Transcriptome analysis reveals an Atoh1b-dependent gene set downstream of Dlx3b/4b during early inner ear development in zebrafish.

The vertebrate inner ear is the sensory organ mediating hearing and balance. The entire organ develops from the otic placode, which itself originates from the otic-epibranchial progenitor domain (OEPD). Multiple studies in various species have shown the importance of the forkhead-box and distal-less homeodomain transcription factor families for OEPD and subsequent otic placode formation. However, the transcriptional networks downstream of these factors are only beginning to be understood. Using transcriptome analysis, we here reveal numerous genes regulated by the distal-less homeodomain transcription factors Dlx3b and Dlx4b (Dlx3b/4b). We identify known and novel transcripts displaying widespread OEPD expression in a Dlx3b/4b-dependent manner. Some genes, with a known OEPD expression in other vertebrate species, might be members of a presumptive vertebrate core module required for proper otic development. Moreover, we identify genes controlling early-born sensory hair cell formation as well as regulating biomineral tissue development, both consistent with defective sensory hair cell and otolith formation observed in dlx3b/4b mutants. Finally, we show that ectopic Atoh1b expression can rescue early sensorigenesis even in the absence of Dlx3b/4b. Taken together, our data will help to unravel the gene regulatory network underlying early inner ear development and provide insights into the molecular control of vertebrate inner ear formation to restore hearing loss in humans ultimately.

P90 ribosomal S6 kinases: A bona fide target for novel targeted anticancer therapies?

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases. They are downstream effectors of the Ras/ERK/MAPK signaling cascade. ERK1/2 activation directly results in the phosphorylation of RSKs, which further, through interaction with a variety of different downstream substrates, activate various signaling events. In this context, they have been shown to mediate diverse cellular processes like cell survival, growth, proliferation, EMT, invasion, and metastasis. Interestingly, increased expression of RSKs has also been demonstrated in various cancers, such as breast, prostate, and lung cancer. This review aims to present the most recent advances in the field of RSK signaling that have occurred, such as biological insights, function, and mechanisms associated with carcinogenesis. We additionally present and discuss the recent advances but also the limitations in the development of pharmacological inhibitors of RSKs, in the context of the use of these kinases as putative, more efficient targets for novel anticancer therapeutic approaches.

Bioinformatic Analysis of the BCL-xL/BCL2L1 Interactome in Patients with Pancreatic Cancer.

Objectives: The aim of the present study was to analyze the differential gene expression of BCL-xL/BCL2L and the associated genetic, molecular, and biologic functions in pancreatic ductal adenocarcinoma (PDAC) by employing advanced bioinformatics to investigate potential candidate genes implicated in the pathogenesis of PDAC. Materials and Methods: Bioinformatic techniques were employed to build the gene network of BCL-xL, to assess the translational profile of BCL-xL in PDAC, assess its role in predicting PDAC, and investigate the associated biologic functions and the regulating miRNA families. Results: Microarray data extracted from one dataset was incorporated, including 130 samples (PDAC: 69; Control: 61). In addition, the expression level of BCL-xL was higher in PDAC compared to control samples (p < 0.001). Furthermore, BCL-xL demonstrated excellent discrimination (AUC: 0.83 [95% Confidence Intervals: 0.76, 0.90]; p < 0.001) and calibration (R squared: 0.31) traits for PDAC. A gene set enrichment analysis (GSEA) demonstrated the molecular functions and miRNA families (hsa-miR-4804-5p, hsa-miR-4776-5p, hsa-miR-6770-3p, hsa-miR-3619-3p, and hsa-miR-7152-3p) related to BCL-xL. Conclusions: The current findings unveil the biological implications of BCL-xL in PDAC and the related molecular functions and miRNA families.

Effect of Direct Current Electric Fields on Cone Like Retinal Photoreceptor Cells.

