Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

UV-C LED-induced cyclobutane pyrimidine dimer formation, lesion repair and mutagenesis in the biofilm-forming diatom, Navicula incerta.

The use of ultraviolet-C (UV-C) irradiation in marine biofouling control is a relatively new and potentially disruptive technology. This study examined effects of UV-C exposure on the biofilm-forming diatom, Navicula incerta. UV-C-induced mutations were identified via Illumina HiSeq. A de novo genome was assembled from control sequences and reads from UV-C-exposed treatments were mapped to this genome, with a quantitative estimate of mutagenesis then derived from the frequency of single nucleotide polymorphisms. UV-C exposure increased cyclobutane pyrimidine dimer (CPD) abundance with a direct correlation between lesion formation and fluency. Cellular repair mechanisms gradually reduced CPDs over time, with the highest UV-C fluence treatments having the fastest repair rates. Mutation abundances were, however, negatively correlated with CPD abundance suggesting that UV-C exposure may influence lesion repair. The threshold fluence for CPD formation exceeding CPD repair was >1.27 J cm-2. Fluences >2.54 J cm-2 were predicted to inhibit repair mechanisms. While UV-C holds considerable promise for marine antifouling, diatoms are just one, albeit an important, component of marine biofouling communities. Determining fluence thresholds for other representative taxa, highlighting the most resistant, would allow UV-C treatments to be specifically tuned to target biofouling organisms, whilst limiting environmental effects and the power requirement.

A computational model for microbial colonization of an antifouling surface.

Biofouling of marine surfaces such as ship hulls is a major industrial problem. Antifouling (AF) paints delay the onset of biofouling by releasing biocidal chemicals. We present a computational model for microbial colonization of a biocide-releasing AF surface. Our model accounts for random arrival from the ocean of microorganisms with different biocide resistance levels, biocide-dependent proliferation or killing, and a transition to a biofilm state. Our computer simulations support a picture in which biocide-resistant microorganisms initially form a loosely attached layer that eventually transitions to a growing biofilm. Once the growing biofilm is established, immigrating microorganisms are shielded from the biocide, allowing more biocide-susceptible strains to proliferate. In our model, colonization of the AF surface is highly stochastic. The waiting time before the biofilm establishes is exponentially distributed, suggesting a Poisson process. The waiting time depends exponentially on both the concentration of biocide at the surface and the rate of arrival of resistant microorganisms from the ocean. Taken together our results suggest that biofouling of AF surfaces may be intrinsically stochastic and hence unpredictable, but immigration of more biocide-resistant species, as well as the biological transition to biofilm physiology, may be important factors controlling the time to biofilm establishment.

Total Synthesis and Antimalarial Activity of 2-(p-Hydroxybenzyl)-prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine MYP1 Isolated from Marine Bacteria.

Highly efficient and straightforward synthetic routes toward the first total synthesis of 2-(p-hydroxybenzyl)-prodigiosins (2-5), isoheptylprodigiosin (6), and geometric isomers of tambjamine MYP1 ((E/Z)-7) have been developed. The crucial steps involved in these synthetic routes are the construction of methoxy-bipyrrole-carboxaldehydes (MBCs) and a 20-membered macrocyclic core and a regioselective demethylation of MBC analogues. These new synthetic routes enabled us to generate several natural prodiginines 24-27 in larger quantity. All of the synthesized natural products exhibited potent asexual blood-stage antiplasmodial activity at low nanomolar concentrations against a panel of Plasmodium falciparum parasites, with a great therapeutic index. Notably, prodiginines 6 and 24-27 provided curative in vivo efficacy against erythrocytic Plasmodium yoelii at 25 mg/kg × 4 days via oral route in a murine model. No overt clinical toxicity or behavioral change was observed in any mice treated with prodiginines and tambjamines.

Common light chain chickens produce human antibodies of high affinity and broad epitope coverage for the engineering of bispecifics.

