A novel formamidase is required for riboflavin biosynthesis in invasive bacteria.

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Specialization in a Nitrogen-Fixing Symbiosis: Proteome Differences Between Sinorhizobium medicae Bacteria and Bacteroids.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

De novo heterozygous missense and loss-of-function variants in CDC42BPB are associated with a neurodevelopmental phenotype.

CDC42BPB encodes MRCKß (myotonic dystrophy-related Cdc42-binding kinase beta), a serine/threonine protein kinase, and a downstream effector of CDC42, which has recently been associated with Takenouchi-Kosaki syndrome, an autosomal dominant neurodevelopmental disorder. We identified 12 heterozygous predicted deleterious variants in CDC42BPB (9 missense, 2 frameshift, and 1 nonsense) in 14 unrelated individuals (confirmed de novo in 11/14) with neurodevelopmental disorders including developmental delay/intellectual disability, autism, hypotonia, and structural brain abnormalities including cerebellar vermis hypoplasia and agenesis/hypoplasia of the corpus callosum. The frameshift and nonsense variants in CDC42BPB are expected to be gene-disrupting and lead to haploinsufficiency via nonsense-mediated decay. All missense variants are located in highly conserved and functionally important protein domains/regions: 3 are found in the protein kinase domain, 2 are in the citron homology domain, and 4 in a 20-amino acid sequence between 2 coiled-coil regions, 2 of which are recurrent. Future studies will help to delineate the natural history and to elucidate the underlying biological mechanisms of the missense variants leading to the neurodevelopmental and behavioral phenotypes.

A question of food intake: The impact of living arrangement and meal factors on total daily caloric intake.

There has been dramatic weight gain among college students during their collegiate years. A food diary can give much insight of a college student's life. The purpose of this study is to analyze the food intake of college-aged students taking in factors such as the size of meal, the foods being eaten, the location of the meal, and if the meal was eaten with others. The results of this study suggest that being male, eating breakfast, and eating more snacks relative to the number meals increases daily caloric intake. On the other hand, being female and eating more meals at home will result in a lower daily caloric intake.

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis.

Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multi-step pathway. The early intermediates of this pathway are notoriously reactive and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. In the present paper, we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA respectively). We present cheminformatic, biochemical, genetic and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multi-enzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites.

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts.

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 µg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 µg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk.

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)20-IC80) of 0.1-0.9, 3-129 and 12-26 ng/ml, whilst linear range in milk was 0.13-0.74, 11-376 and 2-12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1%CV when measured on all three calibration curves. Graphical Abstract MBio SnapEsi reader and cartridge.

Small RNA binding-site multiplicity involved in translational regulation of a polycistronic mRNA.

In animal systems, mRNAs subject to posttranscriptional regulation by small RNAs (sRNAs) often possess multiple binding sites with imperfect complementarity to a given sRNA. In contrast, small RNA-mRNA interactions in bacteria and plants typically involve a single binding site. In a previous study, we demonstrated that the Escherichia coli sRNA SgrS base pairs with a site in the coding region of the first gene of a polycistronic message, manXYZ. This interaction was shown to be responsible for translational repression of manX and to contribute to destabilization of the manXYZ mRNA. In the current study, we report that translational repression of the manY and manZ genes by SgrS requires a second binding site located in the manX-manY intergenic region. Pairing at this site can repress translation of manY and manZ even when mRNA degradation is blocked. Base pairing between SgrS and the manX site does not affect translation of manY or manZ. Pairing at both sites is required for optimal SgrS-mediated degradation of the full-length manXYZ mRNA and for a particular stress phenotype. These results suggest that bacterial sRNAs may use target-site multiplicity to enhance the efficiency and stringency of regulation. Moreover, use of multiple binding sites may be particularly important for coordinating regulation of multiple genes encoded in operons.

Validation of a Minor Modification to the Soleris® Direct Yeast and Mold Vial and Selective Supplement.

Here we describe results of a study to validate minor reagent formulation changes to the Soleris Direct Yeast and Mold (DYM) automated growth-based method for semi-quantitative detection of yeast and mold in food products. In order to reduce the maximum concentration of the selective agent chloramphenicol in the Soleris reagents, chloramphenicol was removed from the selective supplement and added to the vial growth medium itself. Therefore, both the vial medium and supplement have been reformulated in an alternative version of the method. A probability of detection (POD) statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with plate counts determined using the U.S. Food and Drug Administration Bacteriological Analytical Manual dilution plating procedure. Three matrixes were tested; yogurt, tomato juice, and cocoa powder. POD analysis showed that the percentage of positive Soleris tests at various test thresholds were within the limits predicted by the reference method plate counts for all matrixes evaluated. Real-time stability data on three manufactured lots showed that the modified Soleris vial and supplement are stable for at a minimum of 10 months when stored at 2-8°C. In sum, results presented here demonstrate that the modifications to the Soleris DYM vial and supplement do not impact method performance. The modified Soleris DYM method can be used as an accurate alternative to conventional dilution plating procedures for semi-quantitative determination of yeast and mold at threshold levels, while saving as much as 3 days in analysis time.

Validation of Modifications to the ANSR® Salmonella Method for Improved Ease of Use.

This paper describes the results of a study to validate minor reagent formulation and procedural changes to the ANSR® Salmonella method, AOAC Performance Tested Method™ 061203. In order to improve ease of use and diminish risk of amplicon contamination, the lyophilized reagent components were reformulated for increased solubility, thus eliminating the need to mix by pipetting. In the alternative procedure, an aliquot of the lysate is added to lyophilized ANSR reagents, immediately capped, and briefly mixed by vortex. Results of the validation study with ice cream, peanut butter, dry dog food, raw ground turkey, raw ground beef, and sponge samples from a stainless steel surface showed no statistically significant differences in performance between the ANSR method and the U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture-Food Safety and Inspection Services Microbiology Laboratory Guidebook reference culture procedures. Results of inclusivity and exclusivity testing were unchanged from those of the original validation study; exclusivity was 100% and inclusivity was 99.1% with only a single strain of Salmonella Weslaco testing negative. Robustness testing was also conducted, with variations to lysis buffer volume, lysis time, and sample volume having no demonstrable effect on assay results.

Validation of the NeoFilm for Yeast and Mold Method for Enumeration of Yeasts and Molds in Select Foods.

NeoFilm Yeast and Mold (Y&M), also known as Sanita-kun Yeasts and Molds, is a simple, effective device used for the enumeration of yeasts and molds. It consists of a nonwoven fabric on which a layer of microbial nutrients is deposited in a film. A 1 mL sample homogenate is applied to the membrane and this, in turn, is incubated for 48-72 h at 25°C. Sample homogenates were prepared using two different diluents for customer convenience: phosphate buffered saline (PBS) and 0.1% peptone water. In comparative testing of breaded chicken nuggets, dry pet food, orange juice concentrate, yogurt, and cake mix, there were statistically significant differences in the counts obtained by the NeoFilm Y&M and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture methods only in the following instances: medium level for orange juice with PBS as diluent and low level for pet food with 0.1% peptone water as diluent, where reference method counts were higher than those of NeoFilm; medium level for cake mix with PBS, and low and medium levels for cake mix with 0.1% peptone water, where NeoFilm produced higher counts than the reference method. In addition to the method comparison study with five matrixes, robustness and stability/lot-to-lot testing were also performed. Results of robustness testing showed no significant effect on results even with perturbation to three assay parameters simultaneously. Results of testing of three lots of devices ranging in age from 2 to 26 months post-manufacture showed no significant differences in performance.

Validation of the ANSR® Listeria Method for Detection of Listeria spp. in Selected Foods.

ANSR® Listeria was previously certified as Performance Tested Method(SM) 101202 for detection of Listeria spp. on selected environmental surfaces. This study proposes a matrix extension to the method for detection of Listeria spp. in selected food matrixes. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in less than 50 min, requiring only simple instrumentation. Inclusivity testing was performed using a panel of 51 strains of Listeria spp., representing the species L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. All strains tested were detected by the ANSR assay. Exclusivity testing of 30 strains representing non-Listeria Gram-positive bacteria yielded no evidence of cross-reactivity. Performance of the ANSR method for detection of Listeria spp. was compared to that of reference culture procedures for pasteurized liquid egg, pasteurized 2% milk, Mexican-style cheese, ice cream, smoked salmon, lettuce, cantaloupe, and guacamole. Data obtained in these unpaired studies and analyzed using a probability of detection model demonstrated that there were no statistically significant differences in results between the ANSR and reference culture methods, except for milk at 16 h and cantaloupe. In milk and smoked salmon, ANSR sensitivity was low at 16 h and therefore the recommended incubation time is 24 h. In cantaloupe, ANSR was found to be more sensitive than the reference culture method at both 16 and 24 h in independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in selected food types.

Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein.

Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago spp. and other legumes, secretes a considerable amount of riboflavin. This precursor of the cofactors flavin mononucleotide and flavin adenine dinucleotide is a bioactive molecule that has a beneficial effect on plant growth. The ribBA gene of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone phosphate synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect the ability of S. meliloti to establish an effective symbiosis with the host plant but does affect the ability of the bacteria to secrete flavins, colonize host-plant roots, and compete for nodulation. A strain missing the RibBA protein retains considerable GTP cyclohydrolase II activity. Based on these results, we hypothesize that S. meliloti has two partly interchangeable modules for biosynthesis of riboflavin, one fulfilling the internal need for flavins in bacterial metabolism and the other producing riboflavin for secretion. Our data also indicate that bacteria-derived flavins play a role in communication between rhizobia and the legume host and that the RibBA protein is important in this communication process even though it is not essential for riboflavin biosynthesis and symbiosis.

Semiquantitative determination of mesophilic, aerobic microorganisms in cocoa products using the Soleris NF-TVC method.

The Soleris Non-fermenting Total Viable Count method was previously validated for a wide variety of food products, including cocoa powder. A matrix extension study was conducted to validate the method for use with cocoa butter and cocoa liquor. Test samples included naturally contaminated cocoa liquor and cocoa butter inoculated with natural microbial flora derived from cocoa liquor. A probability of detection statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with aerobic plate counts determined using the AOAC Official Method 966.23 dilution plating method. Results of the two methods were not statistically different at any dilution level in any of the three trials conducted. The Soleris method offers the advantage of results within 24 h, compared to the 48 h required by standard dilution plating methods.

Validation of the Soleris direct yeast and mold method for semiquantitative determination of yeast and mold in a variety of foods.

A study was carried out to determine the efficacy of the Soleris Direct Yeast and Mold (DYM) automated growth-based method for semiquantitative detection of yeast and mold in a variety of food products. A probability of detection (POD) statistical model was used to compare Soleris results at multiple test thresholds (dilutions) with plate counts determined using the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 18, dilution plating procedure. Fourteen naturally contaminated food products were tested, with Soleris testing performed at three or more threshold levels for each food. Using the POD model, the majority of Soleris test results were in statistical agreement with the reference plating procedures. The exceptions included a single threshold level in yogurt, black pepper, dried fruit, and dry pet food, and two levels in nonfat dry milk and saw palmetto powder. In all but one of these instances, the exception being pet food, the statistical disagreement was due to Soleris estimating a higher level of contamination than the reference method. Results of ruggedness testing showed that the Soleris method produced accurate results even when significant variances in a critical operating parameter, incubation temperature, were introduced. Results of the internal and independent laboratory validation studies showed that the Soleris DYM method can be used as an accurate alternative to conventional dilution plating procedures for evaluation of yeast and mold counts at threshold levels, while saving as much as 72 h in analysis time.

Validation of the ANSR Salmonella method for detection of Salmonella spp. in a variety of foods. Performance Tested Method 061203.

This study represents a proposal to extend the matrix claims for the ANSR Salmonella test, Performance Tested Method 061203. The test is based on the nicking enzyme amplification reaction (NEAR) isothermal nucleic acid amplification technology. The assay platform features simple instrumentation, minimal labor, and following a single-step 16-24 h enrichment (depending on sample type), an extremely short assay time of 30 min including sample preparation. Detection is real-time using fluorescent molecular beacon probes. ANSR Salmonella was originally validated for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, and oat cereal, and on stainless steel, plastic, sealed concrete, ceramic tile, and rubber surfaces. The matrixes tested in this study include pet food, ice cream, soy flour, raw almonds, peanut butter, spinach, black pepper, raw frozen shrimp, cocoa powder, and pasteurized dried egg. In unpaired comparative testing there were no statistically significant differences in the number of positive results obtained with the ANSR and the reference culture methods. Enrichment for 16 h was effective for all commodities tested except ice cream, black pepper, dried pasteurized egg, and 375 g samples of dry pet food, for which enrichment for 24 h is indicated.

Evaluation of the ANSR for Salmonella assay for identification of Salmonella spp. from colony picks from selective/differential agar media: first action 2013.14.

A collaborative study was conducted to evaluate performance of the ANSR for Salmonella assay for identification of Salmonella spp. from colony picks taken from selective/differential agar media. The ANSR Salmonella assay is an isothermal nucleic acid amplification test based on the nicking enzyme amplification reaction chemistry. The test can be completed in less than 40 min including sample preparation. A total of 18 laboratories representing industry, government, academic, and commercial testing laboratories participated in the study. Each collaborator tested up to 84 samples, comprised of colony picks of six Salmonella spp. and six non-salmonellae taken from six selective/differential agar media as well as tryptic soy agar. A total of 1441 analyses were performed, 1416 of which gave the correct identification, for overall accuracy of 98.3%. For identification of Salmonella spp., 755 of 756 tests (99.9%) produced the correct result. For identification of non-salmonellae as such, 661 of 685 assays (96.5%) produced the correct result. Of the 18 laboratories, 15 produced data sets with 99-100% accuracy. The majority of false-positive results were clustered in three laboratories; analysis of raw data suggests procedural difficulties in at least two cases, which may explain the atypical data from these collaborators. The ANSR Salmonella assay can be used as a rapid, accurate adjunct or alternative to biochemical testing for identification of presumptive Salmonella spp. isolates.

Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer.

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90 % along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

Delivery of siRNA to the mouse lung via a functionalized lipopolyamine.

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.