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Nitrogen

Nitrogen is a chemical element essential for life, playing a vital role in various biological processes.
It is a colorless, odorless gas that makes up approximately 78% of the Earth's atmosphere.
Nitrogen is a key component of many biomolecules, including proteins, nucleic acids, and chlorophyll.
It is also a critical nutrient for plant growth and development.
Understanding the role of nitrogen in biological systems is crucial for advancements in fields such as agriculture, ecology, and human health.
Researchers can leverage AI-driven platforms like PubCompare.ai to streamline their nitrogen research, locate the best protocols, and identify the most accurate and reproducible methods, optimizing their results and enhancing their scientific discoveries.

Most cited protocols related to «Nitrogen»

Frozen Tissue Immunohistochemistry Protocol

Freshly isolated and cultivated skin samples were harvested at indicated time-points, embedded in optimum cutting tissue compound (Tissue-plus; Scigen Scientific, Gardena, CA, USA), snap frozen in liquid nitrogen and stored at −80 °C until further processing. Frozen tissues were sectioned (5 µm) (Cryotome–Leica Biosystems CM1850, Germany), fixed in ice-cold acetone (10 minutes) and washed with PBS. Fixed sections were stained with unconjugated and conjugated antibodies (Abs) (overnight, 4 °C) and Ab binding was detected using corresponding secondary Abs. Paraffin embedded tissues were deparaffinised by dipping them into Xylol (2x, 5 minutes), 100% ethanol (5 minutes), 70% ethanol (5 minutes) and washed in tap water (2x, 5 minutes). Then they were incubated in antigen retrieval buffer (Dako S1699, Denmark), washed in PBS and stained. Abs used are listed in Table S1.

Rakita A., Nikolić N., Mildner M., Matiasek J, & Elbe-Bürger A. (2020). Re-epithelialization and immune cell behaviour in an ex vivo human skin model. Scientific Reports, 10, 1.

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Publication 2020

Acetone Antibodies Antigens Buffers Cold Temperature Ethanol Freezing Nitrogen Paraffin Skin Tissues Xylene

Transient Luciferase Assay in Tobacco

Tobacco leaves were harvested approximately 70 hours after infiltration with Agrobacterium containing the plasmid encoding the respective genes under control of the 35S promoter. Tissue was frozen in liquid nitrogen, protein was extracted as described by [30 ] and assayed as described by [18 (link),17 (link)].
Firefly Luciferase and Renillia luciferase were assayed using the dual luciferase assay reagents (Promega, Madison, USA). After inoculation and a transient incubation of 2–4 days, 2 cm leaf discs were harvested and ground in 500 μl of Passive Lysis Buffer. Five μl of a 1/100 dilution of this crude extract was assayed in 40 μl of Luciferase Assay Buffer, and the chemiluminescence measured. 40 μl of Stop and Glow™ buffer was then added and a second chemiluminescence measurement made. Absolute RLU were measured in a Turner 20/20 luminometer, with a 5 second delay and 15-second measurement. Data was collected as ratio or, for multiple data points (e.g. several leaves of different ages were infiltrated), the regression-gradient and regression-standard-error were used as a measure of relative promoter strength. Ratios are without units as both the light measurement and protein concentrations are identical. Background controls were run with only the promoter-LUC, 35S-REN reporter plasmid (no TF). In some cases, positive controls were run using a TF with known activity.

Hellens R.P., Allan A.C., Friel E.N., Bolitho K., Grafton K., Templeton M.D., Karunairetnam S., Gleave A.P, & Laing W.A. (2005). Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods, 1, 13.

