Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Post-marketing surveillance of the safety and effectiveness of naldemedine in the management of opioid-induced constipation in patients with cancer pain in Japan.

PURPOSE: This prospective post-marketing surveillance (PMS) was designed to collect data on the safety and effectiveness of naldemedine in routine clinical practice in patients with opioid-induced constipation (OIC) and cancer pain in Japan and explore the characteristics of patients prone to diarrhea. METHODS: The enrolled patients received naldemedine (0.2 mg, once a day) orally for up to 12 weeks. In the safety analysis, adverse drug reactions (ADRs), including diarrhea as a special interest, were assessed. Effectiveness was evaluated, especially regarding the frequency and condition of bowel movement. RESULTS: In the safety analysis set (n = 1177), 145 ADRs occurred in 133 (11.30%) patients, and diarrhea was the most frequent event (n = 107, 9.09%). Most cases of diarrhea were non-serious (98.1%). Most ADRs were non-serious (93.8%), and they resolved within 2 weeks (75.9%). No patient characteristics influenced the risk of diarrhea development or aggravation. Both the frequency (75.0% and 83.2%) and condition of bowel movement (80.0% and 88.0%) were improved at 2 and 12 weeks, respectively in the effectiveness analysis set (n = 953). Frequency and condition of bowel movement were also improved in patients excluded (e.g., Eastern Cooperative Oncology Group performance status was ≥ 3) or with very small numbers (e.g., received weak opioid) in the clinical trials. CONCLUSIONS: This PMS indicates that naldemedine is well tolerated and effective in patients of various backgrounds in routine clinical practice who have OIC and cancer pain. TRIAL REGISTRATION: UMIN000042851.

Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Safety and effectiveness of baloxavir marboxil for the treatment of influenza in Japanese clinical practice: A postmarketing surveillance of more than 3000 patients.

Baloxavir marboxil is an oral anti-influenza drug that inhibits the cap-dependent endonuclease of the virus polymerase acidic protein. In clinical trials, baloxavir reduced the time to alleviation of influenza symptoms and time to resolution of fever in adults, adolescents, and children. The purpose of this study is to collect data on the safety and effectiveness of baloxavir when used in clinical practice. This postmarketing surveillance (clinicaltrials.jp; JapicCTI-183882), conducted at 688 Japanese hospitals or clinics (March 2018 to March 2019), enrolled patients of any age with influenza A or B infection who received a single, weight-based dose of baloxavir. Adverse drug reactions (ADRs) were seen in 11.2% of 3094 patients during the 7-day observation period; the most common ADR was diarrhea (6.1%). ADRs were more common in children aged <12 years (14.1%) than in adults (10.0%). Almost all ADRs were non-serious (98.9%) and were recovered or recovering (96.7%). Median time to alleviation of symptoms (N = 2884) was 2.5 days (overall, influenza A, and influenza B groups). Median time to resolution of fever (N = 2946) was 1.5 days (overall, influenza A, and influenza B groups). Biphasic fever (increased temperature after previous fever resolution) was seen in 6.7% of patients overall and 28.6% of patients <6 years infected with influenza B, similar to rates published elsewhere with other influenza drugs and in untreated influenza. This postmarketing surveillance of >3000 patients suggests that baloxavir is well tolerated and effective regardless of patient age or influenza virus type.

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions.

In Euglena gracilis, pyruvate:NADP+ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP+ oxidoreductase. In contrast, pyruvate:NADP+ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.

C2 metabolism in Euglena.

Euglenoids are able to assimilate fatty acids and alcohols with various carbon-chain lengths, and ethanol is known to be one of the best carbon sources to support the growth of Euglena gracilis. Ethanol is first oxidized to acetate by the sequential reactions of alcohol dehydrogenase and acetaldehyde dehydrogenase in the mitochondria, and then converted to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is metabolized through the glyoxylate cycle which is a modified tricarboxylic acid (TCA) cycle in which isocitrate lyase (ICL) and malate synthase (MS) function to bypass the two decarboxylation steps of the TCA cycle, enabling the net synthesis of carbohydrates from C2 compounds. ICL and MS form a unique bifunctional enzyme localized in Euglena mitochondria, not in glyoxysome as in other eukaryotes. The unique glyoxylate and glycolate metabolism during photorespiration is also discussed in this chapter.

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

[Evaluation of Artificial Hip Joint with Radiofrequency Heating Issues during MRI Examination: A Comparison between 1.5 T and 3 T].

