SET1 and p300 act synergistically, through coupled histone modifications, in transcriptional activation by p53.

The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.

ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference.

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.

ASH2L regulates ubiquitylation signaling to MLL: trans-regulation of H3 K4 methylation in higher eukaryotes.

Crosstalk between H2B ubiquitylation (H2Bub) and H3 K4 methylation plays important roles in coordinating functions of diverse cofactors during transcription activation. The underlying mechanism for this trans-tail signaling pathway is poorly defined in higher eukaryotes. Here, we show the following: (1) ASH2L in the MLL complex is essential for H2Bub-dependent H3 K4 methylation. Deleting or mutating K99 of the N-terminal winged helix (WH) motif in ASH2L abrogates H2Bub-dependent regulation. (2) Crosstalk can occur in trans and does not require ubiquitin to be on nucleosomes or histones to exert regulatory effects. (3) trans-regulation by ubiquitin promotes MLL activity for all three methylation states. (4) MLL3, an MLL homolog, does not respond to H2Bub, highlighting regulatory specificity for MLL family histone methyltransferases. Altogether, our results potentially expand the classic histone crosstalk to nonhistone proteins, which broadens the scope of chromatin regulation by ubiquitylation signaling.

The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation.

Past studies have documented a crosstalk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little (if any) direct evidence exists explaining the mechanism underlying H2Bub-dependent H3K4 methylation on chromatin templates. Here, we took advantage of an in vitro histone methyltransferase assay employing a reconstituted yeast Set1 complex (ySet1C) and a recombinant chromatin template containing fully ubiquitylated H2B to gain valuable insights. Combined with genetic analyses, we demonstrate that the n-SET domain within Set1, but not Swd2, is essential for H2Bub-dependent H3K4 methylation. Spp1, a homolog of human CFP1, is conditionally involved in this crosstalk. Our findings extend to the human Set1 complex, underscoring the conserved nature of this disease-relevant crosstalk pathway. As not all members of the H3K4 methyltransferase family contain n-SET domains, our studies draw attention to the n-SET domain as a predictor of an H2B ubiquitylation-sensing mechanism that leads to downstream H3K4 methylation.

Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries.

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone post-translational modification (PTM) signatures remains a daunting task in the epigenetics field. We introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semisynthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries, once they have been treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome, is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how preexisting PTMs, alone or synergistically, affect further PTM deposition via cross-talk mechanisms. We anticipate that the high throughput and sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

Syndecan-1 (CD138) deficiency increases Staphylococcus aureus infection but has no effect on pathology in a mouse model of peritoneal dialysis.

BACKGROUND: Technique failure in peritoneal dialysis (PD) due to fibrosis and angiogenesis is complicated by peritonitis. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. The heparan sulfate proteoglycan, syndecan-1 (CD138), was reported to regulate fibrosis, angiogenesis, inflammation and S. aureus infection. The objectives of this study were to examine the effects of syndecan-1 on S. aureus infection and histopathology in a PD model. RESULTS: Syndecan-1(-/-) and wild type mice were dialyzed for 4 weeks and infected intraperitoneally with S. aureus. Tissues were collected after 4 h for histomorphometric analysis. Intravital microscopy was used to observe leukocyte recruitment and to quantify syndecan-1 in the parietal peritoneum microcirculation. The dialyzed syndecan-1(-/-) mice were more susceptible to S. aureus infection than undialyzed syndecan-1(-/-) controls and wild type animals. However, peritoneal fibrosis and neovascularization due to PD did not differ between syndecan-1(-/-) and wild type mice. Intravital microscopy showed that in S. aureus infection, syndecan-1 was removed from the subendothelial layer of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment. CONCLUSIONS: This study indicates that, while syndecan-1 is important for providing a barrier to acute S. aureus infection in PD, it does not affect peritoneal fibrosis and angiogenesis.

Role of PBPD1 in stimulation of Listeria monocytogenes biofilm formation by subminimal inhibitory ß-lactam concentrations.

