor-binding properties leading to ligand-specific modulation of the downstream signaling cascade or additional components participating in the signaling activation complex allow diversification of the encoded signal in a ligand-dependent manner at all cellular levels. In this review we focus on signal specification of TGFβ members, particularly of BMPs and GDFs addressing the role of binding affinities, specificities, and kinetics of individual ligand-receptor interactions for the assembly of specific receptor complexes with potentially distinct signaling properties.

HIC-5 in cancer-associated fibroblasts contributes to esophageal squamous cell carcinoma progression

Esophageal squamous cell carcinoma (ESCC) remains one of the most common malignancies in China and has a high metastasis rate and poor prognosis. Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, can affect tumor progression and metastasis, but the underlying mechanism remains unclear. There are no studies that explore the role of hydrogen peroxide-inducible clone 5 (HIC-5) in ESCC or compare the role of HIC-5 in CAFs and adjacent noncancerous normal fibroblasts (NFs). In this study, we isolated primary CAFs and NFs from ESCC patients. HIC-5 was highly expressed in CAFs from the tumor stroma of human ESCC patients. HIC-5 knockdown in CAFs inhibited the migration and invasion of ESCC cells in vitro. Supernatant CCL2 levels of CAFs were significantly higher after TGF-β stimulation and lower after knocking down HIC-5 expression, independent of TGF-β treatment. HIC-5 knockdown in CAFs led xenograft tumors derived from ESCC cells mixed with CAFs to present more regular morphology, express higher CDH1, and lower CCL2. Further RNA-seq data showed that HIC-5 has distinct biological functions in CAFs vs. NFs, especially in cell movement and the Rho GTPase signaling kinase pathway, which was verified by wound-healing assays and western blotting. An ESCC tissue microarray revealed that increased HIC-5 expression in the tumor stroma was associated with positive lymph node metastasis and a higher TNM stage. In summary, we identified that stromal HIC-5 was a predictive risk factor for lymph node metastasis in human ESCC and that CAF-derived HIC-5 regulated ESCC cell migration and invasion by regulating cytokines and modifying the ECM.

The differential distributions of ASPM isoforms and their roles in Wnt signaling, cell cycle progression, and pancreatic cancer prognosis

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and treatment‐resistant malignancy. The lack of pathway‐informed biomarkers hampers the development of rational diagnostics or therapies. Recently, the protein abnormal spindle‐like microcephaly‐associated (ASPM) was identified as a novel Wnt and stemness regulator in PDAC, while the pathogenic roles of its protein isoforms remain unclarified. We developed novel isoform‐specific antibodies and genetic knockdown (KD) of putative ASPM isoforms, whereby we uncovered that the levels of ASPM isoform 1 (iI) and ASPM‐iII are variably upregulated in PDAC cells. ASPM isoforms show remarkably different subcellular locations; specifically, ASPM‐iI is exclusively localized to the cortical cytoplasm of PDAC cells, while ASPM‐iII is predominantly expressed in cell nuclei. Mechanistically, ASPM‐iI co‐localizes with disheveled‐2 and active β‐catenin as well as the stemness marker aldehyde dehydrogenase‐1 (ALDH‐1), and its expression is indispensable for the Wnt activity, stemness, and the tumorigenicity of PDAC cells. By contrast, ASPM‐iII selectively regulates the expression level of cyclin E and cell cycle progression in PDAC cells. The expression of ASPM‐iI and ASPM‐iII displays considerable intratumoral heterogeneity in PDAC tissues and only that of ASPM‐iI was prognostically significant; it outperformed ALDH‐1 staining and clinico‐pathological variables in a multivariant analysis. Collectively, the distinct expression patterns and biological functions of ASPM isoforms may illuminate novel molecular mechanisms and prognosticators in PDAC and may pave the way for the development of therapies targeting this novel oncoprotein. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Selenium Uptake and Biotransformation in Brassica rapa Supplied with Selenite and Selenate: A Hydroponic Work with HPLC Speciation and RNA-Sequencing.

