A nanoscale photonic thermal transistor for sub-second heat flow switching.

Control of heat flow is critical for thermal logic devices and thermal management and has been explored theoretically. However, experimental progress on active control of heat flow has been limited. Here, we describe a nanoscale radiative thermal transistor that comprises of a hot source and a cold drain (both are ~250 nm-thick silicon nitride membranes), which are analogous to the source and drain electrodes of a transistor. The source and drain are in close proximity to a vanadium oxide (VOx)-based planar gate electrode, whose dielectric properties can be adjusted by changing its temperature. We demonstrate that when the gate is located close ( < ~1 µm) to the source-drain device and undergoes a metal-insulator transition, the radiative heat transfer between the source and drain can be changed by a factor of three. More importantly, our nanomembrane-based thermal transistor features fast switching times ( ~ 500 ms as opposed to minutes for past three-terminal thermal transistors) due to its small thermal mass. Our experiments are supported by detailed calculations that highlight the mechanism of thermal modulation. We anticipate that the advances reported here will open new opportunities for designing thermal circuits or thermal logic devices for advanced thermal management.

Characterization of Dichloroisoeverninic Acid Biosynthesis and Chemoenzymatic Synthesis of New Orthosomycins.

The orthosomycins are highly modified oligosaccharide natural products with a broad spectrum and potent antimicrobial activities. These include everninomicins and avilamycins, which inhibit protein translation by binding a unique site on the bacterial ribosome. Notably, ribosomal bound structures reveal a network of interactions between the 50S subunit and dichloroisoeverninic acid (DCIE), the aromatic A1-ring conserved across orthosomycins, but the relationship of these interactions to their antimicrobial activity remains undetermined. Genetic functional analysis of three genes putatively associated with DCIE biosynthesis in the everninomicin producer Micromonospora carbonacea delineates the native biosynthetic pathway and provides previously unreported advanced biosynthetic intermediates. Subsequent in vitro biochemical analyses demonstrate the complete DCIE biosynthetic pathway and provide access to novel everninomicin analogs. In addition to the orsellinate synthase EvdD3 and a flavin-dependent halogenase EvdD2, our results identified a key acyltransferase, EvdD1, responsible for transferring orsellinate from the acyl carrier protein domain of EvdD3 to a heptasaccharide orthosomycin biosynthetic intermediate. We have also shown that EvdD1 is able to transfer unnatural aryl groups via their N-acyl cysteamine thioesters to the everninomicin scaffold and used this as a biocatalyst to generate a panel of unnatural aryl analogs. The impact of diverse aryl functional group substitution on both ribosome inhibition and antibacterial activities demonstrates the importance of the DCIE moiety in the pharmacology of orthosomycins, notably revealing an uncoupling between ribosomal engagement and antibiotic activity. Control of A1-ring functionality in this class of molecules provides a potential handle to explore and address pharmacological roles of the DCIE ring in this potent and unique class of antibiotics.

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.

The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

Strongly enhanced THz generation enabled by a graphene hot-carrier fast lane.

Semiconductor photoconductive switches are useful and versatile emitters of terahertz (THz) radiation with a broad range of applications in THz imaging and time-domain spectroscopy. One fundamental challenge for achieving efficient ultrafast switching, however, is the relatively long carrier lifetime in most common semiconductors. To obtain picosecond ultrafast pulses, especially when coupled with waveguides/transmission lines, semiconductors are typically engineered with high defect density to reduce the carrier lifetimes, which in turn lowers the overall power output of the photoconductive switches. To overcome this fundamental trade-off, here we present a new hybrid photoconductive switch design by engineering a hot-carrier fast lane using graphene on silicon. While photoexcited carriers are generated in the silicon layer, similar to a conventional switch, the hot carriers are transferred to the graphene layer for efficient collection at the contacts. As a result, the graphene-silicon hybrid photoconductive switch emits THz fields with up to 80 times amplitude enhancement compared to its graphene-free counterpart. These results both further the understanding of ultrafast hot carrier transport in such hybrid systems and lay the groundwork toward intrinsically more powerful THz devices based on 2D-3D hybrid heterostructures.

Neural network based 3D tracking with a graphene transparent focal stack imaging system.

Recent years have seen the rapid growth of new approaches to optical imaging, with an emphasis on extracting three-dimensional (3D) information from what is normally a two-dimensional (2D) image capture. Perhaps most importantly, the rise of computational imaging enables both new physical layouts of optical components and new algorithms to be implemented. This paper concerns the convergence of two advances: the development of a transparent focal stack imaging system using graphene photodetector arrays, and the rapid expansion of the capabilities of machine learning including the development of powerful neural networks. This paper demonstrates 3D tracking of point-like objects with multilayer feedforward neural networks and the extension to tracking positions of multi-point objects. Computer simulations further demonstrate how this optical system can track extended objects in 3D, highlighting the promise of combining nanophotonic devices, new optical system designs, and machine learning for new frontiers in 3D imaging.

Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues.

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A1 , C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A1 - and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A1 -ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.

Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome.

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca, we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

Fixing the Unfixable: The Art of Optimizing Natural Products for Human Medicine.

Molecules isolated from natural sources including bacteria, fungi, and plants are a long-standing source of therapeutics that continue to add to our medicinal arsenal today. Despite their potency and prominence in the clinic, complex natural products often exhibit a number of liabilities that hinder their development as therapeutics, which may be partially responsible for the current trend away from natural product discovery, research, and development. However, advances in synthetic biology and organic synthesis have inspired a new generation of natural product chemists to tackle powerful undeveloped scaffolds. In this Perspective, we will present case studies demonstrating the historical and current focus on making targeted, but significant, changes to natural product scaffolds via biosynthetic gene cluster manipulation, total synthesis, semisynthesis, or a combination of these methods, with a focus on increasing activity, decreasing toxicity, or improving chemical and pharmacological properties.

