Neurotensin and neurotensin receptor 1 mRNA expression in song‐control regions changes during development in male zebra finches

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine‐related measures change throughout development in song‐control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song‐control regions of male zebra finches in four stages of the song learning process: pre‐subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre‐subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018     

Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin

MicroRNAs (miRNAs) regulate the expression of many mammalian genes and play key roles in embryonic hair follicle development; however, little is known of their functions in postnatal hair growth. We compared the effects of deleting the essential miRNA biogenesis enzymes Drosha and Dicer in mouse skin epithelial cells at successive postnatal time points. Deletion of either Drosha or Dicer during an established growth phase (anagen) caused failure of hair follicles to enter a normal catagen regression phase, eventual follicular degradation and stem cell loss. Deletion of Drosha or Dicer in resting phase follicles did not affect follicular structure or epithelial stem cell maintenance, and stimulation of anagen by hair plucking caused follicular proliferation and formation of a primitive transient amplifying matrix population. However, mutant matrix cells exhibited apoptosis and DNA damage and hair follicles rapidly degraded. Hair follicle defects at early time points post-deletion occurred in the absence of inflammation, but a dermal inflammatory response and hyperproliferation of interfollicular epidermis accompanied subsequent hair follicle degradation. These data reveal multiple functions for Drosha and Dicer in suppressing DNA damage in rapidly proliferating follicular matrix cells, facilitating catagen and maintaining follicular structures and their associated stem cells. Although Drosha and Dicer each possess independent non-miRNA-related functions, the similarity in phenotypes of the inducible epidermal Drosha and Dicer mutants indicates that these defects result primarily from failure of miRNA processing. Consistent with this, Dicer deletion resulted in the upregulation of multiple direct targets of the highly expressed epithelial miRNA miR-205.     

lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis

To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (CySC). Here, we show that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led us to examine the developmental relationship between CySCs and hub cells. In contrast to a previous report, we did not observe significant conversion of steady-state CySC progeny to hub fate. However, we found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, lines mutant embryos exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and we found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche.     

The carbon monoxide releasing molecule CORM-2 attenuates Pseudomonas aeruginosa biofilm formation

Chronic infections resulting from biofilm formation are difficult to eradicate with current antimicrobial agents and consequently new therapies are needed. This work demonstrates that the carbon monoxide-releasing molecule CORM-2, previously shown to kill planktonic bacteria, also attenuates surface-associated growth of the gram-negative pathogen Pseudomonas aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm. CORM-2 treatment has an additive effect when combined with tobramycin, a drug commonly used to treat P. aeruginosa lung infections. CORM-2 inhibited biofilm formation and planktonic growth of the majority of clinical P. aeruginosa isolates tested, for both mucoid and non-mucoid strains. While CORM-2 treatment increased the production of reactive oxygen species by P. aeruginosa biofilms, this increase did not correlate with bacterial death. These data demonstrate that CO-RMs possess potential novel therapeutic properties against a subset of P. aeruginosa biofilm related infections.     

Fibrinogen-like protein 1, a hepatocyte derived protein is an acute phase reactant

Fibrinogen-like protein 1 (FGL1) is a hepatocyte derived protein that is upregulated in regenerating rodent livers following partial hepatectomy. It has been implicated as a mitogen for liver cell proliferation. In this study, we show that recombinant human IL-6 induces FGL1 expression in Hep G2 cells in a pattern similar to those of acute phase reactants. Following induction of acute inflammation in rats by subcutaneous injection of turpentine oil, serum FGL1 levels are also enhanced. Although, a recent report suggests that FGL1 associates almost exclusively with the fibrin matrix, we report here that approximately 20% of the total plasma FGL1 remains free. The enhancement of FGL1 levels in vitro by IL-6 and its induction after turpentine oil injection suggest that it is an acute phase reactant. Its presence in bound and free forms in the blood also implies biological roles that extend beyond the proposed autocrine effect it has on hepatocytes during regeneration.     

p21-dependent inhibition of colon cancer cell growth by mevastatin is independent of inhibition of G1 cyclin-dependent kinases

Mevastatin arrested HCT116 colon cancer cells at the G1/S transition and increased cellular levels of p21CIP1/WAF1. p21-deficient colon cancer cells continued to proliferate in the presence of mevastatin. Although p21 was necessary for the G1/S block, the G1 cyclin-dependent kinases (Cdks) cyclin E-Cdk2 and cyclin D-Cdk4 remained active. Despite the activity of the G1 Cdks the retinoblastoma protein was hypophosphorylated due to unknown mechanisms that were dependent on the p21 protein. The resulting decrease in cyclin A mRNA and protein led to a decrease in the activity of cyclin A-Cdk2. Therefore, although p21 was required for the G1/S arrest of HCT116 colon cancer cells by mevastatin, its mode of action was more complicated than the simple formation of a physical complex with cyclin-Cdk2. This mechanism of inhibition is different from that seen in prostate cancer cells (Ukomadu, C., and Dutta, A. (2003) J. Biol. Chem. 278, 4840-4846) where the activating phosphorylation of cyclin E-Cdk2 is suppressed and p21 is not required, suggesting the existence of cell line-specific differences in the mechanism by which statins arrest the cell cycle.     

Inhibition of cdk2 activating phosphorylation by mevastatin

Phosphorylation of cdk2 on threonine 160 is essential for kinase activity. Mevastatin, an inhibitor of cholesterol synthesis, inhibits cell growth through inhibition of cdk2 and this has been suggested to be due to enhancement of p21 levels. In a prostate cancer cell line, PC3, mevastatin treatment led to elevated levels of p21 and caused a small increase in the p21 associated with cdk2. However, this increase in the associated p21 appeared out of proportion with the resulting dramatic inhibition of kinase activity. Using RNA interference we show that mevastatin inhibits cdk2 activity despite lack of induction of p21, p27, and p57. Instead the kinase was inhibited due to a decrease in activating phosphorylation. Phosphorylation of cdk2 from mevastatin-treated cells with exogenous cyclin-dependent kinase (cdk)-activating enzymes restored its functional activity. The only known mammalian cyclin H.cdk7.mat1 complex (cdk2-activating kinase, Cak), was not inhibited by mevastatin, suggesting either that a different CAK is responsible for cdk2 phosphorylation in vivo or that the regulation is at the level of substrate accessibility or of cdk2 dephosphorylation. These results suggest that mevastatin inhibits cdk2 activity in PC3 cells through the inhibition of Thr-160 phosphorylation of cdk2, providing a novel example of regulation of cdk2 at this level.     

Targeted Deletion of Fibrinogen Like Protein 1 Reveals a Novel Role in Energy Substrate Utilization

Fibrinogen like protein 1(Fgl1) is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.     

Candida albicans Ethanol Stimulates Pseudomonas aeruginosa WspR-Controlled Biofilm Formation as Part of a Cyclic Relationship Involving Phenazines

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.