Disinfectant-tolerant Listeria monocytogenes biofilms can colonize surfaces that come into contact with food, leading to contamination and, potentially, food-borne illnesses. To better understand the process of L. monocytogenes biofilm formation and dispersal, we screened 1,120 off-patent FDA-approved drugs and identified several that modulate Listeria biofilm development. Among the hits were more than 30 ß-lactam antibiotics, with effects ranging from inhibiting (≤50%) to stimulating (≥200%) biofilm formation compared to control. Most ß-lactams also dispersed a substantial proportion of established biofilms. This phenotype did not necessarily involve killing, as >50% dispersal could be achieved with concentrations as low as 1/20 of the MIC of some cephalosporins. Penicillin-binding protein (PBP) profiling using a fluorescent penicillin analogue showed similar inhibition patterns for most ß-lactams, except that biofilm-stimulatory drugs did not bind PBPD1, a low-molecular-weight d,d-carboxypeptidase. Compared to the wild type, a pbpD1 mutant had an attenuated biofilm response to stimulatory ß-lactams. The cephalosporin-responsive CesRK two-component regulatory system, whose regulon includes PBPs, was not required for the response. The requirement for PBPD1 activity for ß-lactam stimulation of L. monocytogenes biofilms shows that the specific set of PBPs that are inactivated by a particular drug dictates whether a protective biofilm response is provoked.

Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.

Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA·GDP with unexpectedly high affinity (K(d) = 5 pm). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (K(d) = 3 nm). The 2.8-Å structure of the RhoA·guanosine 5'-[ß,γ-imido] triphosphate (GMPPNP)·RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI.

Flexible and general synthesis of functionalized phosphoisoprenoids for the study of prenylation in vivo and in vitro.

Protein modification with isoprenoid lipids affects hundreds of signaling proteins in eukaryotic cells. Modification of isoprenoids with reporter groups is the main approach for the creation of probes for the analysis of protein prenylation in vitro and in vivo. Here, we describe a new strategy for the synthesis of functionalized phosphoisoprenoids that uses an aminederivatized isoprenoid scaffold as a starting point for the synthesis of functionalized phosphoisoprenoid libraries. This overcomes a long-standing problem in the field, where multistep synthesis had to be carried out for each individual isoprenoid analogue. The described approach enabled us to synthesize a range of new compounds, including two novel fluorescent isoprenoids that previously could not be generated by conventional means. The fluorescent probes that were developed using the described approach possess significant spectroscopic advantages to all previously generated fluorescent isoprenoid analogue. Using these analogues for flow cytometry and cell imaging, we analyzed the uptake of isoprenoids by mammalian cells and zebrafish embryos. Furthermore, we demonstrate that derivatization of the scaffold can be coupled in a one-pot reaction to enzymatic incorporation of the resulting isoprenoid group into proteins. This enables rapid evaluation of functional groups for compatibility with individual prenyltransferases and identification of the prenyltransferase specific substrates.

Small-molecule modulators of Listeria monocytogenes biofilm development.

Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (≤ 50% of vehicle control) or increased (≥ 200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 µM, d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C(9:0) and C(14:0) were Listeria biofilm inhibitors, whereas fatty acids of C(16:0) or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging.

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Solar-powered hearing aids for children with impaired hearing in Vietnam: a pilot study.

BACKGROUND: Hearing loss is a barrier to speech and social and cognitive development. This can be especially pronounced in children living in low- and middle-income countries with limited resources. AIM: To determine the feasibility, durability and social impact of ComCare GLW solar-powered hearing aids provided for Vietnamese children with hearing impairment. METHODS: A retrospective review of data from an international, multi-discipline humanitarian visit was performed. Hearing aids were given to 28 children enrolled at the Khoai Chau Functional Rehabilitation School, Hung Yen Province, Vietnam. Device inspection and observational assessments were performed by teachers using a modified Parents' Evaluation of Aural/Oral Performance of Children and an Infant Hearing Program Amplification Benefit Questionnaire. Qualitative interviews were undertaken to assess the study aims. RESULTS: Hearing aids were well tolerated for use during regular school hours. All units remained functional during the study period (12 months). Teachers noted increased student awareness and responsiveness to surrounding sounds, but the degree of response to amplification varied between children. There was no significant improvement in speech development as all subjects had prelingual deafness. Teachers felt confident in troubleshooting any potential device malfunction. CONCLUSIONS: A solar-powered hearing aid may be a viable option for children in low- and middle-income countries. This study demonstrates that device distribution, maintenance and function can be established in countries with limited resources, while providing feasibility data to support future studies investigating how similar devices may improve the quality of life of those with hearing loss.

The influence of pressure on crude oil biodegradation in shallow and deep Gulf of Mexico sediments.