Response of Secondary Metabolism of Hypogean Actinobacterial Genera to Chemical and Biological Stimuli.

Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses under different stimulus conditions. Here, we surveyed the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics, with over 30% of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via nuclear magnetic resonance (NMR), revealing several known secondary metabolite families, including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate of Streptosporangium when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with Bacillus species. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, Streptosporangium sp. strain KDCAGE35, which facilitated a deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies.IMPORTANCE Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Here, we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low-abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.

Acid-responsive nanospheres from an asparagine-derived amphiphile.

We describe the synthesis and self-assembly of an asparagine-derived amphiphile. The self-assembled systems formulated with the inclusion of cholesterol (0-50 mol%) show encapsulation for a hydrophobic model drug and rapidly disintegrate in response to mild acidic conditions.

Induction of invasive transitional cell bladder carcinoma in immune intact human MUC1 transgenic mice: a model for immunotherapy development.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

Antitumor effects of L-BLP25 antigen-specific tumor immunotherapy in a novel human MUC1 transgenic lung cancer mouse model.

BACKGROUND: L-BLP25 antigen-specific cancer immunotherapeutic agent is currently in phase III clinical trials for non-small cell lung cancer. Using a novel human MUC1 transgenic (hMUC1.Tg) lung cancer mouse model, we evaluated effects of L-BLP25 combined with low-dose cyclophosphamide (CPA) pretreatment on Th1/Th2 cytokine production and antitumor activity. METHODS: A chemically-induced lung tumor model was developed in hMUC1.Tg C57BL/6 mice by administering 10 weekly 0.75-mg/g doses of the chemical carcinogen urethane by intraperitoneal injection. Serum cytokines associated with Th1/Th2 polarization and inflammation were measured by multiplex cytokine assay during tumorigenesis. Antitumor activity of L-BLP25 (10 µg) with CPA (100 mg/kg) pretreatment was evaluated following either one or two eight-week cycles of treatment by preparing lung whole mounts and counting tumor foci, and assessing IFN-γ production by ELISpot assay. RESULTS: During the carcinogenesis phase, no detectable Th1- or Th2-associated cytokine responses were observed, but levels of pro-inflammatory cytokines were increased with distinctive kinetics. A single cycle of L-BLP25 consisting of eight weekly doses was ineffective, whereas adding a second cycle given during tumor progression showed a significant reduction in the incidence of tumor foci. Administering two cycles of L-BLP25 induced Th1 cytokines IL-12, IL-2 and IFNγ at 24 h after the last dose, while Th2 and inflammatory cytokines were elevated to a lesser extent. CONCLUSIONS: Urethane-induced lung tumors in hMUC1.Tg mice can be used as a model to assess the efficacy of the MUC1 antigen-specific cancer immunotherapeutic agent L-BLP25. The results indicate that the antitumor response to L-BLP25 requires at least two cycles and pre-treatment with CPA. In addition, monitoring pro-inflammatory serum cytokines may be useful as a biomarker of L-BLP25 response. Taken together, the preclinical lung tumor model can be utilized for determining effective combinations of L-BLP25 with chemotherapy and/or other immunotherapies.

L-BLP25 vaccine plus letrozole for breast cancer: Is translation possible?

We have recently reported immunomodulatory effects for tamoxifen and letrozole on the L-BLP25 (Stimuvax(®))-induced immune response in a MUC1-expressing breast cancer mouse model. While neither tamoxifen nor letrozole appeared to interfere with the Th1-polarized cytokine response induced by L-BLP25, only letrozole increased the survival advantage of L-BLP25.

L-BLP25 vaccine plus letrozole induces a TH1 immune response and has additive antitumor activity in MUC1-expressing mammary tumors in mice.

PURPOSE: In this study, we examine the immunomodulatory effects and antitumor activity of tamoxifen and letrozole when combined with the human epithelial mucin (hMUC1)-specific vaccine, L-BLP25, in the hMUC1-expressing mammary tumor (MMT) mouse model. EXPERIMENTAL DESIGN: Dose-finding studies were conducted for both tamoxifen and letrozole. Letrozole and L-BLP25 combination studies used 69 MMT female mice assigned to five groups: untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, letrozole 0.8 mg/kg, and cyclophosphamide + L-BLP25 + letrozole. Tamoxifen and L-BLP25 combination studies used 48 MMT female mice assigned to five treatment groups: untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, tamoxifen 50 mg/kg, and cyclophosphamide + L-BLP25 + tamoxifen 50 mg/kg group. Mice were injected subcutaneously with L-BLP25 (10 µg) weekly for 8 weeks. Serum cytokines were serially measured using a Luminex assay, whereas splenocytes at termination were analyzed by ELISpot to determine T-helper (T(H))1/T(H)2 polarization of immune response. RESULTS: Daily oral doses of 50 and 0.8 mg/kg of tamoxifen and letrozole, respectively, resulted in a significant survival advantage over controls (P < 0.05). A predominant T(H)1-polarized immune response in vaccinated mice was seen with or without tamoxifen or letrozole treatments. In the L-BLP25 plus letrozole treatment group, statistically significant (P < 0.05) additive antitumor activity was observed, whereas tamoxifen plus L-BLP25 was not significantly different (P > 0.05). CONCLUSION: The results of this study show that hormonal therapy does not interfere with L-BLP25-induced predominant T(H)1 response, and the combination of L-BLP25 with letrozole has additive antitumor activity in the MMT mouse model.