Cell Kinetic Studies Fail to Identify Sequentially Proliferating Progenitors as the Major Source of Epithelial Renewal in the Adult Murine Prostate

There is evidence that stem cells and their progeny play a role in the development of the prostate. Although stem cells are also considered to give rise to differentiated progeny in the adult prostate epithelium ex vivo, the cohort of adult prostate stem cells in vivo as well as the mechanisms by which the adult prostate epithelium is maintained and regenerated remain highly controversial. We have attempted to resolve this conundrum by performing in vivo tracing of serially replicating cells after the sequential administration of two thymidine analogues to mice. Our results show that, during normal prostate homeostasis, sequentially proliferating cells are detected at a rate that is consistent with a stochastic process. These findings indicate that in vivo, under steady-state conditions, most adult prostate epithelial cells do not represent the progeny of a small number of specialized progenitors that generate sequentially replicating transit-amplifying (TA) cells but are formed by stochastic cell division. Similarly, no rapidly cycling TA cells were detected during regeneration following one cycle of androgen-mediated involution/regeneration of the prostate epithelium. These findings greatly enhance our understanding of the mechanisms regulating prostate epithelial cell renewal and may have significant implications in defining the cell of origin of proliferative prostatic diseases.     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.     

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [¹³C₆]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [¹³C₆]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.     

Jasmonate and ppHsystemin Regulate Key Malonylation Steps in the Biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides,an Abundant and Effective Direct Defense against Herbivores in Nicotiana attenuata

We identified 11 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) that occur in concentrations equivalent to starch (mg/g fresh mass) in aboveground tissues of coyote tobacco (Nicotiana attenuata) and differ in their sugar moieties and malonyl sugar esters (0-2). Concentrations of HGL-DTGs, particularly malonylated compounds, are highest in young and reproductive tissues. Within a tissue, herbivore elicitation changes concentrations and biosynthetic kinetics of individual compounds. Using stably transformed N. attenuata plants silenced in jasmonate production and perception, or production of N. attenuata Hyp-rich glycopeptide systemin precursor by RNA interference, we identified malonylation as the key biosynthetic step regulated by herbivory and jasmonate signaling. We stably silenced N. attenuata geranylgeranyl diphosphate synthase (ggpps) to reduce precursors for the HGL-DTG skeleton, resulting in reduced total HGL-DTGs and greater vulnerability to native herbivores in the field. Larvae of the specialist tobacco hornworm (Manduca sexta) grew up to 10 times as large on ggpps silenced plants, and silenced plants suffered significantly more damage from herbivores in N. attenuata''s native habitat than did wild-type plants. We propose that high concentrations of HGL-DTGs effectively defend valuable tissues against herbivores and that malonylation may play an important role in regulating the distribution and storage of HGL-DTGs in plants.     

Biophysical Characterization of an Ensemble of Intramolecular i-Motifs Formed by the Human c-MYC NHE III1 P1 Promoter Mutant Sequence

i-Motif-forming sequences are present in or near the regulatory regions of >40% of all genes, including known oncogenes. We report here the results of a biophysical characterization and computational study of an ensemble of intramolecular i-motifs that model the polypyrimidine sequence in the human c-MYC P1 promoter. Circular dichroism results demonstrate that the mutant sequence (5′-CTT TCC TAC CCTCCC TAC CCT AA-3′) can adopt multiple “i-motif-like,” classical i-motif, and single-stranded structures as a function of pH. The classical i-motif structures are predominant in the pH range 4.2-5.2. The “i-motif-like” and single-stranded structures are the most significant species in solution at pH higher and lower, respectively, than that range. Differential scanning calorimetry results demonstrate an equilibrium mixture of at least three i-motif folded conformations with T_m values of 38.1, 46.6, and 49.5°C at pH 5.0. The proposed ensemble of three folded conformations includes the three lowest-energy conformations obtained by computational modeling and two folded conformers that were proposed in a previous NMR study. The NMR study did not report the most stable conformer found in this study.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Membrane Damage Elicits an Immunomodulatory Program in Staphylococcus aureus

The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating ΔhrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host.