Vegetables are an ideal source of human Se intake; it is important to understand selenium (Se) speciation in plants due to the distinct biological functions of selenocompounds. In this hydroponic study, the accumulation and assimilation of selenite and selenate in pak choi (Brassica rapa), a vastly consumed vegetable, were investigated at 1-168 h with HPLC speciation and RNA-sequencing. The results showed that the Se content in shoots and Se translocation factors with selenate addition were at least 10.81 and 11.62 times, respectively, higher than those with selenite addition. Selenite and selenate up-regulated the expression of SULT1;1 and PHT1;2 in roots by over 240% and 400%, respectively. Selenite addition always led to higher proportions of seleno-amino acids, while SeO42- was dominant under selenate addition (>49% of all Se species in shoots). However, in roots, SeO42- proportions declined substantially by 51% with a significant increase of selenomethionine proportions (63%) from 1 to 168 h. Moreover, with enhanced transcript of methionine gamma-lyase (60% of up-regulation compared to the control) plus high levels of methylselenium in shoots (approximately 70% of all Se species), almost 40% of Se was lost during the exposure under the selenite treatment. This work provides evidence that pak choi can rapidly transform selenite to methylselenium, and it is promising to use the plant for Se biofortification.

9-Azido Analogues of Three Sialic Acid Forms for Metabolic Remodeling of Cell-Surface Sialoglycans.

Neu5Ac, Neu5Gc, and KDN are three forms of sialic acids in vertebrates that possess distinct biological functions. Herein, we report the synthesis and metabolic incorporation of the 9-azido analogues of three sialic acid forms in mammalian cells. The incorporated sialic acid analogues enable fluorescent imaging of cell-surface sialoglycans and proteomic profiling of sialoglycoproteins. Furthermore, we apply them to metabolically engineer cell surfaces with sialoglycans terminated with distinct sialic acids or their 9-azido analogues. The remodeled cells expressing specific cell-surface sialoglycoforms show distinct binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted by Shiga toxigenic Escherichia coli. The 9-azido analogues of sialic acid forms developed in this work provide a versatile tool for metabolic remodeling of cell-surface properties and modulating pathogen-host interactions.

CDK4/6 Inhibition Suppresses Myeloma Cell Growth and Viability via Perturbation of E2F Proliferative Program

Cancer cells are characterized by perturbed transcriptional profiles. These dysregulations depend on transcriptional regulators unpredicted by genetic changes. Multiple myeloma cells also tend to develop striking dependencies on super-enhancer regulatory elements. Also, promoter proximal transcription factors like E2F1 and SP1 play prominent roles in myeloma cell biology by controlling proliferative and survival genes. Importantly, our genome-wide profiling of the MM epigenome revealed that promoter and enhancer-associated factors govern distinct biological functions providing a non-overlapping control of the myeloma transcriptome.

Identifying functions and prognostic biomarkers of network motifs marked by diverse chromatin states in human cell lines

Epigenetic modifications play critical roles in modulating gene expression, yet their roles in regulatory networks in human cell lines remain poorly characterized. We integrated multiomics data to construct directed regulatory networks with nodes and edges labeled with chromatin states in human cell lines. We observed extensive association of diverse chromatin states and network motifs. The gene expression analysis showed that diverse chromatin states of coherent type-1 feedforward loop (C1-FFL) and incoherent type-1 feedforward loops (I1-FFL) contributed to the dynamic expression patterns of targets. Notably, diverse chromatin state compositions could help C1- or I1-FFL to control a large number of distinct biological functions in human cell lines, such as four different types of chromatin state compositions cooperating with K562-associated C1-FFLs controlling “regulation of cytokinesis,” “G1/S transition of mitotic cell cycle,” “DNA recombination,” and “telomere maintenance,” respectively. Remarkably, we identified six chromatin state-marked C1-FFL instances (HCFC1-NFYA-ABL1, THAP1-USF1-BRCA2, ZNF263-USF1-UBA52, MYC-ATF1-UBA52, ELK1-EGR1-CCT4, and YY1-EGR1-INO80C) could act as prognostic biomarkers of acute myelogenous leukemia though influencing cancer-related biological functions, such as cell proliferation, telomere maintenance, and DNA recombination. Our results will provide novel insight for better understanding of chromatin state-mediated gene regulation and facilitate the identification of novel diagnostic and therapeutic biomarkers of human cancers.