Bispecific antibodies are an important and growing segment in antibody therapeutics, particularly in the immuno-oncology space. Manufacturing of a bispecific antibody with two different heavy chains is greatly simplified if the light chains can be the same for both arms of the antibody. Here, we introduce a strain of common light chain chickens, called OmniClic®, that produces antibody repertoires largely devoid of light chain diversity. The antibody repertoire in these chickens is composed of diverse human heavy chain variable regions capable of high-affinity antigen-specific binding and broad epitope diversity when paired with the germline human kappa light chain. OmniClic birds can be used in immunization campaigns for discovery of human heavy chains to different targets. Subsequent pairing of the heavy chain with a germline human kappa light chain serves to facilitate bispecific antibody production by increasing the efficiency of correct pairing. Abbreviations: AID: activation-induced cytidine deaminase; bsAb: bispecific antibody; CDR: complementarity-determining region; CL: light chain constant region; CmLC: common light chain; D: diversity region; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment crystallizable; FcRn: neonatal Fc receptor; FR: framework region; GEM: gel-encapsulated microenvironment; Ig: immunoglobulin; IMGT: the international ImMunoGeneTics information system®; J: joining region; KO: knockout; mAb: monoclonal antibody; NGS: next-generation sequencing; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PGC: primordial germ cell; PGRN: progranulin; TCR: T cell receptor; V: variable region; VK: kappa light chain variable region; VL: light chain variable region; VH: heavy chain variable region.

Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Using Quality Improvement Methodology to Reduce Costs while Improving Efficiency and Provider Satisfaction in a Busy, Academic Musculoskeletal Radiology Division.

Within an everchanging healthcare system, continuous evaluation of standard operating procedures must be performed to ensure optimization of system level organization, communication, and efficiency. Using the Lean management approach, our institution introduced modifications to our musculoskeletal (MSK) radiology workflow in order to facilitate beneficial change that improved clinical workflow efficiency, reduced moonlighting costs, and improved radiologist satisfaction without sacrificing quality of care. The scope of our study included the MSK division of adult inpatient and outpatient populations at three hospitals in a single academic medical center. A root cause analysis was executed to determine the causative factors contributing to clinical inefficiency. Five main factors were identified, and appropriate countermeasures were introduced. Efficiency was measured via the turnaround time (TAT) for radiographic examinations, measured from exam completion to final report submission. Moonlighting expenses were monitored for the fiscal year in which the modifications were implemented. Surveys were administered to MSK radiologists before and after the countermeasures were introduced to determine subjective ratings of efficiency and satisfaction. The average TAT within our MSK division decreased from 40 h to 12 h after introducing changes to our workflow. During one fiscal year, moonlighting expenses decreased from $26,000 to $5000. Post-study survey results indicated increased efficiency of and satisfaction with our implemented modifications to the scheduling and clinical workflow. Optimization of our radiology department's workflow led to increased productivity, efficiency, and radiologist satisfaction, as well as a reduction in moonlighting costs. This project leveraged Lean management principles to combat clinical inefficiency, waste time, and high costs.

Thermoplastic, rubber-like marine antifouling coatings with micro-structures via mechanical embossing.

New processing routes and materials for non-biocidal, antifouling (AF) coatings with an improved performance are currently much sought after for a range of marine applications. Here, the processing, physical properties and marine AF performance of a fluorinated coating based on a thermoplastic (non-crosslinked) fluorinated polymer are reported. It was found that the addition of lubricating oil and hydrodynamic drag reducing microstructures improved the AF properties substantially, i.e. the settlement of a marine biofilm, containing mixed microalgae including diatoms, was reduced to low levels. More importantly, the remaining fouling was removed from the coatings at low hydrodynamic shear rates and promising AF properties were obtained. Moreover, additional potential benefits were revealed originating from the thermoplastic nature of the coating material which might result in significant cost reductions.

Nonfreezable Water and Polymer Swelling Control the Marine Antifouling Performance of Polymers with Limited Hydrophilic Content.

Zwitterionic chemical groups have well-documented resistance to marine fouling species when presented as homogeneous polymer brushes. These model formulations are not, however, suitable for practical fouling-control applications. It is presently unknown if a uniform film of zwitterions is required to elicit nonfouling character via the binding of interfacial water or if the incorporation of zwitterionic functionality into a more practical bulk polymer system will suffice. Here, copolymers of n-butyl methacrylate were synthesized with low incorporation levels (up to 20 mol %) of hydrophilic functionality, including zwitterionic moieties. Their antifouling (AF) properties were evaluated using barnacle cyprids (Balanus improvisus), diatom cells (Navicula incerta), and a multispecies biofilm. The laboratory assays revealed higher resistance of ionic copolymers toward cyprid settlement, which was attributed to their swelling and the presence of nonfreezable water molecules bound tightly to the polymer chains. Additionally, cells of N. incerta and the multispecies biofilm were removed more effectively on polymers containing sulfobetaine methacrylate and sulfopropyl methacrylate moieties. The results indicate that the presence of tightly bound interfacial water is not limited to model systems of pure hydrophilic homopolymers, but that this mechanism can also reduce the settlement and adhesion of fouling species via bulk copolymer systems with limited hydrophilic content. The swelling of polymers with hydrophilic content may also contribute to their AF efficacy, and such materials may therefore represent a route to translation of the well-documented nonfouling character of zwitterions into practical, industrially relevant coating formulations.