In magnetic resonance imaging (MRI), when radiofrequency (RF) is irradiated to a subject with metallic implant, it can generate heat by RF irradiation. Recently 3 T MRI scanner has spread widely and imaging for any regions of whole body has been conducted. However specific absorption rate (SAR) of 3 T MRI becomes approximately four times as much as the 1.5 T, which can significantly affect the heat generation of metallic implants. So, we evaluated RF heating of artificial hip joints in different shapes and materials in 1.5 T and 3 T MRI. Three types of artificial hip joints made of stainless alloy, titanium alloy and cobalt chrome alloy were embedded in the human body-equivalent phantom respectively and their temperature change were measured for twenty minutes by 1.5 T and 3 T MRI. The maximum temperature rise was observed at the bottom head in all of three types of artificial hip joints, the rise being 12°C for stainless alloy, 11.9°C for titanium alloy and 6.1°C for cobalt chrome alloy in 1.5 T. The temperature rise depended on SAR and the increase of SAR had a good linear relationship with the temperature rise. It was found from the result that the RF heating of metallic implants can take place in various kinds of material and the increase of SAR has a good linear relationship with the temperature rise. This experience shows that reduction of SAR can decrease temperature of metallic implants.

Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

Crystal structures of the catalytic domain of a novel glycohydrolase family 23 chitinase from Ralstonia sp. A-471 reveals a unique arrangement of the catalytic residues for inverting chitin hydrolysis.

Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.

Exploration of polar lipid accumulation profiles in Euglena gracilis using LipidBlast, an MS/MS spectral library constructed in silico.

A rapid protocol for polar lipid profiling was applied to Euglena gracilis lipid metabolism by LipidBlast, an MS/MS spectral similarity search tool. The similarity search results suggested anoxia-induced polar lipid metabolism in Euglena characterized by the accumulation of differential lipid classes, carbon chain lengths, and unsaturated bond numbers. The informatics-supported MS spectral search provides an alternative option for global lipid profiling studies.

Genome editing-based mutagenesis stably modifies composition of wax esters synthesized by Euglena gracilis under anaerobic conditions.

Microalgal oil production represents a promising renewable biofuel source. Metabolic engineering can enhance its utility, transforming it into an improved biofuel and expanding its applications as a feedstock for commodity chemicals, thereby increasing their value in biorefineries. This study focused on anaerobic wax ester production by the microalga Euglena gracilis, aiming to develop stable mutant strains with altered wax ester profiles through genome editing. Two enzymes in the fatty acid beta-oxidation pathway involved in wax ester production were targeted-3-ketoacyl-CoA thiolase and acyl-CoA dehydrogenase-using clustered regularly interspaced short palindromic repeats/Cas9. The results revealed one genetic mutation that lengthened and three that shortened the distribution of wax ester compositions compared to the wild-type (WT). The triple-knockout mutant, combining mutations that shorten wax ester chains, produced wax esters with acyl chains two carbons shorter than WT. This study established a methodology to stably modify wax ester composition in E. gracilis.

Fractionation of cassava pectins and their detailed structural analyses using various pectinolytic enzymes.

In this study, we analyzed the pectin structure within the pulp of cassava. Cassava pectin, derived from cassava pulp treatment at 120 °C for 90 min, was separated into four fractions (CP-P, CP-SD1, CP-SD2F, and CP-SD2R) based on variations in water solubility, electrical properties, and molecular weights. Sugar composition analysis demonstrated an abundance of homogalacturonan (HG) in CP-P and CP-SD2F, rhamnogalacturonan I (RG-I) in CP-SD2R, and neutral sugars in CP-SD1. Because RG-I possesses a complex structure, we analyzed CP-SD2R using various pectinolytic enzymes. Galactose was the major sugar in CP-SD2R accounting for 49 %, of which 65 % originated from arabinogalactan I, 9 % from galactose and galactooligosaccharides, 5 % from arabinogalactan II, and 11 % from galactoarabinan. Seventy-four percent of arabinose in CP-SD2R was present as galactoarabinan. The methylation (DM) and acetylation (DAc) degrees of cassava pectin were 11 and 15 %, respectively. The HG and RG-I regions exhibited DAc values of 5 and 44 %, respectively, signifying the high DAc of RG-I compared to HG. Information derived from the structural analysis of cassava pectin will enable efficient degradation of pectin and cellulose, leading to the use of cassava pulp as a raw material for biorefineries.

Crystal structure of endo-1,4-ß-glucanase from Eisenia fetida.

The saccharification process is essential for bioethanol production from woody biomass including celluloses. Cold-adapted cellulase, which has sufficient activity at low temperature (<293 K), is capable of reducing heating costs during the saccharification process and is suitable for simultaneous saccharification and fermentation. Endo-1,4-ß-glucanase from the earthworm Eisenia fetida (EF-EG2) belonging to glycoside hydrolase family 9 has been shown to have the highest activity at 313 K, and also retained a comparatively high activity at 283 K. The recombinant EF-EG2 was purified expressed in Pichia pastoris, and then grew needle-shaped crystals with dimensions of 0.02 × 0.02 × 1 mm. The crystals belonged to the space group P3221 with unit-cell parameters of a = b = 136 Å, c = 55.0 Å. The final model of EF-EG2, including 435 residues, two ions, seven crystallization reagents and 696 waters, was refined to a crystallographic R-factor of 14.7% (free R-factor of 16.8%) to 1.5 Å resolution. The overall structure of EF-EG2 has an (α/α)6 barrel fold which contains a putative active-site cleft and a negatively charged surface. This structural information helps us understand the catalytic and cold adaptation mechanisms of EF-EG2.