INTRODUCTION: Studies show that electric fields are used as therapy during nerve and tissue injuries along with trans-retinal stimulation. However, cellular and molecular changes induced by such treatments remain largely unknown especially in retinal photoreceptor cells. In vitro studies show that direct current electric fields (dcEF) were known to influence cell division, polarity, shape, and motility. Here we could characterize for the first time the reactions of 661W, a retinal cone photoreceptor especially regarding organelle polarization, membrane polarization of mitochondria, O2 consumption, ATP/ADP ratio and gene expression. METHODS: The 661W cells were stimulated with a constant dcEF of field strength 5 V/cm during 30 min or 5 h depending on the parameters studied. RESULTS: In response to dcEF, the cells aligned perpendicular to the field by forming a leading edge with extended membrane protrusions towards the cathode. Using immunofluorescence and live cell imaging, we show that the cell membrane depolarized at the cathodal side. The microtubules spread into the direction of migration. Also, the microtubule organization center re-oriented into this direction. Concomitantly with the microtubules, actin filaments reorganized in an asymmetrical fashion mainly at the cathodal side. The Golgi apparatus, which is involved in many steps of actin synthesis, moved to the cathodal side. In the last 2 h of the 5 h experiment, microtubules positioned themselves at the rear (anodal side), like the nucleus. The averaged displacement of the whole cells under dcEF was 155% of control for 3 V/cm and 235% for 5 V/cm. The average speed increased by 142% and 243% respectively. Inside the cells mitochondria moved to the cathodal side, where the energy consuming producing processes take place. In this line, we measured an increase in ATP production and O2 consumption. Mitochondrial calcium was found more on the anodal side, at the site of the nucleus with its calcium delivering endoplasmic reticulum. In addition, oxymetry studies reveal an increased ATP synthesis by 115.2% and oxygen consumption by 113.3% 3 h after dcEF stimulation. An analysis of differentially expressed genes by RNA sequencing revealed an upregulation of genes involved in cellular movement, cell to cell and intracellular signaling, molecular transport, assembly and organization. CONCLUSIONS: The mechanisms found can enhance our understanding regarding the beneficial effects of EF treatment in retinal diseases.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Epigenetic modifier balances Mapk and Wnt signalling in differentiation of goblet and Paneth cells.

Differentiation and lineage specification are controlled by cooperation of growth factor signalling. The involvement of epigenetic regulators in lineage specification remains largely elusive. Here, we show that the histone methyltransferase Mll1 prevents intestinal progenitor cells from differentiation, whereas it is also involved in secretory lineage specification of Paneth and goblet cells. Using conditional mutagenesis in mice and intestinal organoids, we demonstrate that loss of Mll1 renders intestinal progenitor cells permissive for Wnt-driven secretory differentiation. However, Mll1-deficient crypt cells fail to segregate Paneth and goblet cell fates. Mll1 deficiency causes Paneth cell-determined crypt progenitors to exhibit goblet cell features by unleashing Mapk signalling, resulting in increased numbers of mixed Paneth/goblet cells. We show that loss of Mll1 abolishes the pro-proliferative effect of Mapk signalling in intestinal progenitor cells and promotes Mapk-induced goblet cell differentiation. Our data uncover Mll1 and its downstream targets Gata4/6 as a regulatory hub of Wnt and Mapk signalling in the control of lineage specification of intestinal secretory Paneth and goblet cells.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation.

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

Clonal Analysis Delineates Transcriptional Programs of Osteogenic and Adipogenic Lineages of Adult Mouse Skeletal Progenitors.

Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.

Zebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells.

The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34+ cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34+ cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.

Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Aldehyde dehydrogenase activity is necessary for beta cell development and functionality in mice.

AIMS/HYPOTHESIS: Pancreatic beta cells maintain glucose homeostasis and beta cell dysfunction is a major risk factor in developing diabetes. Therefore, understanding the developmental regulatory networks that define a fully functional beta cell is important for elucidating the genetic origins of the disease. Aldehyde dehydrogenase activity has been associated with stem/progenitor cells and we have previously shown that Aldh1b1 is specifically expressed in pancreas progenitor pools. Here we address the hypothesis that Aldh1b1 may regulate the timing of the appearance and eventual functionality of beta cells. METHODS: We generated an Aldh1b1-knockout mouse line (Aldh1b1 (tm1lacZ)) and used this to study pancreatic development, beta cell functionality and glucose homeostasis in the absence of Aldh1b1 function. RESULTS: Differentiation in the developing pancreas of Aldh1b1 (tm1lacZ) null mice was accelerated. Transcriptome analyses of newborn and adult islets showed misregulation of key beta cell transcription factors and genes crucial for beta cell function. Functional analyses showed that glucose-stimulated insulin secretion was severely compromised in islets isolated from null mice. Several key features of beta cell functionality were affected, including control of oxidative stress, glucose sensing, stimulus-coupling secretion and secretory granule biogenesis. As a result of beta cell dysfunction, homozygous mice developed glucose intolerance and age-dependent hyperglycaemia. CONCLUSIONS/INTERPRETATION: These findings show that Aldh1b1 influences the timing of the transition from the pancreas endocrine progenitor to the committed beta cell and demonstrate that changes in the timing of this transition lead to beta cell dysfunction and thus constitute a diabetes risk factor later in life. Gene Expression Omnibus (GEO) accession: GSE58025.

Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming.

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

SAMHD1 prevents autoimmunity by maintaining genome stability.