System-Level Process Change Improves Communication and Follow-Up for Emergency Department Patients With Incidental Radiology Findings.

The appropriate communication and management of incidental findings on emergency department (ED) radiology studies is an important component of patient safety. Guidelines have been issued by the ACR and other medical associations that best define incidental findings across various modalities and imaging studies. However, there are few examples of health care facilities designing ways to manage incidental findings. Our institution aimed to improve communication and follow-up of incidental radiology findings in ED patients through the collaborative development and implementation of system-level process changes including a standardized loop-closure method. We assembled a multidisciplinary team to address the nature of these incidental findings and designed new workflows and operational pathways for both radiology and ED staff to properly communicate incidental findings. Our results are based on all incidental findings received and acknowledged between November 1, 2016, and May 30, 2017. The total number of incidental findings discovered was 1,409. Our systematic compliance fluctuated between 45% and 95% initially after implementation. However, after overcoming various challenges through optimization, our system reached a compliance rate of 93% to 95%. Through the implementation of our new, standardized communication system, a high degree of compliance with loop closure for ED incidental radiology findings was achieved at our institution.

Marine antifouling performance of polymer coatings incorporating zwitterions.

Zwitterionic materials display antifouling promise, but their potential in marine anti-biofouling is still largely unexplored. This study evaluates the effectiveness of incorporating small quantities (0-20% on a molar basis) of zwitterions as sulfobetaine methacrylate (SBMA) or carboxybetaine methacrylate (CBMA) into lauryl methacrylate-based coatings whose relatively hydrophobic nature encourages adhesion of the diatom Navicula incerta, a common microfouling organism responsible for the formation of 'slime'. This approach allows potential enhancements in antifouling afforded by zwitterion incorporation to be easily quantified. The results suggest that the incorporation of CBMA does provide a relatively minor enhancement in fouling-release performance, in contrast to SBMA which does not display any enhancement. Studies with coatings incorporating mixtures of varying ratios of the cationic monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride and the anionic monomer (3-sulfopropyl)methacrylate, which offer a potentially lower cost approach to the incorporation of anionic and cationic charge, suggest these monomers impart little significant effect on biofouling.

MarH, a Bifunctional Enzyme Involved in the Condensation and Hydroxylation Steps of the Marineosin Biosynthetic Pathway.

A novel bifunctional enzyme, MarH, has been identified, and its key functional role in the marineosin biosynthesis successfully probed. MarH catalyzes (1) a condensation step between 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (UP) to form undecylprodiginine (UPG) and (2) hydroxylation of the alkyl chain of UPG to form the (S)-23-hydroxyundecylprodiginine (HUPG), which is essential for MarG catalyzed bicyclization toward the formation of an unusual spiro-tetrahydropyran-aminal ring of marineosins. The final enigmatic steps in the marineosin biosynthesis have now been deciphered.

Stenotrophomonas maltophilia OleC-Catalyzed ATP-Dependent Formation of Long-Chain Z-Olefins from 2-Alkyl-3-hydroxyalkanoic Acids.

The bacterial pathway of olefin biosynthesis starts with OleA catalyzed "head-to-head" condensation of two CoA-activated long-chain fatty acids to generate (R)-2-alkyl-3-ketoalkanoic acids. A subsequent OleD-catalyzed reduction generates (2R,3S)-2-alkyl-3-hydroxyalkanoic acids. We now show that the final step in the pathway is an OleC-catalyzed ATP-dependent decarboxylative dehydration to form the corresponding Z olefins. Higher kcat /Km values were seen for substrates with longer alkyl chains. All four stereoisomers of 2-hexyl-3-hydroxydecanoic acid were shown to be substrates, and GC-MS and NMR analyses confirmed that the product in each case was (Z)-pentadec-7-ene. LC-MS analysis supported the formation of AMP adduct as an intermediate. The enzymatic and stereochemical course of olefin biosynthesis from long-chain fatty acids by OleA, OleD and OleC is now established.

Identification and Characterization of FabA from the Type II Fatty Acid Synthase of Streptomyces coelicolor.

FabA is proposed to catalyze the dehydration step of chain elongation in fatty acid and undecylprodiginine biosynthesis in Streptomyces coelicolor. Analysis of the S. coelicolor genome has revealed a fabA gene (SCO4636-SCO4637, encoding a heterodimer 3-hydroxyacyl-ACP dehydratase). Herein, we report the identification and characterization of the corresponding gene products. Kinetic analysis has demonstrated that FabA is capable of utilizing various chain lengths of straight- and branched-chain 3-hydroxyacyl-NAC substrates. Additionally, FabA does not discriminate between acyl carrier proteins (ACPs) from primary and secondary metabolism. These data provide the first experimental evidence that FabA has 3-hydroxyacyl-ACP dehydratase activity and processes intermediates for both biosynthetic pathways.