A significant portion of oil released during the Deepwater Horizon disaster reached the Gulf of Mexico (GOM) seafloor. Predicting the long-term fate of this oil is hindered by a lack of data about the combined influences of pressure, temperature, and sediment composition on microbial hydrocarbon remineralization in deep-sea sediments. To investigate crude oil biodegradation by native GOM microbial communities, we incubated core-top sediments from 13 GOM sites at water depths from 60-1500 m with crude oil under simulated aerobic seafloor conditions. Biodegradation occurred in all samples and followed a predictable compound class sequence dictated by molecular weight and structure. 45 to ~100% of total n-alkane and 3 to 60% of total polycyclic aromatic hydrocarbons (PAH) were depleted. In reactors incubated at 4°C and at pressures of 6-15 MPa, the depletion in total n-alkane was inversely correlated to pressure (R2 ~ 0.85), equivalent to a 4% decrease in total n-alkane depletion for every 1 MPa increase. Our results indicated a modest inhibitory effect of pressure on biodegradation over our experimental range. However, the expansion of oil exploration to deeper waters (e.g., 5000 m) opens the risk of spills at conditions at which pressure might have a more pronounced effect.

New Class of Antitrypanosomal Agents Based on Imidazopyridines.

The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and 12 compounds exhibited an in vitro EC50 ≤ 1 µM against Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) parasites, respectively. Based on promising results of in vitro activity (EC50 < 100 nM), cytotoxicity, metabolic stability, protein binding, and pharmacokinetics (PK) properties, compound 20 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi (Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 20 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 20 represents a potential lead for the development of drugs to treat trypanosomiasis.

Prevention of atherosclerosis by bioactive palmitoleate through suppression of organelle stress and inflammasome activation.

De novo lipogenesis (DNL), the conversion of glucose and other substrates to lipids, is often associated with ectopic lipid accumulation, metabolic stress, and insulin resistance, especially in the liver. However, organ-specific DNL can also generate distinct lipids with beneficial metabolic bioactivity, prompting a great interest in their use for the treatment of metabolic diseases. Palmitoleate (PAO), one such bioactive lipid, regulates lipid metabolism in liver and improves glucose utilization in skeletal muscle when it is generated de novo from the obese adipose tissue. We show that PAO treatment evokes an overall lipidomic remodeling of the endoplasmic reticulum (ER) membranes in macrophages and mouse tissues, which is associated with resistance of the ER to hyperlipidemic stress. By preventing ER stress, PAO blocks lipid-induced inflammasome activation in mouse and human macrophages. Chronic PAO supplementation also lowers systemic interleukin-1ß (IL-1ß) and IL-18 concentrations in vivo in hyperlipidemic mice. Moreover, PAO prevents macrophage ER stress and IL-1ß production in atherosclerotic plaques in vivo, resulting in a marked reduction in plaque macrophages and protection against atherosclerosis in mice. These findings demonstrate that oral supplementation with a product of DNL such as PAO can promote membrane remodeling associated with metabolic resilience of intracellular organelles to lipid stress and limit the progression of atherosclerosis. These findings support therapeutic PAO supplementation as a potential preventive approach against complex metabolic and inflammatory diseases such as atherosclerosis, which warrants further studies in humans.

Loss of membrane-bound lytic transglycosylases increases outer membrane permeability and ß-lactam sensitivity in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections. Its relatively impermeable outer membrane (OM) limits antibiotic entry, and a chromosomally encoded AmpC ß-lactamase inactivates ß-lactam antibiotics. AmpC expression is linked to peptidoglycan (PG) recycling, and soluble (sLT) or membrane-bound (mLT) lytic transglycosylases are responsible for generating the anhydromuropeptides that induce AmpC expression. Thus, inhibition of LT activity could reduce AmpC-mediated ß-lactam resistance in P. aeruginosa. Here, we characterized single and combination LT mutants. Strains lacking SltB1 or MltB had increased ß-lactam minimum inhibitory concentrations (MICs) compared to wild type, while only loss of Slt decreased MICs. An sltB1 mltB double mutant had elevated ß-lactam MICs compared to either the sltB1 or mltB single mutants (96 vs. 32 µg/mL cefotaxime), without changes to AmpC levels. Time-kill assays with ß-lactams suggested that increased MIC correlated with a slower rate of autolysis in the sltB1 mltB mutant - an antisuicide phenotype. Strains lacking multiple mLTs were more sensitive to ß-lactams and up to 16-fold more sensitive to vancomycin, normally incapable of crossing the OM. Multi-mLT mutants were also sensitive to bile salts and osmotic stress, and were hyperbiofilm formers, all phenotypes consistent with cell envelope compromise. Complementation with genes encoding inactive forms of the enzymes - or alternatively, overexpression of Braun's lipoprotein - reversed the mutants' cell envelope damage phenotypes, suggesting that mLTs help to stabilize the OM. We conclude that P. aeruginosa mLTs contribute physically to cell envelope stability, and that Slt is the preferred target for future development of LT inhibitors that could synergize with ß-lactams.

DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100µg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100µg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25µg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms.