Changes in carbohydrate metabolism and soil microorganisms under the stress of polyamide and polyethylene nanoplastics during rice (Oryza sativa L.) growth.

Nanoplastics (NPs) presence in agricultural soils can affect plant growth and impact the quality of agricultural products. To investigate the effect of polyamide (PA) NPs and polyethylene (PE) NPs on carbohydrate metabolism and soil microorganisms during rice growth, rice seedlings were exposed to soil containing 2 g/kg of 100 nm PA or 100 nm PE powder for 33 d. The results revealed that 100 nm PE reduced shoot length and dry weight of rice by 4.14 % and 15.68 %, respectively. Analyzing the expression of hexokinase-2 (HXK), phosphofructokinase-1 (PFK), pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), which are four genes related to carbohydrate metabolism, 100 nm PA decreased the expression of PFK and increased the expression of PK and IDH. 100 nm PE increased the expression of HXK, PFK, PK, and IDH. The results of soil microorganisms showed that 100 nm PA significantly effects on 3 bacterial phyla (Bacteroidota, Deinococcota, and Desulfobacterota), whereas 100 nm PE significantly effects on phylum Rozellomycota, class Umbelopsidomycetes, and an unclassified Firmicutes. Our study provides direct evidence of the negative effects of PA and PE on rice, which may be important for assessing the risk of NPs on agroecosystems.

Metabolic engineering of Corynebacterium glutamicum for the high-level production of valerolactam, a nylon-5 monomer.

Valerolactam (VL) is an important precursor chemical for nylon-5 and nylon 6,5. It has been produced by petroleum-based route involving harsh reaction conditions and generating toxic wastes. Here, we report the complete biosynthesis of VL by metabolically engineered Corynebacterium glutamicum overproducing L-lysine. The pathway comprising L-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) from Pseudomonas putida, and ß-alanine CoA transferase (act) from Clostridium propionicum was introduced into the C. glutamicum GA16 strain. To increase the VL flux, competitive pathways predicted from sRNA knockdown target screening were deleted. This engineered C. glutamicum strain produced VL as a major product, but still secreted significant amount of its precursor, 5-aminovaleric acid (5AVA). To circumvent this problem, putative 5AVA transporter genes were screened and engineered in the genome, thereby reuptaking 5AVA excreted. Also, multiple copies of the act gene were integrated into the genome to strengthen the conversion of 5AVA to VL. The final VL10 (pVL1) strain was constructed by enhancing glucose uptake system, which produced 9.68 g/L of VL in flask culture. Fed-batch fermentation of the VL10 (pVL1) strain produced 76.1 g/L of VL from glucose with the yield and productivity of 0.28 g/g and 0.99 g/L/h, respectively, showcasing a high potential for bio-based production of VL from renewable resources.

High-Throughput Characterization and Optimization of Polyamide Hydrolase Activity Using Open Port Sampling Interface Mass Spectrometry.

Enzymatic biodegradation of polymers, such as polyamides (PA), has the potential to cost-effectively reduce plastic waste, but enhancements in degradation efficiency are needed. Engineering enzymes through directed evolution is one pathway toward identification of critical domains needed for improving activity. However, screening such enzymatic libraries (100s-to-1000s of samples) is time-consuming. Here we demonstrate the use of robotic autosampler (PAL) and immediate drop on demand technology (I.DOT) liquid handling systems coupled with open-port sampling interface-mass spectrometry (OPSI-MS) to screen for PA6 and PA66 hydrolysis by 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC) in a high-throughput (8-20 s/sample) manner. The OPSI-MS technique required minimal sample preparation and was amenable to 96-well plate formats for automated processing. Enzymatic hydrolysis of PA characteristically produced soluble linear oligomer products that could be identified by OPSI-MS. Incubation temperatures and times were optimized for PA6 (65 °C, 24 h) and PA66 (75 °C, 24 h) over 108 experiments. In addition, the I.DOT/OPSI-MS quantified production of PA6 linear dimer (8.3 ± 1.6 µg/mL) and PA66 linear monomer (13.5 ± 1.5 µg/mL) by NylC with a lower limit of detection of 0.029 and 0.032 µg/mL, respectively. For PA6 and PA66, linear oligomer production corresponded to 0.096 ± 0.018% and 0.204 ± 0.028% conversion of dry pellet mass, respectively. The developed methodology is expected to be utilized to assess enzymatic hydrolysis of engineered enzyme libraries, comprising hundreds to thousands of individual samples.

Toxic effects of sub-acute microplastic (polyamide) exposure on the accumulation, hematological, and antioxidant responses in crucian carp, Carassius carassius.

The purpose of this study is to investigate the impact of microplastics (MPs) on fish and to confirm the toxic effects of MPs on fish, as well as to clarify the standard indicators. MPs are present in a large amount in the aquatic environment and can have various adverse effects on aquatic animals. Crucian carp, Carassius carassius (mean weight, 23.7 ± 1.6 g; mean length, 13.9 ± 1.4 cm), were exposed to PA (Polyamide) concentrations of 0, 4, 8, 16, 32 and 64 mg/L for 2 weeks. The PA accumulation profile in C. carassius decreased from the intestine to the gill to the liver. Hematological parameters such as red blood cell (RBC) counts, hemoglobin (Hb), and hematocrit (Ht) notably decreased at high levels of PA exposure. Plasma components such as calcium, magnesium, glucose, cholesterol, total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were significantly altered by PA exposure. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione (GSH) of liver, gill and intestine significantly increased following PA exposure. The results of this study suggest that MP exposure affects the hematological physiology and antioxidant responses in C. carassius as well as accumulation in specific tissues.

Ingestion of Nylon 11 Polymers by the Mealworm (Tenebrio molitor) Beetle and Subsequent Enrichment of Monomer-Metabolizing Bacteria in Fecal Microbiome.

BACKGROUND: Nylon 11 is a synthetic plastic widely used in commercial products such as tubing for automobiles, offshore oilfields, and medical devices. An increasing amount of nylon and other plastic wastes have been released into various environments, posing ecological threats. The biodegradation of bundled nylon polymers has been considered impossible due to their crystalline structures. METHODS: Nylon 11 film was created and incubated with adult mealworms. The mass, as well as structures, of nylon 11 films at pre- and post-incubation with beetles were compared. The number of nylon 11 monomer degrading bacteria in feces were determined by culture-dependent approach. The t-test was utilized to examine the statistical significance. RESULTS: We discovered that adult mealworm (Tenebrio molitor) beetle can ingest nylon 11 when stretched thin. The microscopic observation of their feces did not identify the presence of large fragments of nylon 11. The analysis of fecal bacteria revealed that while the total number of culturable bacteria did not change significantly, the number of 11-aminoundecanoic acid-metabolizing bacteria increased by 10,000-fold. CONCLUSIONS: Our results suggest that bundled nylon 11 polymers were fragmented into smaller pieces, including monomeric units (11-aminoundecanoic acid) by adult mealworm. The monomers seem to have supported the proliferation of gut microbial communities capable of utilizing 11-aminoundecanoic acid as a carbon and nitrogen source. Our work implies the potential use of the mealworm beetle as a means to fragment nylon polymers for remediation applications.

X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

Recent advances in microbial production of diamines, aminocarboxylic acids, and diacids as potential platform chemicals and bio-based polyamides monomers.

Recently, bio-based manufacturing processes of value-added platform chemicals and polymers in biorefineries using renewable resources have extensively been developed for sustainable and carbon dioxide (CO2) neutral-based industry. Among them, bio-based diamines, aminocarboxylic acids, and diacids have been used as monomers for the synthesis of polyamides having different carbon numbers and ubiquitous and versatile industrial polymers and also as precursors for further chemical and biological processes to afford valuable chemicals. Until now, these platform bio-chemicals have successfully been produced by biorefinery processes employing enzymes and/or microbial host strains as main catalysts. In this review, we discuss recent advances in bio-based production of diamines, aminocarboxylic acids, and diacids, which has been developed and improved by systems metabolic engineering strategies of microbial consortia and optimization of microbial conversion processes including whole cell bioconversion and direct fermentative production.

Use of DNA-alkylating pyrrole-imidazole polyamides for anti-cancer drug sensitivity screening in pancreatic ductal adenocarcinoma.

BACKGROUND: Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. METHODS: KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. RESULT: KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. CONCLUSION: These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.

Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs.

BACKGROUND: Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI. METHODS: Long bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 µM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining. RESULTS: Our studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI. CONCLUSIONS: Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.

Investigation of microplastic accumulation in Rastrelliger kanagurta fish gut and microplastic degradation behaviour of existing gut bacteria Pseudomonas sp.

Microplastic is a minute particle of chemical pollutant in marine environment and classified as less than 5 mm size. The microplastics could not degrade for long years and they are ingested, incorporated, and accumulated in tissues of living organisms, particularly can cause various ecotoxicological effects for their behavioural change, cytotoxicity, neuro-toxicity effects, liver stress, etc. This preliminary study was investigated the abundance and accumulation of microplastic in marine fish of Indian mackerel (Rastrelliger kanagurta) gut region. Further, we identified the microplastic through stereomicroscope in Indian mackerel fish size up to 0.02 mm. In FT-IR analysis were identified the chemical group which were represents as nylon. In GC-MS analysis were identified that hexa decanoic acid and methyl ester plastic compounds as well as identify and screened the microplastic degrading bacteria from fish gut through partial 16S rRNA gene sequencing analysis it was shows that the isolate reveals a Pseudomonas sp. As a result, it is possible that gut bacteria have a probiotic role in fish gut to may degrade microplastics.

Intestinal permeability and gene expression after polyethylene and polyamide microplastic ingestion in Wistar rats.

Microplastic particles are ubiquitous in the environment. However, little is known about their uptake and effects in humans or mammalian model organisms. Here, we studied the effects of pristine polyamide (15-20 µm) and polyethylene (40-48 µm) particles after oral ingestion in rats. The animals received feed containing microplastic particles (0.1% polyamide or polyethylene, or a mixture of both polymers) or a control diet without microplastic particles, for 5 weeks. The permeability of the duodenum was investigated in an Ussing chamber, whereas gene expression and concentration of tight junction proteins were measured in gut tissue and plasma. Microplastic particles were quantified by pyrolysis-gas chromatography/mass spectrometry in rats' feces. Rats fed with microplastic particles had higher duodenal permeability. Expression of gene coding for the tight junction protein occludin (OCLN) was higher in PE treated animals compared to control or the PA group. No changes in the expression of the gene coding for zonula occludens protein 1 were detected. Occludin protein concentrations were below the limit of detection of the applied method in both gut and plasma. Zonula occludens protein 1 concentrations in the gut were significantly higher in groups exposed to PA and PE as compared to control, while zonula occludens protein 1 concentrations in plasma did not show significant changes. These results demonstrated that short-term exposure to a dose of 0.1% (w/w) microplastic particles in feed had limited effects on duodenal permeability, expression of pro-inflammatory protein genes and tight junction protein genes in the duodenum.

Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors.

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

Diverse Impacts on Prokaryotic and Eukaryotic Membrane Activities from Hydrophobic Subunit Variation Among Nylon-3 Copolymers.

Synthetic, sequence-random polymers that feature a wide range of backbone and side chain structures have been reported to function as mimics of natural host-defense peptides, inhibiting bacterial growth while exerting little or no toxicity toward eukaryotic cells. The common themes among these materials are net positive charge, which is thought to confer preferential action toward prokaryotic vs eukaryotic cells, and the presence of hydrophobic components, which are thought to mediate membrane disruption. This study is based on a set of new binary cationic-hydrophobic nylon-3 copolymers that was designed to ask whether factors beyond net charge and net hydrophobicity influence the biological activity profile. In previous work, we found that nonpolar subunits preorganized by a ring led to copolymers with a diminished tendency to disrupt human cell membranes (as measured via lysis of red blood cells) relative to copolymers containing more flexible nonpolar subunits. An alternative mode of conformational restriction, involving geminal substitution, also minimized hemolysis. Here, we asked whether combining a cyclic constraint and geminal substitution would be synergistic; the combination was achieved by introducing backbone methyl groups to previously described cyclopentyl and cyclohexyl subunits. The new cyclic subunits containing two quaternary backbone carbons (i.e, two sites of geminal substitution) were comparable or slightly superior in terms of antibacterial potency but markedly superior in terms of low hemolytic activity, relative to cyclic subunits lacking the quaternary carbons. However, new cyclic units containing only one quaternary carbon were very hemolytic, which was unanticipated. Variations in net hydrophobicity cannot explain the trend in hemolysis, in contrast to the standard perspective in this field. The impact of each new polymer on live E. coli cells was evaluated via fluorescence microscopy. All new polymers moved rapidly across the outer membrane without large-scale disruption of barrier function. Increasing the number of quaternary carbons in the nonpolar subunit correlated with an increased propensity to permeabilize the cytoplasmic membrane of E. coli cells. Collectively, these findings show that relationships between nonpolar subunit identity and biological activity are influenced by factors in addition to hydrophobicity and charge. We propose that the variation of subunit conformational properties may be one such factor.

A novel amino cellulose derivative using ATRP method: Preparation, characterization, and investigation of its antibacterial activity.

In this study, we prepared a novel amino cellulose derivative (benzyl cellulose-g-poly [2-(N,N-Dimethylamino)ethyl methacrylate]) via a homogeneous ATRP method. The successful synthesis of the novel amino cellulose was confirmed by FT-IR and 1H NMR. This study addressed the different characteristics of the prepared polymer including the thermal stability, solubility, and X-ray diffraction pattern. The antibacterial activity of the synthesized cellulose derivative was investigated using the diffusion disk method against both gram-negative (Escherichia coli, Salmonella enterica) and gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria. Based on the inhibition zone, it was confirmed that the prepared benzyl cellulose-g-PDMAEMA possesses acceptable antibacterial activity against Escherichia coli, Salmonella enterica, and Staphylococcus aureus while Bacillus subtilis is resistant to the prepared polymer. Also according to the inhibition zone, it was shown that benzyl cellulose-g-PDMAEMA has more impact on E. coli and Salmonella enterica than Staphylococcus aureus. Molecular dynamics simulation was also used to study the interaction of the synthesized cellulose derivative with a model membrane which presented atomistic details of the polymer-lipid interactions. According to the results obtained from the molecular dynamics simulation, the polymer was able to destabilize the structure of the membrane and clearly express its signs of degradation.

An Integrated Cofactor/Co-Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines.

We report a highly atom-efficient integrated cofactor/co-product recycling cascade employing cycloalkylamines as multifaceted starting materials for the synthesis of nylon building blocks. Reactions using E. coli whole cells as well as purified enzymes produced excellent conversions ranging from >80 and 95 % into desired ω-amino acids, respectively with varying substrate concentrations. The applicability of this tandem biocatalytic cascade was demonstrated to produce the corresponding lactams by employing engineered biocatalysts. For instance, ϵ-caprolactam, a valuable polymer building block was synthesized with 75 % conversion from 10 mM cyclohexylamine by employing whole-cell biocatalysts. This cascade could be an alternative for bio-based production of ω-amino acids and corresponding lactam compounds.

Bacterial biopolymers: from pathogenesis to advanced materials.

Bacteria are prime cell factories that can efficiently convert carbon and nitrogen sources into a large diversity of intracellular and extracellular biopolymers, such as polysaccharides, polyamides, polyesters, polyphosphates, extracellular DNA and proteinaceous components. Bacterial polymers have important roles in pathogenicity, and their varied chemical and material properties make them suitable for medical and industrial applications. The same biopolymers when produced by pathogenic bacteria function as major virulence factors, whereas when they are produced by non-pathogenic bacteria, they become food ingredients or biomaterials. Interdisciplinary research has shed light on the molecular mechanisms of bacterial polymer synthesis, identified new targets for antibacterial drugs and informed synthetic biology approaches to design and manufacture innovative materials. This Review summarizes the role of bacterial polymers in pathogenesis, their synthesis and their material properties as well as approaches to design cell factories for production of tailor-made bio-based materials suitable for high-value applications.

Surface-Modified G4 PAMAM Dendrimers Cross the Blood-Brain Barrier Following Multiple Tail-Vein Injections in C57BL/6J Mice.

Intracranial injections are currently used to deliver drugs into the brain, as most drugs cannot cross the blood-brain barrier (BBB) following systemic injections. Moreover, multiple dosing is difficult with invasive techniques. Therefore, viable systemic techniques are necessary to facilitate treatment paradigms that require multiple dosing of therapeutics across the BBB. In this study, we show that mixed-surface fourth-generation poly(amidoamine) (PAMAM) dendrimers containing predominantly biocompatible hydroxyl groups and a few amine groups are taken up by cultured primary cortical neurons derived from mouse embryo. We also show that these dendrimers cross the BBB following their administration to healthy mice in multiple doses via tail-vein injections and are taken up by neurons and the glial cells as evidenced by appropriate staining methods. Besides the brain, the dendrimers were found mostly in the kidneys compared to other peripheral organs, such as liver, lungs, and spleen, implying that they may be readily excreted, thereby preventing potential toxic accumulation in the body. Our findings provide a proof-of-concept that appropriate surface modifications of dendrimers provide safe, biocompatible nanomaterial with the potential to deliver therapeutic cargo across the BBB into the brain via multiple tail-vein injections.

PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration.

INTRODUCTION: We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. METHODS: Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)2. Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)3(Bdiol)110. Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30 min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1 h and 24 h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. RESULTS: Intravenous administration of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of 89ZrNB(DFO)2 dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24 h. In contrast, IA infusion of 89ZrG4(DFO)3(Bdiol)110 resulted in poor initial accumulation in the brain, with complete clearance within 1 h of administration. Ex-vivo biodistribution results reflected those on PET-CT. CONCLUSIONS: IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary.

Sequence-Selective Minor Groove Recognition of a DNA Duplex Containing Synthetic Genetic Components.

The structural basis of minor groove recognition of a DNA duplex containing synthetic genetic information by hairpin pyrrole-imidazole polyamides is described. Hairpin polyamides induce a higher melting stabilization of a DNA duplex containing the unnatural P·Z base-pair when an imidazole unit is aligned with a P nucleotide. An NMR structural study showed that the incorporation of two isolated P·Z pairs enlarges the minor groove and slightly narrows the major groove at the site of this synthetic genetic information, relative to a DNA duplex consisting entirely of Watson-Crick base-pairs. Pyrrole-imidazole polyamides bind to a P·Z-containing DNA duplex to form a stable complex, effectively mimicking a G·C pair. A structural hallmark of minor groove recognition of a P·Z pair by a polyamide is the reduced level of allosteric distortion induced by binding of a polyamide to a DNA duplex. Understanding the molecular determinants that influence minor groove recognition of DNA containing synthetic genetic components provides the basis to further develop unnatural base-pairs for synthetic biology applications.