Synthesis and Structure-Activity Relationships of Tambjamines and B-Ring Functionalized Prodiginines as Potent Antimalarials.

Synthesis and antimalarial activity of 94 novel bipyrrole tambjamines (TAs) and a library of B-ring functionalized tripyrrole prodiginines (PGs) against a panel of Plasmodium falciparum strains are described. The activity and structure-activity relationships demonstrate that the ring-C of PGs can be replaced by an alkylamine, providing for TAs with retained/enhanced potency. Furthermore, ring-B of PGs/TAs can be substituted with short alkyl substitutions at either 4-position (replacement of OMe) or 3- and 4-positions without impacting potency. Eight representative TAs and two PGs have been evaluated for antimalarial activity against multidrug-resistant P. yoelii in mice in the dose range of 5-100 mg/kg × 4 days by oral administration. The KAR425 TA offered greater efficacy than previously observed for any PG, providing 100% protection to malaria-infected mice until day 28 at doses of 25 and 50 mg/kg × 4 days, and was also curative in this model in a single oral dose (80 mg/kg). This study presents the first account of antimalarial activity in tambjamines.

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A.

The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

Characterization of FabG and FabI of the Streptomyces coelicolor dissociated fatty acid synthase.

Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain ß-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis.

Stereospecific synthesis of 23-hydroxyundecylprodiginines and analogues and conversion to antimalarial premarineosins via a Rieske oxygenase catalyzed bicyclization.

Facile and highly efficient synthetic routes for the synthesis of (S)- and (R)-23-hydroxyundecylprodiginines ((23S)-2, and (23R)-2), 23-ketoundecylprodiginine (3), and deuterium-labeled 23-hydroxyundecylprodiginine ([23-d]-2) have been developed. We demonstrated a novel Rieske oxygenase MarG catalyzed stereoselective bicyclization of (23S)-2 to premarineosin A (4), a key step in the tailoring process of the biosynthesis of marineosins, using a marG heterologous expression system. The synthesis of various A-C-ring functionalized prodiginines 32-41 was achieved to investigate the substrate promiscuity of MarG. The two analogues 32 and 33 exhibit antimalarial and cytotoxic activities stronger than those of the marineosin intermediate 2, against Plasmodium falciparum strains (CQ(S)-D6, CQ(R)-Dd2, and 7G8) and hepatocellular HepG2 cancer cell line, respectively. Feeding of 34-36 to Streptomyces venezuelae expressing marG led to production of novel premarineosins, paving a way for the production of marineosin analogues via a combinatorial synthetic/biosynthetic approach. This study presents the first example of oxidative bicyclization mediated by a Rieske oxygenase.

Elucidation of final steps of the marineosins biosynthetic pathway through identification and characterization of the corresponding gene cluster.

The marine Streptomyces sp. CNQ-617 produces two diastereomers, marineosins A and B. These are structurally related to alkyl prodiginines, but with a more complex cyclization and an unusual spiroaminal skeleton. We report the identification of the mar biosynthetic gene cluster and demonstrate production of marineosins through heterologous expression in a S. venezuelae host named JND2. The mar cluster shares the same gene organization and has high homology to the genes of the red cluster (which directs the biosynthesis of undecylprodiginine) but contains an additional gene, named marA. Replacement of marA in the JND2 strain leads to the accumulation of premarineosin, which is identical to marineosin with the exception that the middle pyrrole (Ring B) has not been reduced. The final step of the marineosin pathway is thus a MarA catalyzed reduction of this ring. Replacement of marG (a homologue of redG that directs undecylprodiginine cyclization to give streptorubin B) in the JND2 strain leads to the loss of all spiroaminal products and the accumulation of 23-hydroxyundecylprodiginine and a shunt product, 23-ketoundecylprodiginine. MarG thus catalyzes the penultimate step of the marineosin pathway catalyzing conversion of 23-hydroxyundecylprodiginine to premarineosin. The preceding steps of the biosynthetic marineosin pathway likely mirror that in the red-directed biosynthetic process, with the exception of the introduction of the hydroxyl functionality required for spiroaminal formation. This work presents the first experimentally supported scheme for biosynthesis of marineosin and provides a new biologically active molecule, premarineosin.