Distinct Phenotypic and Genomic Signatures Underlie Contrasting Pathogenic Potential of Staphylococcus epidermidis Clonal Lineages.

Background: Staphylococcus epidermidis is a common skin commensal that has emerged as a pathogen in hospitals, mainly related to medical devices-associated infections. Noteworthy, infection rates by S. epidermidis have the tendency to rise steeply in next decades together with medical devices use and immunocompromized population growth. Staphylococcus epidermidis population structure includes two major clonal lineages (A/C and B) that present contrasting pathogenic potentials. To address this distinction and explore the basis of increased pathogenicity of A/C lineage, we performed a detailed comparative analysis using phylogenetic and integrated pangenome-wide-association study (panGWAS) approaches and compared the lineages’s phenotypes in in vitro conditions mimicking carriage and infection.Results: Each S. epidermidis lineage had distinct phenotypic signatures in skin and infection conditions and differed in genomic content. Combination of phenotypic and genotypic data revealed that both lineages were well adapted to skin environmental cues. However, they appear to occupy different skin niches, perform distinct biological functions in the skin and use different mechanisms to complete the same function: lineage B strains showed evidence of specialization to survival in microaerobic and lipid rich environment, characteristic of hair follicle and sebaceous glands; lineage A/C strains showed evidence for adaption to diverse osmotic and pH conditions, potentially allowing them to occupy a broader and more superficial skin niche. In infection conditions, A/C strains had an advantage, having the potential to bind blood-associated host matrix proteins, form biofilms at blood pH, resist antibiotics and macrophage acidity and to produce proteases. These features were observed to be rare in the lineage B strains. PanGWAS analysis produced a catalog of putative S. epidermidis virulence factors and identified an epidemiological molecular marker for the more pathogenic lineage.Conclusion: The prevalence of A/C lineage in infection is probably related to a higher metabolic and genomic versatility that allows rapid adaptation during transition from a commensal to a pathogenic lifestyle. The putative virulence and phenotypic factors associated to A/C lineage constitute a reliable framework for future studies on S. epidermidis pathogenesis and the finding of an epidemiological marker for the more pathogenic lineage is an asset for the management of S. epidermidis infections.

CD23 provides a noninflammatory pathway for IgE-allergen complexes

Type I hypersensitivity is mediated by allergen-specific IgE, which sensitizes the high-affinity IgE receptor FcεRI on mast cells and basophils and drives allergic inflammation upon secondary allergen contact. CD23/FcεRII, the low-affinity receptor for IgE, is constitutively expressed on B cells and has been shown to regulate immune responses. Simultaneous binding of IgE to FcεRI and CD23 is blocked by reciprocal allosteric inhibition, suggesting that the 2 receptors exert distinct roles in IgE handling. We aimed to study how free IgE versus precomplexed IgE-allergen immune complexes (IgE-ICs) target the 2 IgE receptors FcεRI and CD23, and we investigated the functional implications of the 2 pathways. We performed binding and activation assays with human cells invitro and IgE pharmacokinetics and anaphylaxis experiments invivo. We demonstrate that FcεRI preferentially binds free IgE and CD23 preferentially binds IgE-ICs. We further show that those different binding properties directly translate to distinct biological functions: free IgE initiated allergic inflammation through FcεRI on allergic effector cells, while IgE-ICs were noninflammatory because of reduced FcεRI binding and enhanced CD23-dependent serum clearance. We propose that IgE-ICs are noninflammatory through reduced engagement by FcεRI but increased targeting of the CD23 pathway.

Functional annotation of the cattle genome through systematic discovery and characterization of chromatin states and butyrate-induced variations

BackgroundThe functional annotation of genomes, including chromatin accessibility and modifications, is important for understanding and effectively utilizing the increased amount of genome sequences reported. However, while such annotation has been well explored in a diverse set of tissues and cell types in human and model organisms, relatively little data are available for livestock genomes, hindering our understanding of complex trait variation, domestication, and adaptive evolution. Here, we present the first complete global landscape of regulatory elements in cattle and explore the dynamics of chromatin states in rumen epithelial cells induced by the rumen developmental regulator—butyrate.ResultsWe established the first global map of regulatory elements (15 chromatin states) and defined their coordinated activities in cattle, through genome-wide profiling for six histone modifications, RNA polymerase II, CTCF-binding sites, DNA accessibility, DNA methylation, and transcriptome in rumen epithelial primary cells (REPC), rumen tissues, and Madin-Darby bovine kidney epithelial cells (MDBK). We demonstrated that each chromatin state exhibited specific enrichment for sequence ontology, transcription, methylation, trait-associated variants, gene expression-associated variants, selection signatures, and evolutionarily conserved elements, implying distinct biological functions. After butyrate treatments, we observed that the weak enhancers and flanking active transcriptional start sites (TSS) were the most dynamic chromatin states, occurred concomitantly with significant alterations in gene expression and DNA methylation, which was significantly associated with heifer conception rate and stature economic traits.ConclusionOur results demonstrate the crucial role of functional genome annotation for understanding genome regulation, complex trait variation, and adaptive evolution in livestock. Using butyrate to induce the dynamics of the epigenomic landscape, we were able to establish the correlation among nutritional elements, chromatin states, gene activities, and phenotypic outcomes.

Subclinical endometritis in dairy cattle is associated with distinct mRNA expression patterns in blood and endometrium.

Cattle with subclinical endometritis (SCE) are sub-fertile and diagnosing subclinical uterine disease remains a challenge. The hypothesis for this study was that endometrial inflammation is reflected in mRNA expression patterns of peripheral blood leucocytes. Transcriptome profiles were evaluated in healthy cows and in cows with SCE using circulating white blood cells (WBC) and endometrial biopsy samples collected from the same animals at 45–55 days postpartum. Bioinformatic analyses of microarray-based transcriptional data identified gene profiles associated with distinct biological functions in circulating WBC and endometrium. In circulating WBC, SCE promotes a pro-inflammatory environment, whereas functions related to tissue remodeling are also affected in the endometrium. Nineteen differentially expressed genes associated with SCE were common to both circulating WBC and the endometrium. Among these genes, transcript abundance of immune factors C3, C2, LTF, PF4 and TRAPPC13 were up-regulated in SCE cows at 45–55 days postpartum. Moreover, mRNA expression of C3, CXCL8, LTF, TLR2 and TRAPPC13 was temporally regulated during the postpartum period in circulating WBC of healthy cows compared with SCE cows. This observation might indicate an advantageous modulation of the immune system in healthy animals. The transcript abundance of these genes represents a potential source of indicators for postpartum uterine health.

Quantitative molecular tissue atlas of Bis(monoacylglycero)phosphate and phosphatidylglycerol membrane lipids in rodent organs generated by methylation assisted high resolution mass spectrometry

Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10 PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.

Physiological and Transcriptomic Changes during the Early Phases of Adventitious Root Formation in Mulberry Stem Hardwood Cuttings.

The initiation and induction of root primordia are of great importance for adventitious root (AR) formation in cutting propagation of horticultural and forestry crops. However, the underlying mechanisms orchestrating these early phases of AR formation remain largely unexplored. Here, we investigated the physiological and transcriptomic changes during the early AR phases in mulberry stem hardwood cuttings. The results showed that the concentrations of soluble proteins increased, whereas concentrations of soluble sugars and starch were decreased. Indole-3-acetic acid (IAA) and zeatin had a rapid transit peak at 6 h after planting (hAP) and declined thereafter. The activities of peroxidase and catalase persistently increased and indole-3-acetic acid oxidase was maintained at a higher stable level from 0 hAP, while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels. The comparative transcriptome identified 4276 common genes that were differentially regulated at −6, 0 and 54 hAP. They were separated into five clusters with distinct biological functions such as defense response and photosynthesis. Considerable common genes were assigned to pathways of sugar metabolism, mitogen-activated protein kinase, and circadian rhythm. The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses, hormone signaling, energy metabolism, starch metabolism, and circadian rhythm. These findings demonstrate the positive effect of auxin on AR induction, and uncovered the crucial roles of stress responses, hormone signaling and circadian rhythm in coordinating the physiological changes during the early phases of AR formation in mulberry stem hardwood cuttings.

