The myosuppressin structure-activity relationship for cardiac contractility and its receptor interactions support the presence of a ligand-directed signaling pathway in heart.

The structural conservation and activity of the myosuppressin cardioinhibitory peptide across species suggests it plays an important role in physiology, yet much remains unknown regarding its signaling. We previously reported Drosophila melanogaster myosuppressin (dromyosuppressin, DMS; TDVDHVFLRF-NH2) decreases cardiac contractility through a G protein-coupled receptor, DMS-R2. Our study showed the DMS N-terminus amino acids influence its structure-activity relationship (SAR), yet how they act is not established. We predicted myosuppressin N-terminal amino acids played a role in signaling. Here, we tested our hypothesis in the beetle, Zophobas atratus, using a semi-isolated heart bioassay to explore SAR in a different Order and focus on cardiac signaling. We generated a series of myosuppressin truncated analogs by removing the N-terminal residue and measuring the activity of each structure on cardiac contractility. While DVDHVFLRF-NH2 decreased cardiac contractility, we found VDHVFLRF-NH2, DHVFLRF-NH2, and HVFLRF-NH2 increased activity. In contrast, VFLRF- NH2 decreased activity and FLRF-NH2 was inactive. Next, we analyzed molecular docking data and found the active truncated analogs interacted with the 3-6 lock in DMS-R2, the myosuppressin cardiac receptor, disrupting the salt bridge between H114 and E369, and K289 and Q372. Further, the docking results showed the inhibitory effect on contractility may be associated with contact to Y78, while the analogs that increased contractility lacked this interaction. The data from our study demonstrated N-terminal amino acids played a role in myosuppressin activity and signaling suggesting the cardiac receptor can be targeted by biased agonists. Our myosuppressin cardiac contractility data and predicted receptor interactions describe the presence of functional selectivity in a ligand-directed signaling pathway in heart.

Luminescent properties of a 3,5-diphenylpyrazole bridged Pt(ii) dimer.

We report the synthesis, electrochemistry, photophysical properties and electroluminescence of a highly luminescent pyrazolate-bridged platinum(ii) complex. The complex has the general formula of [((N^C^N)Pt)2(µ-pz)][PF6] where N^C^N = 1,3-di(2-pyridyl)benzene and µ-pz = 3,5-diphenylpyrazolate. The X-ray structure shows that the bridging pyrazolate ligand causes a close Pt-Pt interaction of 3.05(7) Å. The emission profile of the complex was determined in solution, glassy 2-methyltetrahydrofurane at 77 K, and the solid state at both room temperature and 77 K. Each emission profile displayed a strong red metal-metal-to-ligand charge transfer while the solution and glassy 2-methyltetrahydrofurane emission profiles also displayed a ligand-centred transition. The absolute quantum yields of the complex in solution and the solid state at room temperate are 86% and 39%, respectively. Light-emitting electrochemical cells (LEECs) of [((N^C^N)Pt)2(µ-pz)][PF6] were fabricated which displayed appreciable electroluminescence, among the brightest and most efficient LEECs from dinuclear compounds to date.

Copper Selective Polymeric Extractant Synthesized by Ring-Opening Metathesis Polymerization.

Novel polymers bearing pendant picolinic acid functionalities have been synthesized by ring-opening metathesis polymerization (ROMP) for applications in separations-based purification protocols. These polymers and their corresponding monomer were shown to be selective for Cu2+ over a variety of other divalent metal cations as inferred from pH dependent studies carried out under both liquid-liquid and solid-liquid extraction conditions. The polymer system of this study also showed high selectivity for Cu2+ over Ni2+ in mock protocols that could be relevant to the purification of Cu radioisotopes. Separation factors as high as 290 were achieved for extractions from solutions containing a 100-fold excess of Ni2+ relative to Cu2+.

Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA.

The Rett-syndrome-associated methyl-CpG-binding protein 2 (MeCP2) selectively binds methylated DNA to regulate transcription during the development of mature neurons. Like other members of the methyl-CpG-binding domain (MBD) family, MeCP2 functions through the recognition of symmetrical 5-methylcytosines in CpG (mCG) dinucleotides. Advances in base-level resolution epigenetic mapping techniques have revealed, however, that MeCP2 can bind asymmetrically methylated and hydroxymethylated CpA dinucleotides and that this alternative binding selectivity modifies gene expression in the developing mammalian brain. The structural determinants of binding to methylated CpA (mCA) and hydroxymethylated DNA have not been previously investigated. Here, we employ isothermal titration calorimetry and NMR spectroscopy to characterize MeCP2 binding to methylated and hydroxymethylated mCG and mCA DNA, examine the effects of Rett-syndrome-associated missense mutations, and make comparisons to the related and evolutionarily most ancient protein, MBD2. These analyses reveal that MeCP2 binds mCA with high affinity in a strand-specific and orientation-dependent manner. In contrast, MBD2 does not show high affinity or methyl-specific binding to mCA. The Rett-associated missense mutations (T158M, R106W, and P101S) destabilize the MeCP2 MBD and disrupt the recognition of mCG and mCA equally. Finally, hydroxymethylation of a high-affinity mCA site does not alter the binding properties, whereas hemi-hydroxylation of the equivalent cytosine in an mCG site decreases affinity and specificity. Based on these findings, we suggest that MeCP2 recognition of methylated/hydroxymethylated CpA dinucleotides functions as an epigenetic switch redistributing MeCP2 among mCG and mCA loci.

Functional Disorders of Constipation: Paradoxical Puborectalis Contraction and Increased Perineal Descent.

Paradoxical puborectalis contraction (PPC) and increased perineal descent (IPD) are subclasses of obstructive defecation. Often these conditions coexist, which can make the evaluation, workup, and treatment difficult. After a thorough history and examination, workup begins with utilization of proven diagnostic modalities such as cinedefecography and anal manometry. Advancements in technology have increased the surgeon's diagnostic armamentarium. Biofeedback and pelvic floor therapy have proven efficacy for both conditions as first-line treatment. In circumstances where PPC is refractory to biofeedback therapy, botulinum toxin injection is recommended. Historically, pelvic floor repair has been met with suboptimal results. In IPD, surgical therapy now is directed toward the potentially attendant abnormalities such as rectoanal intussusception and rectal prolapse. When these associated abnormalities are not present, an ostomy should be considered in patients with IPD as well as medically refractory PPC.

The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists.

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists.

Conserved molecular switch interactions in modeled cardioactive RF-NH2 peptide receptors: Ligand binding and activation.

Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. We hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, we investigated RF-NH2 receptor (RFa-R) molecular switches in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding.

Changing the Way We Manage Rectal Cancer-Standardizing TME from Open to Robotic (Including Laparoscopic).

Standardizing total mesorectal excision (TME) has been a topic of interest since 1979 when Professor Richard J. Heald first described TME and a new approach to rectal cancer. The procedure is optimized only if every one of the relevant factors is tackled with precise attention to detail, so that the preoperative, operative, and postoperative practice is standardized completely. The same concept of TME standardization applies today regardless of technique chosen, that is, open laparoscopic, single-incision laparoscopic surgery, or robotic. This article reviews the relevant operative factors in performing a quality TME, looking at both the oncologic and nononcologic advantages and disadvantages. It supports TME as the standard of care in obtaining a negative circumferential margin for mid and lower-third rectal cancers, and discusses the role of tumor-specific mesorectal excision for upper-third rectal cancers. It discusses the new options and challenges each operative technique holds, and identifies the same standardized principles each must obey to provide the highest quality of oncologic resection. The operative documentation of these critical features from diagnostic workup to pathological reporting is also emphasized.

An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.

The MBD2-NuRD (Nucleosome Remodeling and Deacetylase) complex is an epigenetic reader of DNA methylation that regulates genes involved in normal development and neoplastic diseases. To delineate the architecture and functional interactions of the MBD2-NuRD complex, we previously solved the structures of MBD2 bound to methylated DNA and a coiled-coil interaction between MBD2 and p66α that recruits the CHD4 nucleosome remodeling protein to the complex. The work presented here identifies novel structural and functional features of a previously uncharacterized domain of MBD2 (MBD2IDR). Biophysical analyses show that the MBD2IDR is an intrinsically disordered region (IDR). However, despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg(286) and Leu(287). Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.

Occurrence of methicillin-resistant Staphylococci in surgically treated dogs and the environment in a Swedish animal hospital.

OBJECTIVES: To investigate whether hospitalised dogs treated surgically may become culture positive for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. METHODS: Surgically treated dogs (n=45) were sampled for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus on admission, before and after surgery and at the time of removal of surgical stitches. The hospital environment (n=57), including healthy dogs in the veterinary hospital environment (n=34), were sampled for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. Genetic variations among methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus isolates were identified through detection of restriction fragment polymorphisms. RESULTS: No dogs developed a wound infection due to methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. However, there was a significant increase in the number of dogs carrying methicillin-resistant Staphylococcus pseudintermedius after hospitalisation compared to admission (P<0·001). No methicillin-resistant Staphylococcus aureus was isolated from dogs, but was present in the environment. Methicillin-resistant Staphylococcus pseudintermedius isolates were recovered from environmental surfaces and hospitalised animals, but not from healthy dogs. Methicillin-resistant Staphylococcus pseudintermedius isolates representing nine different restriction endonuclease digestion patterns were found, with two of these occurring in both the environment and on dogs. CLINICAL SIGNIFICANCE: Dogs may contract methicillin-resistant Staphylococcus pseudintermedius in association with surgery and hospitalisation. Resistant bacteria may be transmitted between dogs, staff and the environment. Dogs colonised with methicillin-resistant Staphylococcus pseudintermedius may be a source for hospital- and community-acquired infections.

Results of improvement in adequacy of intermittent hemodialysis in uremic patients.

PURPOSE: Increasing number of patients, who need intermittent hemodialysis (IHD), is a great challenge for every society. The aim of study is to look if small increase in IHD adequacy is able to improve standard medical parameters. MATERIAL AND METHODS: In 40 patients, Kt/V was monitored on-line during the middle IHD session in the week, 4 times in each of 6 consecutive months. In the first month of observation Kt/V was lower (1.09 +/- 0.02) than in the further months, in which Kt/V was increasing to 1.17 +/- 0.01. Blood count was estimated every month. At the beginning of study period, after 3 months and at the end of studies, dry body mass, body mass index (BMI), the blood pH and serum concentration of calcium, phosphate, intact parathormone (iPTH), total protein, albumin, cholesterol, iron, ferritin, urea and creatinine were determined. RESULTS: The increase in Kt/V was accompanied by rising values of hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume, iron, blood pH before and after IHD session as well as by decreasing values of PTH. Statistically unchanged parameters included dry body mass, BMI, serum concentration of total protein, phosphate, cholesterol and ferritin as well as white blood cells and platelet count. There were correlations between Kt/V and serum concentrations of phosphate, PTH, ferritin, Hb and Hct, indicating that higher IHD doses were provided to patients in more advanced uremic state. CONCLUSIONS: Even small increase in IHD adequacy leads to beneficial changes in management of uremic patients.

Lymphocyte subsets in the course of continuous ambulatory peritoneal dialysis.

The present study evaluated whether estimation of lymphocyte subset counts can be more helpful than total lymphocyte count (TLC) in earlier diagnosis of immune and nutritional changes in the course of continuous ambulatory peritoneal dialysis (CAPD). For the study, 50 CAPD patients were divided into four groups depending on dialysis duration. Group I consisted of patients treated for 6-12 months (n = 15); group II, for 13-24 months (n = 16); group III, for 25-36 months (n = 12); and group IV, for more than 36 months (n = 7). Thirteen patients, being 8 +/- 7 days before CAPD initiation, were included in group 0. Flow cytometry was used for estimation of lymphocyte subsets (determination of CD3, CD4, CD8, CD19, and CD16 + 56 antigens). Our uremic patients started CAPD therapy with decreased TLC and lymphocyte subset (excluding CD16 + 56) counts. After 6-12 months of CAPD therapy, a significant increase in TLC, CD4:CD8 ratio, and all examined lymphocyte subset counts was observed. In the next years of CAPD therapy, TLC, CD3, CD4, CD8, and CD19 cell counts decreased. In patients on CAPD for more than 36 months, CD3, CD4, CD8, and CD19 cell counts were below the normal range, but mean TLC was maintained in the normal range, and CD16 + 56 exceeded the upper limit of normal. A significant negative correlation between CD19 cell count and dialysis duration was seen (r = -0.298, p = 0.035, n = 50). In conclusion, the first months of CAPD therapy see an improvement in immune and nutritional status as expressed by an increase in TLC, lymphocyte subset counts, and CD4:CD8 ratio. Repeat determinations of CD3, CD4, CD8, and CD19 cell counts indicate that these counts decrease earlier than an evaluation of TLC indicates. We recommend lymphocyte subset determinations for detection of immune and nutritional abnormalities in the course of CAPD treatment. An increase in natural killer cells above the normal range may reflect chronic sterile or infectious inflammatory response, which deteriorates nutritional status.

Polyglucose dialysis solution induces changes in blood chemistry.

In peritoneal dialysis patients, polyglucose dialysis solution (PG-DS) influences serum levels of sodium, amylase, and lipase, and of iron parameters. We aimed to examine, in the blood or serum of continuous ambulatory peritoneal dialysis (CAPD) patients treated with PG-DS, changes in the concentrations of Na+, K+, Ca++, total Ca, phosphorus, urea nitrogen, creatinine, uric acid, total protein, albumin, and intact parathyroid hormone (iPTH); in lipid profile [total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, HDL:total cholesterol ratio]; and in acid-base status. We started studies in 14 CAPD patients in whom 7.5% PG-DS was applied for the overnight 2-L exchange (duration: about 10 hours). Determinations of blood chemistry were carried out at 1.6 +/- 0.8 months before the introduction of PG-DS (period I, n = 14); after 1.2 +/- 0.6 months (period II, n = 14), 4.4 +/- 0.8 months (period III, n = 11), and 8.8 +/- 2.4 months (period IV, n = 9) of PG-DS administration; and 2.0 +/- 0.6 months after PG-DS discontinuation (period V, n = 11). The most pronounced (significant) differences in the examined parameters were seen between periods I and III or periods I and IV for Na+ (140 +/- 3 mmol/L vs 136 +/- 4 mmol/L), K+ (4.2 +/- 0.6 mmol/L vs 4.8 +/- 0.6 mmol/L), total Ca (9.4 +/- 1.1 mg/dL vs 10.5 +/- 1.3 mg/dL), urea nitrogen (61.3 +/- 25.9 mg/dL vs 79.4 +/- 20.9 mg/dL), creatinine (10.7 +/- 2.6 mg/dL vs 12.8 +/- 4.3 mg/dL), uric acid (4.8 +/- 2.3 mg/dL vs 7.1 +/- 1.7 mg/dL), and total protein (61.7 +/- 10.8 g/L vs 70.5 +/- 8.0 g/L). Serum lipid levels were stable during PG-DS administration, but they increased after discontinuation of the PG-DS. Other studied parameters usually returned to pre-treatment values after PG-DS discontinuation. All patients were in good clinical status during the study. The changes in blood chemistry did not cause clinical intervention. Our results indicate that PG-DS influences blood chemistry. The observed differences need to be clinically analyzed.

Amplification of putative chlorocatechol dioxygenase gene fragments from alpha- and beta-Proteobacteria.

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and cholorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.