Macrophage-Colony-Stimulating Factor Receptor Enhances Prostate Cancer Cell Growth and Aggressiveness In Vitro and In Vivo and Increases Osteopontin Expression.

Prostate cancer is a major public health concern and one of the most prevalent forms of cancer worldwide. The definition of altered signaling pathways implicated in this complex disease is thus essential. In this context, abnormal expression of the receptor of Macrophage Colony-Stimulating Factor-1 (M-CSF or CSF-1) has been described in prostate cancer cells. Yet, outcomes of this expression remain unknown. Using mouse and human prostate cancer cell lines, this study has investigated the functionality of the wild-type CSF-1 receptor in prostate tumor cells and identified molecular mechanisms underlying its ligand-induced activation. Here, we showed that upon CSF-1 binding, the receptor autophosphorylates and activates multiple signaling pathways in prostate tumor cells. Biological experiments demonstrated that the CSF-1R/CSF-1 axis conferred significant advantages in cell growth and cell invasion in vitro. Mouse xenograft experiments showed that CSF-1R expression promoted the aggressiveness of prostate tumor cells. In particular, we demonstrated that the ligand-activated CSF-1R increased the expression of spp1 transcript encoding for osteopontin, a key player in cancer development and metastasis. Therefore, this study highlights that the CSF-1 receptor is fully functional in a prostate cancer cell and may be a potential therapeutic target for the treatment of prostate cancer.

Expression of CD300lf by microglia contributes to resistance to cerebral malaria by impeding the neuroinflammation.

Genetic mapping and genome-wide studies provide evidence for the association of several genetic polymorphisms with malaria, a complex pathological disease with multiple severity degrees. We have previously described Berr1and Berr2 as candidate genes identified in the WLA/Pas inbreed mouse strain predisposing to resistance to cerebral malaria (CM) induced by P. berghei ANKA. We report in this study the phenotypic and functional characteristics of a congenic strain we have derived for Berr2WLA allele on the C57BL/6JR (B6) background. B6.WLA-Berr2 was found highly resistant to CM compared to C57BL/6JR susceptible mice. The mechanisms associated with CM resistance were analyzed by combining genotype, transcriptomic and immune response studies. We found that B6.WLA-Berr2 mice showed a reduced parasite sequestration and blood-brain barrier disruption with low CXCR3+ T cell infiltration in the brain along with altered glial cell response upon P. berghei ANKA infection compared to B6. In addition, we have identified the CD300f, belonging to a family of Ig-like encoding genes, as a potential candidate associated with CM resistance. Microglia cells isolated from the brain of infected B6.WLA-Berr2 mice significantly expressed higher level of CD300f compared to CMS mice and were associated with inhibition of inflammatory response.

Pyrrolomycins Are Potent Natural Protonophores.

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

N-confused porphyrin tautomers: lessons from density functional theory.

Using first-principle calculations, we characterize the properties of N-confused porphyrins (NCP), with a focus on the differences between the 2H and 3H tautomers. We find that NCP-3H is almost as strongly aromatic as porphyrin, and about twice as aromatic, i.e., remarkably more stable, than NCP-2H, due to the less efficient π-conjugation in the latter form. The deprotonation of the NH-group at the external side of the inverted ring of NCP-2H, adds a lone pair to the π-system, which restores a strong aromaticity, while methylation has no significant effect. Investigating the impact of solvation using a continuum model, we find quite stable solvation energies with a relative dielectric constant, εr, in the 5-40 range, for both tautomers. NCP-3H presents a slightly lower energy than its NCP-2H counterpart in all solvents. However, the energy differences between the two species are of the order of the error margin of the method, hence too small to discuss the experimentally observed stabilization of NCP-3H in dichloromethane (DCM, a poorly polar solvent) and NCP-2H in N,N-dimethylformamide (DMF, a strongly polar solvent) or to extract the population ratios between the two forms in the different solvents. Therefore, the vibronic absorption spectra are also investigated in an effort to rationalize the complex absorption profiles of these NCP derivatives. We find very distinct spectra for the 2H and 3H forms in DMF and DCM, respectively, each fairly reproducing the experiment. We also find that, in the same solvent, the two species exhibit very different signatures, which allows us to conclude that the 2H and 3H tautomers are largely dominant in DMF and DCM, respectively. Interestingly, the vibrational motions that strongly participate in the shoulder of the Soret band and the multiple maxima of the Q-bands largely differ in the two tautomers.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Assessment of adherence to at-home oral anti-infective therapy among paediatric patients discharged from a Quebec hospital.

OBJECTIVES: Non-adherence to anti-infective therapy contributes to treatment failure and the emergence of bacterial resistance. This study aimed to assess at-home adherence, by paediatric patients, to oral anti-infective (OAI) therapy prescribed for treatment of acute infections and to explore the factors contributing to non-adherence. METHODS: This prospective descriptive study involved French-speaking patients under 16 years of age who were discharged with one or more OAIs prescribed for home administration for a maximum of 30 days. Telephone surveys were used to assess overall adherence, which consisted of primary adherence (patient's ability to procure the medication) and secondary adherence (patient's ability to take the treatment as prescribed). RESULTS: Overall, 51.7% (30/58) of patients were adherent to OAI therapy, with 100% primary adherence (n=69/69) and 51.7% secondary adherence (n=30/58). On average, patients took 98% of the total number of doses prescribed, and non-adherence was related mostly to not following medication administration schedules (63.3% of patients followed the exact schedule). Indeed, the adherence rate for patients taking one or two doses per day was twice the rate for patients taking more than two doses per day (81.8% vs 44.7%, p=0.043). CONCLUSIONS: Half of the paediatric patients treated for acute infections were non-adherent to OAI therapy at home. Interventions are needed to improve this situation.

Flow Cytometry-based Method for Efficient Sorting of Senescent Cells.

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-ß-galactosidase (SA-ß-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-ß-D-galactopyranoside (C12FDG), a fluorigenic substrate of ß-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract.

The simplest method for in vitro ß-cell production from human adult stem cells.

Diabetes mellitus is a challenging autoimmune disease. Biomedical researchers are currently exploring efficient and effective ways to solve this challenge. The potential of stem cell therapies for treating diabetes represents one of the major focuses of current research on diabetes treatment. Here, we have attempted to differentiate adult stem cells from umbilical cord blood-derived mesenchymal cells (UCB-MSC), Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) and amniotic epithelial stem cells (AE-SC) into insulin-producing cells. The serum-free protocol developed in this study resulted in the differentiation of cells into definitive endoderm, pancreatic foregut, pancreatic endoderm and, finally, pancreatic endocrine cells, which expressed the marker genes SOX17, PDX1, NGN3, NKX6.1, INS, GCG, and PPY, respectively. Detection of the expression of the gap junction-related gene connexin-36 (CX36) using RT-PCR provided conclusive evidence for insulin-producing cell differentiation. In addition to this RT-PCR result, insulin and C-peptide protein were detected by immunohistochemistry and ELISA. Glucose stimulation test results showed that significantly greater amounts of C-peptide and insulin were released from differentiated cells than from undifferentiated cells. In conclusion, the methods investigated in this study can be considered an effective and efficient means of obtaining insulin-producing cells from adult stem cells within a week.

Exploration de l'association possible entre la consommation d'antibiotiques et l'émergence de résistance dans un centre hospitalier universitaire mère-enfant.

Background: The emergence of antibiotic resistance has contributed to the development of multidrug-resistant bacteria, which is a major concern. Objectives: The primary objective was to explore the possible association between antibiotic use and the emergence of resistance in a mother-child university hospital. Method: This retrospective study was conducted in a university hospital centre. Antibiotic-bacteria pairs were established, taking into account the number of isolates, actual antibiotic use, and clinical relevance. For each pair, a comparison of 2 variables (antibiotic utilization and rate of resistance) was quantified with the Pearson coefficient. Three analyses were conducted: no lag between utilization and resistance, 1-year lag, and 2-year lag. Results: Thirty antibiotic-bacteria pairs were selected from hematology-oncology and 18 from neonatology. In hematology-oncology, 6 pairs had a positive correlation (Pearson coefficient > 0.7): 2 pairs involving meropenem, 2 involving ceftazidime, and 2 involving piperacillin-tazobactam. In 3 of these cases, there was no lag between consumption of antibiotics and presence of resistance. In neonatology, 3 antibiotic-bacteria pairs had a positive correlation, 1 each involving vancomycin, cloxacillin, and meropenem. Conclusions: It is possible to explore the potential association between consumption of antibiotics and emergence of resistance in a particular centre. Our exploratory approach was based on manual data processing. It would be interesting to consider a continuous systematic approach, allowing automatic generation of correlations.

A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.

Developmental outcome after a single episode of status epilepticus.

Consequences of status epilepticus (SE) on psychomotor development and the specific impact of the convulsive event on emerging executive functions remain controversial. Infants treated for a single episode of SE, those treated for a single febrile seizure, and healthy infants were tested with respect to motor development, language, personal, and social skills and self-regulation. The children were divided into two age groups to investigate the impact of the convulsive event at different windows of brain maturation. We found that infants who had had SE were inferior to healthy controls on the development scales. Age differentiated SE impact on visuomotor development versus sociolinguistic development. Children who had been treated for SE had significantly more difficulties delaying a response to an attractive stimulus in one of the long-delay conditions. A single episode of SE can interfere with psychomotor and cognitive development in children without previous developmental delay, and it seems that the functions that are emerging at the time of insult are most vulnerable.

Profile of Antimicrobial Use in the Pediatric Population of a University Hospital Centre, 2015/16 to 2018/19.

BACKGROUND: Antimicrobial stewardship is a standard practice in health facilities to reduce both the misuse of antimicrobials and the risk of resistance. OBJECTIVE: To determine the profile of antimicrobial use in the pediatric population of a university hospital centre from 2015/16 to 2018/19. METHODS: In this retrospective, descriptive, cross-sectional study, the pharmacy information system was used to determine the number of days of therapy (DOTs) and the defined daily dose (DDD) per 1000 patient-days (PDs) for each antimicrobial and for specified care units in each year of the study period. For each measure, the ratio of 2018/19 to 2015/16 values was also calculated (and expressed as a proportion); where the value of this proportion was ≤ 0.8 or ≥ 1.2 (indicating a substantial change over the study period), an explanatory rating was assigned by consensus. RESULTS: Over the study period, 94 antimicrobial agents were available at the study hospital: 70 antibiotics (including antiparasitics and antituberculosis drugs), 14 antivirals, and 10 antifungals. The total number of DOTs per 1000 PDs declined from 904 in 2015/16 to 867 in 2018/19. The 5 most commonly used antimicrobials over the years, expressed as minimum/maximum DOTs per 1000 PDs, were piperacillin-tazobactam (78/105), trimethoprim-sulfamethoxazole (74/84), ampicillin (51/69), vancomycin (53/68), and cefotaxime (55/58). In the same period, the care units with the most antimicrobial use (expressed as minimum/maximum DOTs per 1000 PDs) were hematology-oncology (2529/2723), pediatrics (1006/1408), and pediatric intensive care (1328/1717). CONCLUSIONS: This study showed generally stable consumption of antimicrobials from 2015/16 to 2018/19 in a Canadian mother-and-child university hospital centre. Although consumption was also stable within drug groups (antibiotics, antivirals, and antifungals), there were important changes over time for some individual drugs. Several factors may explain these variations, including disruptions in supply, changes in practice, and changes in the prevalence of infections. Surveillance of antimicrobial use is an essential component of an antimicrobial stewardship program.

CONTEXTE: La gestion des antimicrobiens est une pratique courante dans les centres hospitaliers afin de réduire l'utilisation inappropriée des antimicrobiens et le risque de résistance. OBJECTIF: Décrire l'évolution de l'utilisation des antimicrobiens dans un centre hospitalier universitaire de 2015­16 à 2018­19. MÉTHODES: Dans cette étude rétrospective, descriptive et transversale, les dossiers pharmacologiques ont servi à déterminer le nombre de jours de traitement (NJT) et la dose définie journalière (DDD) par 1000 jours-présence (JP) pour chaque antimicrobien et pour chaque unité de soins par année de l'étude. Pour chaque mesure, on a également comparé le ratio de 2018­19 à celui de 2015­16, qui est exprimé en proportion; lorsque la valeur de cette proportion était ≤ 0,8 ou ≥ 1,2, ce qui indiquait un changement important durant la période de l'étude, une note explicative a été attribuée par consensus. RÉSULTATS: Durant la période à l'étude, 94 antimicrobiens ont été disponibles dans notre centre : 70 antibiotiques (dont les antiparasitaires et les antituberculeux), 14 antiviraux et 10 antifongiques. Le nombre total de NJT par 1000 JP a diminué de 904 en 2015­16 à 867 en 2018­19. Les cinq antimicrobiens utilisés le plus fréquemment et présentés en minimum / maximum de NJT par 1000 JP étaient les suivants : piperacilline-tazobactam (78/105), trimethoprim-sulfamethoxazole (74/84), ampicilline (51/69), vancomycine (53/68) et cefotaxime (55/58). Pendant la même période, les unités de soins qui faisaient la plus grande utilisation d'antimirobiens (exprimée en minimum / maximum de NJT par 1000 JP) étaient hématologie-oncologie (2529/2723), pédiatrie (1006/1408) et soins intensifs pédiatriques (1328/1717). CONCLUSIONS: Cette étude démontre une consommation stable d'antimicrobiens entre 2015­16 et 2018­19 dans un centre hospitalier universitaire mère-enfant canadien. Malgré le fait que la consommation entre les groupes d'antimicrobiens (antibiotiques, antiviraux, antifongiques) était stable, on a constaté d'importantes variations concernant certains médicaments individuels. Plusieurs facteurs peuvent expliquer cette variation, notamment des ruptures d'approvisionnement, des changements de pratique et des changements dans la prévalence d'infections. La surveillance de la consommation des antimicrobiens est une partie essentielle de tout programme d'antibiogouvernance.

Suppression of mucosal Th17 memory responses by acellular pertussis vaccines enhances nasal Bordetella pertussis carriage.

Pertussis has made a spectacular rebound in countries that have switched from whole-cell (wPV) to acellular pertussis vaccines (aPV). Here, we show that, unlike wPV, aPV, while protective against lung colonization by Bordetella pertussis (Bp), did not protect BALB/c mice from nasal colonization, but instead substantially prolonged nasal carriage. aPV prevented the natural induction of nasal interleukin-17 (IL-17)-producing and interferon-γ (IFN-γ)-producing CD103+ CD44+ CD69+ CD4+-resident memory T (TRM) cells. IL-17-deficient, but not IFN-γ-deficient, mice failed to clear nasal Bp, indicating a key role of IL-17+ TRM cells in the control of nasal infection. These cells appeared essential for neutrophil recruitment, crucial for clearance of Bp tightly bound to the nasal epithelium. Transfer of IL-17+ TRM cells from Bp-infected mice to IL-17-deficient mice resulted in neutrophil recruitment and protection against nasal colonization. Thus, aPV may have augmented the Bp reservoir by inhibiting natural TRM cell induction and neutrophil recruitment, thereby contributing to the pertussis resurgence.

s-SHIP Promoter Expression Identifies Mouse Mammary Cancer Stem Cells.

During normal mammary gland development, s-SHIP promoter expression marks a distinct type of mammary stem cells, at two different stages, puberty and early mid-pregnancy. To determine whether s-SHIP is a marker of mammary cancer stem cells (CSCs), we generated bitransgenic mice by crossing the C3(1)-SV40 T-antigen transgenic mouse model of breast cancer, and a transgenic mouse (11.5kb-GFP) expressing green fluorescent protein from the s-SHIP promoter. Here we show that in mammary tumors originating in these bitransgenic mice, s-SHIP promoter expression enriches a rare cell population with CSC activity as demonstrated by sphere-forming assays in vitro and limiting dilution transplantation in vivo. These s-SHIP-positive CSCs are characterized by lower expression of Delta-like non-canonical Notch ligand 1 (DLK1), a negative regulator of the Notch pathway. Inactivation of Dlk1 in s-SHIP-negative tumor cells increases their tumorigenic potential, suggesting a role for DLK1 in mammary cancer stemness.

Feasibility of a Comfort Care Protocol Using Oral Transmucosal Medication Delivery in a Palliative Neonatal Population.

BACKGROUND: The oral transmucosal (OTM) route for administration of comfort medication in infants at the end-of-life has long been favored by our pediatric palliative care team but has rarely been described in the literature. OBJECTIVE: To determine the feasibility of implementing a standardized comfort care protocol using OTM medications in dying neonates. METHOD: A comfort protocol prescribing medication by the OTM route and standardized assessment were established. Each infant included in the study was assessed with the Neonatal Pain, Agitation, and Sedation Scale (N-PASS). Caretakers' satisfaction was assessed using a questionnaire. The feasibility of implementing the protocol was determined by the proportion of assessments done when required, the rate of termination of the protocol, and the feedback from nurses using the protocol. RESULTS: Twelve patients were enrolled. Regular evaluations were performed 85% of the time. When the medication was given as needed, 71% of cases were evaluated before versus 63% when regular doses were given. The as-needed doses were followed by an assessment 30 minutes later in 49% of cases and in 41%, 60 minutes later, for a total of 64% in the hour after medication administration. The protocol was discontinued only for two patients who were discharged to continue end-of-life care at home. There were no significant adverse events reported. Finally, 17 of 18 nurses said they would recommend this protocol to other institutions. CONCLUSION: In the context of neonatal palliative care, the implementation of a standardized protocol for administration of drugs by the OTM route is feasible and safe. However, in the context of this study, adherence was limited because of too-frequent evaluations and misunderstanding of the protocol.

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin- CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells.

Enrichment of Human Stem-Like Prostate Cells with s-SHIP Promoter Activity Uncovers a Role in Stemness for the Long Noncoding RNA H19.

Understanding normal and cancer stem cells should provide insights into the origin of prostate cancer and their mechanisms of resistance to current treatment strategies. In this study, we isolated and characterized stem-like cells present in the immortalized human prostate cell line, RWPE-1. We used a reporter system with green fluorescent protein (GFP) driven by the promoter of s-SHIP (for stem-SH2-domain-containing 5'-inositol phosphatase) whose stem cell-specific expression has been previously shown. We observed that s-SHIP-GFP-expressing RWPE-1 cells showed stem cell characteristics such as increased expression of stem cell surface markers (CD44, CD166, TROP2) and pluripotency transcription factors (Oct4, Sox2), and enhanced sphere-forming capacity and resistance to arsenite-induced cell death. Concomitant increased expression of the long noncoding RNA H19 was observed, which prompted us to investigate a putative role in stemness for this oncofetal gene. Targeted suppression of H19 with siRNA decreased Oct4 and Sox2 gene expression and colony-forming potential in RWPE-1 cells. Conversely, overexpression of H19 significantly increased gene expression of these two transcription factors and the sphere-forming capacity of RWPE-1 cells. Analysis of H19 expression in various prostate and mammary human cell lines revealed similarities with Sox2 expression, suggesting that a functional relationship may exist between H19 and Sox2. Collectively, we provide the first evidence that s-SHIP-GFP promoter reporter offers a unique marker for the enrichment of human stem-like cell populations and highlight a role in stemness for the long noncoding RNA H19.

Usefulness of defined daily dose and days of therapy in pediatrics and obstetrics-gynecology: a comparative analysis of antifungal drugs (2000-2001, 2005-2006, and 2010-2011).

OBJECTIVES: The objective was to describe antifungal drug use by using the number of defined daily doses (DDD)/1000 patient-days per antifungal, the number of days of therapy (DOT)/1000 patient-days per antifungal, and the mean dose in mg/kg/day per antifungal during a 10-year period. METHODS: Retrospective, cross-sectional, descriptive study, in a mother-child university hospital center, with 400 pediatric beds and 100 obstetrics-gynecology beds. All inpatients who received 1 of the 7 authorized antifungals on the institution's local formulary in 2000-2001, 2005-2006, or 2010-2011 were included. Prescriptions for emergency department and outpatient clinics were excluded. The data were extracted from the patients' computerized medication profiles linked to patient admission, discharge, and transfer data. The DDD, DOT, and the mean dose in mg/kg/day were calculated for each antifungal and overall. RESULTS: There was a 2.97-fold increase in the overall number of DDD/1000 patient-days, from 14.8 in 2000-2001 to 37.5 in 2005-2006 and 43.9 in 2010-2011. There was a 2.97-fold increase in the overall number of DOT/1000 patient-days, from 22.8 in 2000-2001 to 50.3 in 2005-2006 and 67.8 in 2010-2011. CONCLUSIONS: It can be difficult to compare the use of antifungal drugs among institutions, owing to numerous factors, but it gives an idea about the consumption outside the studied center. Moreover, these ratios help to evaluate the use of antifungals within a same institution. These data could be correlated among others, with resistance patterns, in order to improve our daily practice concerning antifungal prescription.

Asymmetric distribution of epidermal growth factor receptor directs the fate of normal and cancer keratinocytes in vitro.

Cancer cells are unequal in a tumor mass and in established cultures. This is attributable to cancer stem cells with the unique ability to self-renew and to generate differentiating progeny. This ability is controlled at the level of asymmetric division by mechanisms that are yet not well defined. We found that normal and cancer keratinocyte fate was linked to the asymmetric distribution of epidermal growth factor receptor (EGFR) during mitosis. Although essential for epithelial cell proliferation, differentiation, and survival, this receptor was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability, and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, suggesting that EGFR may be involved in a switch from stem to transient amplifying cell fate. This switch was associated with changes in the expression profile of cell cycle, survival, and mitochondria controlling proteins that varied between normal and cancer cells. In conclusion, it appears that an unequal distribution of EGFR at mitosis controls keratinocyte fate by balancing quiescence and cycling of EGFR(-) cells, clearly malfunctioning in cancer. We believe that our findings provide mechanistic insights into the development of resistance to anti-EGFR therapies.