OBJECTIVES: The HIV restriction factor, SAMHD1 (SAM domain and HD domain-containing protein 1), is a triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs). Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory disorder that shares phenotypic similarity with systemic lupus erythematosus, including activation of antiviral type 1 interferon (IFN). To further define the pathomechanisms underlying autoimmunity in AGS due to SAMHD1 mutations, we investigated the physiological properties of SAMHD1. METHODS: Primary patient fibroblasts were examined for dNTP levels, proliferation, senescence, cell cycle progression and DNA damage. Genome-wide transcriptional profiles were generated by RNA sequencing. Interaction of SAMHD1 with cyclin A was assessed by coimmunoprecipitation and fluorescence cross-correlation spectroscopy. Cell cycle-dependent phosphorylation of SAMHD1 was examined in synchronised HeLa cells and using recombinant SAMHD1. SAMHD1 was knocked down by RNA interference. RESULTS: We show that increased dNTP pools due to SAMHD1 deficiency cause genome instability in fibroblasts of patients with AGS. Constitutive DNA damage signalling is associated with cell cycle delay, cellular senescence, and upregulation of IFN-stimulated genes. SAMHD1 is phosphorylated by cyclin A/cyclin-dependent kinase 1 in a cell cycle-dependent manner, and its level fluctuates during the cell cycle, with the lowest levels observed in G1/S phase. Knockdown of SAMHD1 by RNA interference recapitulates activation of DNA damage signalling and type 1 IFN activation. CONCLUSIONS: SAMHD1 is required for genome integrity by maintaining balanced dNTP pools. dNTP imbalances due to SAMHD1 deficiency cause DNA damage, leading to intrinsic activation of IFN signalling. These findings establish a novel link between DNA damage signalling and innate immune activation in the pathogenesis of autoimmunity.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Identification and expression patterns of novel long non-coding RNAs in neural progenitors of the developing mammalian cortex.

Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis.

KLK11 mRNA expression predicts poor disease-free and overall survival in colorectal adenocarcinoma patients.

BACKGROUND: Dysregulated expression of several KLK family members has been observed in colorectal adenocarcinoma. In the present study, the prognostic value of KLK11 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma was examined. MATERIALS & METHODS: Using quantitative real-time PCR, KLK11 mRNA expression was studied in 120 cancerous and 41 paired noncancerous colorectal specimens obtained from 120 patients with primary colorectal adenocarcinoma. RESULTS: A significant upregulation of KLK11 transcripts in colorectal tumors was observed. KLK11 mRNA expression was associated with the depth of tumor invasion and the histological grade. Furthermore, KLK11 mRNA expression predicted poor disease-free and overall survival, independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy and chemotherapy treatment. CONCLUSION: KLK11 mRNA expression could be considered as a new molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients with highly invasive tumors and/or positive lymph nodes.

Kallikrein-related peptidase-6 (KLK6) mRNA expression is an independent prognostic tissue biomarker of poor disease-free and overall survival in colorectal adenocarcinoma.

Members of the family of tissue kallikrein and kallikrein-related peptidases possess important prognostic value in cancer. Moreover, the oncogenic role of kallikrein-related peptidase-6 (KLK6) in colorectal cancer has been well documented so far. This study investigated the prognostic value of KLK6 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma. For this purpose, KLK6 mRNA expression was studied in 110 primary colorectal adenocarcinomas and 39 paired noncancerous colorectal specimens. A dramatic upregulation of KLK6 mRNA expression was observed in colorectal tumors. KLK6 mRNA overexpression was associated with high depth of tumor invasion, presence of distant metastases, and tumor-node-metastasis (TNM) stage of patients. Furthermore, KLK6 mRNA expression was shown to predict poor disease-free and overall survival independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy, and chemotherapy treatment. Moreover, Kaplan-Meier survival analysis revealed that colorectal adenocarcinoma patients with negative regional lymph nodes (N0) and those without distant metastases (M0) harboring KLK6 mRNA-positive colorectal tumors tended to relapse and die earlier than N0 and M0 patients with KLK6 mRNA-negative colorectal adenocarcinoma. Thus, KLK6 mRNA expression could be considered as an independent, unfavorable molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients without regional or distant metastases.

Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells.

Long-term hematopoietic stem cells (HSCs [LT-HSCs]) are well known to display unpredictable differences in their clonal expansion capacities after transplantation. Here, by analyzing the cellular output after transplantation of stem cells differing in surface expression levels of the Kit receptor, we show that LT-HSCs can be systematically subdivided into two subtypes with distinct reconstitution behavior. LT-HSCs expressing intermediate levels of Kit receptor (Kit(int)) are quiescent in situ but proliferate extensively after transplantation and therefore repopulate large parts of the recipient's hematopoietic system. In contrast, metabolically active Kit(hi) LT-HSCs display more limited expansion capacities and show reduced but robust levels of repopulation after transfer. Transplantation into secondary and tertiary recipient mice show maintenance of efficient repopulation capacities of Kit(int) but not of Kit(hi) LT-HSCs. Initiation of differentiation is marked by the transit from Kit(int) to Kit(hi) HSCs, both of which precede any other known stem cell population.