[Investigation of radio frequency heating of dental implants made of titanium in 1.5 tesla and 3.0 tesla magnetic resonance procedure: measurement of the temperature by using tissue-equivalent phantom].

Titanium (Ti) implants are increasingly being used for dental parts. There is no problem with the attraction of a static magnetic field for Ti in magnetic resonance imaging (MRI), since Ti is paramagnetic. However, there is a risk of radio frequency (RF) heat generation within Ti. 3.0 T-MRI scanners are becoming increasingly common. The specific absorption rate (SAR) of 3.0 T-MRI is quadruple that of SAR compared with 1.5 T-MRI due to its being proportional to the square of the strength of a static magnetic field. The effect of heat generation in 3.0 T-MRI can thus be greater than in 1.5 T-MRI. So, using 1.5 T and 3.0 T-MRI scanners, we measured the temperature of several Ti implants using the same scanning parameters during MRI scanning. Our measurements showed the rise in temperature of the Ti implants to be a maximum of 0.4 degrees C. In this study, however, Ti in a human mouth was not directly measured, so we need to attempt to perform MRI carefully on patients with Ti implants.

Understanding wax ester synthesis in Euglena gracilis: Insights into mitochondrial anaerobic respiration.

Euglena gracilis, photosynthetic protist, has a unique ability to generate wax esters in the absence of oxygen, employing a distinctive fatty acid synthesis mechanism. Through comprehensive inhibitor assays and gene-silencing techniques, our research clearly emphasized the indispensable role of the mitochondrial anaerobic respiratory chain in this biosynthesis. We identified acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) as central molecular components in the pathway. These findings strongly indicated a potential reversal of beta-oxidation occurring within mitochondria for fatty acid production in anaerobic conditions. Furthermore, our analysis revealed the pivotal function of nicotinamide nucleotide transhydrogenase (NNT) in efficiently managing the NADPH/NAD+ conversion essential for sustaining anaerobic metabolism. This review outlines our key findings and provides a comprehensive understanding of the molecular mechanisms that enable E. gracilis to produce wax ester anaerobically.

[Investigation of radiofrequency heating for a closed conducting loop formed in a part of the patient's body in 1.5 Tesla magnetic resonance (MR) imaging and 3.0 Tesla MR imaging-measurement of temperature by use of human body-equivalent phantom].

Thermal injuries have been sometimes reported due to a closed conducting loop formed in a part of the patient's body during magnetic resonance imaging (MRI). In recent years, 3.0 T-MRI scanner has been widely used. However, it is considered that the specific absorption rate (SAR) of 3.0 T-MRI can affect the heat of the loop because its own SAR becomes approximately 4 times as much as that of the1.5 T-MRI scanner. With this, the change in temperature was measured with human body-equivalent loop phantom in both 1.5 T-MRI and 3.0 T-MRI. In the two scanners, the temperature during 20 min of scanning time was measured with three types of sequences such as field echo (FE), spin echo (SE), and turbo SE (TSE) set up with the same scanning condition. It was found from the result that rise in temperature depended on SAR of the scanning condition irrespective of static magnetic field intensity and any pulse sequences. Furthermore, the increase of SAR and rise in temperature were not only in proportion to each other but also were indicated to have good correlation. However, even low SAR can occasionally induce serious thermal injuries. It was found from result that we had to attempt not to form a closed conducting loop with in a part of the patient's body during MRI.

Gelation of konjac glucomannan by acetylmannan esterases from Aspergillus oryzae.

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

Novel acid trehalase belonging to glycoside hydrolase family 37 from Pleurotus sp.: cloning, expression and characterization.

The N-terminal amino acid sequence of the Pleurotus sp. 90 kDa protein was in good agreement with the corresponding sequence of the glycoside hydrolase (GH) family 37 protein (trehalase) from P. ostreatus PC 15 v2.0. The length of the Pleurotus sp. trehalase gene was 2247 bp, encoding a protein of 749 amino acids with a predicted molecular mass of 81.2 kDa. The molecular mass of the recombinant enzyme was estimated to be about 117 kDa by SDS-PAGE. We found that the recombinant enzyme comprised an N-glycosylated sugar chain and that its optimum pH and temperature were 4.5 and 40 ºC, respectively. Moreover, this enzyme exhibited high activity against trehalose exclusively. We found that the enzyme is novel acid trehalase belonging to GH family 37.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.