Compound motor action potentials in transient and persistent phrenic nerve injury - metanalysis.

BACKGROUND: The right phrenic nerve is vulnerable to injury (PNI) during cryoballoon ablation (CBA) isolation of the right pulmonary veins. The complication can be transient or persistent. The reported incidence of PNI fluctuates from 4.73% to 24.7% depending on changes over time, CBA generation, and selected protective methods. M e t h o d s: Through September 2019, a database search was performed on MEDLINE, EMBASE, and Cochrane Database. In the selected articles, the references were also extensively searched. The study provides a comprehensive meta-analysis of the overall prevalence of PNI, assesses the transient to persistent PNI ratio, the outcome of using compound motor action potentials (CMAP), and estimated average time to nerve recovery. R e s u l t s: From 2008 to 2019, 10,341 records from 48 trials were included. Out of 783 PNI retrieved from the studies, 589 (5.7%) and 194 (1.9%) were persistent. CMAP caused a significant reduction in the risk of persistent PNI from 2.3% to 1.1% (p = 0.05; odds ratio [OR] 2.13) in all CBA groups. The mean time to PNI recovery extended beyond the hospital discharge was significantly shorter in CMAP group at three months on average versus non CMAP at six months (p = 0.012). CMAP (in contrast to non-CMAP procedures) detects PNI earlier from 4 to 16 sec (p <0.05; I2 = 74.53%) and 3 to 9o (p <0.05; I2 = 97.24%) earlier. C o n c l u s i o n s: Right PNI extending beyond hospitalization is a relatively rare complication. CMAP use causes a significant decrease in the risk of prolonged injury and shortens the time to recovery.

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial N-acyl homoserine lactones.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A synthetic opioid vaccine attenuates fentanyl-vs-food choice in male and female rhesus monkeys.

AIM: Opioid-targeted vaccines are under consideration as candidate Opioid Use Disorder medications. We recently reported that a fentanyl-targeted vaccine produced a robust and long-lasting attenuation of fentanyl-vs-food choice in rats. In the current study, we evaluated an optimized fentanyl-targeted vaccine in rhesus monkeys to determine whether vaccine effectiveness to attenuate fentanyl choice translated to a species with greater phylogenetic similarity to humans. METHODS: Adult male (2) and female (3) rhesus monkeys were trained to respond under a concurrent schedule of food (1 g pellets) and intravenous fentanyl (0, 0.032-1 µg/kg/injection) reinforcement during daily 2 h sessions. Fentanyl choice dose-effect functions were determined daily and 7-day buprenorphine treatments (0.0032-0.032 mg/kg/h IV; n = 4-5) were determined for comparison to vaccine effects. Subsequently, a fentanyl-CRM197 conjugate vaccine was administered at week 0, 3, 8, 15 over a 29-week experimental period during which fentanyl choice dose-effect functions continued to be determined daily. RESULTS: Buprenorphine significantly decreased fentanyl choice and reciprocally increased food choice. Vaccination eliminated fentanyl choice and increased food choice in four-of-the-five monkeys. A transient and less robust vaccine effect was observed in the fifth monkey. Fentanyl-specific antibody concentrations peaked after the third vaccination to approximately 50 µg/mL while anti-fentanyl antibody affinity increased to a sustained low nanomolar level. CONCLUSION: These results translate fentanyl vaccine effectiveness from rats to rhesus monkeys to decrease fentanyl-vs-food choice, albeit with greater individual differences observed in monkeys. These results support the potential and further clinical evaluation of this fentanyl-targeted vaccine as a candidate Opioid Use Disorder medication.

Synthetic molecules for disruption of the MYC protein-protein interface.

MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.

Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity.

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus-host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo[ b][1,4] dioxin-6-yl)-4'-methoxy-[1,1'-biphenyl]-3-yl)thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop antiarenaviral drugs.

A bioconjugate leveraging xenoreactive antibodies to alleviate cocaine-induced behavior.

A method for potentiating the response to an anti-cocaine vaccine by leveraging xenoreactive antibodies against the carbohydrate epitope Galα1,3-Gal (GAL) was found to result in a highly specific anti-cocaine response that was able to significantly attenuate cocaine-induced locomotion at 20 mg kg-1 with superior efficacy compared to a standard conjugate.

The design and synthesis of an α-Gal trisaccharide epitope that provides a highly specific anti-Gal immune response.

Carbohydrate antigens displaying Galα(1,3)Gal epitopes are recognized by naturally occurring antibodies in humans. These anti-Gal antibodies comprise up to 1% of serum IgG and have been viewed as detrimental as they are responsible for hyperacute organ rejections. In order to model this condition, α(1,3)galactosyltransferase-knockout mice are inoculated against the Galα(1,3)Gal epitope. In our study, two α-Gal trisaccharide epitopes composed of either Galα(1,3)Galß(1,4)GlcNAc or Galα(1,3)Galß(1,4)Glc linked to a squaric acid ester moiety were examined for their ability to elicit immune responses in KO mice. Both target epitopes were synthesized using a two-component enzymatic system using modified disaccharide substrates containing a linker moiety for coupling. While both glycoconjugate vaccines induced the required high anti-Gal IgG antibody titers, it was found that this response had exquisite specificity for the Galα(1,3)Galß(1,4)GlcNAc hapten used, with little cross reactivity with the Galα(1,3)Galß(1,4)Glc hapten. Our findings indicate that while homogenous glycoconjugate vaccines provide high IgG titers, the carrier and adjuvanting factors can deviate the specificity to an antigenic determinant outside the purview of interest.

Studies towards the improvement of an anti-cocaine monoclonal antibody for treatment of acute overdose.

There is currently no clinically-approved antidote for cocaine overdose. Efforts to develop a therapy via passive immunization have resulted in a human monoclonal antibody, GNCgzk, with a high affinity for cocaine (Kd=0.18nM). Efforts to improve the production of antibody manifolds based on this antibody are disclosed. The engineering of an HRV 3C protease cleavage site into the GNCgzk IgG has allowed for increased production of a F(ab')2 with a 20% superior capacity to reduce mortality for cocaine overdose in mice.

Investigations of Enantiopure Nicotine Haptens Using an Adjuvanting Carrier in Anti-Nicotine Vaccine Development.

Despite efforts to produce suitable smoking cessation aids, addiction to nicotine continues to carry a substantive risk of recidivism. An attractive alternative to current therapies is the pharmacokinetic strategy of antinicotine vaccination. A major hurdle in the development of the strategy has been to elicit a sufficiently high antibody concentration to curb nicotine distribution to the brain. Herein, we detail investigations into a new hapten design, which was able to elicit an antibody response of significantly higher specificity for nicotine. We also explore the use of a mutant flagellin carrier protein with adjuvanting properties. These studies underlie the feasibility of improvement in antinicotine vaccine formulations to move toward clinical efficacy.

Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids.

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low-micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in Escherichia coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles.

Pharmacophore reassignment for induction of the immunosurveillance cytokine TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an immunosurveillance cytokine that kills cancer cells but demonstrates little toxicity against normal cells. While investigating the TRAIL-inducing imidazolinopyrimidinone TIC10, a misassignment of its active structure was uncovered. Syntheses of the two isomers, corresponding to the published and reassigned structures, are reported. The ability of each to induce TRAIL expression in macrophages was investigated and it was found that only the compound corresponding to the reassigned structure shows the originally reported activity; the compound corresponding to the published structure is inactive. Importantly, this structural reassignment has furnished a previously unknown antitumor pharmacophore.

Macrocyclization of organo-peptide hybrids through a dual bio-orthogonal ligation: insights from structure-reactivity studies.

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo-biosynthetic strategy for generating macrocyclic organo-peptide hybrids (MOrPHs) through a dual oxime-/intein-mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3-amino-thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2-amino- mercaptomethyl-aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the "i-1" site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side-chain→C-end ligation versus C-end→side-chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo-peptide macrocycles and provide valuable structure-reactivity insights to inform the construction of macrocycle libraries through this chemo-biosynthetic strategy.