Isoform specific editing of the coxsackievirus and adenovirus receptor

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.

Regulation of long non-coding RNAs and circular RNAs in spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are one of the most significant stem cells with the potentials of self-renewal, differentiation, transdifferentiation and dedifferentiation, and thus, they have important applications in reproductive and regenerative medicine. They can transmit the genetic and epigenetic information across generations, which highlights the importance of the correct establishment and maintenance of epigenetic marks. Accurate transcriptional and post-transcriptional regulation is required to support the highly coordinated expression of specific genes for each step of spermatogenesis. Increasing evidence indicates that non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play essential roles in controlling gene expression and fate determination of male germ cells. These ncRNA molecules have distinct characteristics and biological functions, and they independently or cooperatively modulate the proliferation, apoptosis and differentiation of SSCs. In this review, we summarized the features, biological function and fate of mouse and human SSCs, and we compared the characteristics of lncRNAs and circRNAs. We also addressed the roles and mechanisms of lncRNAs and circRNAs in regulating mouse and human SSCs, which would add novel insights into the epigenetic mechanisms underlying mammalian spermatogenesis and provide new approaches to treat male infertility.

Abstract 114: Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype

Abstract Background: Fibronectin-binding αv and α5 integrins mediate homing, adhesive and survival interactions of prostate cancer cells with bone-marrow mesenchymal stromal cells. Monospecific αv integrin antibody therapy slowed progression of bone metastases but failed to impact overall mortality in prostate cancer. Cross-regulation between αv and α5 integrin is a potential source of adaptive resistance to monospecific blockade of either integrin. We assessed the patterns of expression of these partner integrins in bone metastases and the transition from the physiological gland to the malignant phenotype. Methods Formalin-fixed paraffin-embedded (FFPE) radical prostatectomy samples (n=25) from patients with a ≥ Gleason grade 4 component and decalcified FFPE samples of prostate cancer bone metastases (n=10) were obtained from institutional tissue biorepository. Optimized immunohistochemistry methods developed in prostate cancer cell suspensions were applied to assess expression patterns of αv and α5 integrins in benign and malignant glandular elements in primary tumors and bone specimens. Results Integrin αv was universally expressed in the physiological basal layer of benign prostate glands (n=25;100%) but not in the luminal epithelium. With loss of the basal layer in malignant transition, αv expression was recapitulated in 100% of malignant glandular epithelium in distinct patterns including epithelial membranous (24/25;96%), luminal membranous (6/25; 24%), punctate cytoplasmic (14/25;56%), intense foci of membranous staining (single cells or clusters surrounded by αv-negative tumor) (10/25;40%), and rim stromal patterns (14/25;56%). Luminal membranous and rim stromal αv expression patterns were striking in tumors with cribriform morphology. Furthermore, integrin αv was identified in all evaluable bone metastatic samples (7/7:100%). By contrast, integrin α5 was identified infrequently in the physiological basal layer (1/10;10%), was not expressed in malignant glandular epithelium of primary tumors but was paradoxically expressed in malignant epithelium in bone metastases (5/7:71%). Conclusion Integrin αv expression is universally and exclusively found in the physiological basal layer of the normal gland that harbors stem-functions and is recapitulated in high frequency in prostatic adenocarcinomas in diverse patterns suggestive of distinct biological functions that may contribute to disease progression. Co-expression and enrichment of integrins αv and α5 in osseous metastases is supportive of a proposed cooperative role of these fibronectin-binding integrins toward bone colonization. Given the adaptive cross-regulation we have observed with targeting of either integrin, combined αv and α5 integrin targeting in prostate cancer bone metastases may be required for effective therapy. Citation Format: Brendan Connell, Pavel Kopach, Wenying Ren, Raghav Joshi, Steven Naber, Paul Mathew. Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 114.

Phase separation in biology and disease-a symposium report.

Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.