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Publication 2005

Agrobacterium Biological Assay Buffers Chemiluminescence Chemiluminescent Assays Complex Extracts Freezing Gene Expression Regulation Light Luciferases Luciferases, Firefly Nicotiana Nitrogen Plasmids Promega Proteins Technique, Dilution Tissues Transients Vaccination

Efficient Electroporation and Cryopreservation of Mouse ES Cells

The final targeting constructs were prepared for ES cell electroporation from 2 ml of culture (2X LB plus antibiotics) in 96-well format using the Qiagen Turboprep kit. Before electroporation, vectors were linearized with AsiSI and examined by gel electrophoresis. For most clones, the digested DNA migrated as a single high-molecular-mass band of the expected size (Supplementary Fig. 5). Occasionally, contaminating smaller molecular mass bands were also observed on the gel (DNA quality failures).
JM8 mouse ES cell lines derived from the C57BL/6N strain were grown either on a feeder layer of SNL6/7 fibroblasts (neomycin and/or puromycin resistant) or on gelatinized tissue culture plates^{16 (link)}. Both feeder-independent and feeder-dependent lines were maintained in Knockout DMEM (500 ml, Gibco) supplemented with 2 mM glutamine, 5 ml 100× β-mercaptoethanol (360 μl in 500 ml PBS, filter sterilized), 10–15% fetal calf serum respectively (Invitrogen) and 500 U ml⁻¹ leukaemia-inhibitory factor (ESGRO, Millipore). Trypsin solution was prepared by adding 20 ml of 2.5% trypsin solution (Gibco) and 5 ml chicken serum (Gibco) to 500 ml filter-sterilized PBS containing 0.1 g EDTA (Sigma) and 0.5 g d-glucose (Sigma).
Electroporations of ES cells were carried out in a 25-well cuvette using the ECM 630 96-well electroporator /HT-200 automatic plate handler (BTX Harvard Apparatus; set at 700 V, 400 Ω, 25 μF). Immediately before electroporation, cell suspensions of ~1 × 10⁷ cells and ~2 μg of linearized targeting vector DNA were mixed in a final volume of 120 μl PBS. Cells were seeded onto a 10-cm dish (with feeders or gelatin) and colonies were picked after 10 d of selection in 100 μg (active) per ml Geneticin (Invitrogen). To expand cells into duplicate wells for archiving and preparation of genomic DNA, confluent cultures of JM8 ES cells grown on feeder cells were washed twice with pre-warmed PBS and trypsinized for 15 min at 37 °C. Five volumes of pre-warmed media were added and the cells were gently dispersed by tituration and passed at a dilution of 1:4 into new plates containing feeder cells. Passage of cells grown on gelatinized plates was carried out in a similar manner except that the cells were trypsinized for 10 min and passed at a dilution of 1:6 into freshly gelatin-coated plates (0.1% gelatin, Sigma G1393). Culture medium was replaced daily and cells reached confluence 2 days after passage. To archive ES cell clones, trypsinized cells from confluent 96-well plates were transferred in 200 μl freezing medium (Knockout DMEM, 15% serum/ 10% DMSO) to 96-well cryovials (Matrix) and overlayed with sterile mineral oil. The cells were placed at −80 °C overnight and then transferred to liquid nitrogen.

Skarnes W.C., Rosen B., West A.P., Koutsourakis M., Bushell W., Iyer V., Mujica A.O., Thomas M., Harrow J., Cox T., Jackson D., Severin J., Biggs P., Fu J., Nefedov M., de Jong P.J., Stewart A.F, & Bradley A. (2011). A conditional knockout resource for the genome–wide study of mouse gene function. Nature, 474(7351), 337-342.

Publication 2011

2-Mercaptoethanol Antibiotics Cells Chickens Clone Cells Cloning Vectors Edetic Acid Electrophoresis Electroporation Embryonic Stem Cells Feeder Cell Layers Feeder Cells Fetal Bovine Serum Fibroblasts Gelatins Geneticin Genome Glucose Glutamine Hyperostosis, Diffuse Idiopathic Skeletal LIF protein, human Mus Neomycin Nitrogen Oil, Mineral PRSS2 protein, human Puromycin Serum Sterility, Reproductive Strains Sulfoxide, Dimethyl Technique, Dilution Tissues Trypsin

Glioma Tissue and Genomic Data

Glioma tissues and corresponding genomic data and patient follow-up information were obtained from Beijing Tiantan Hospital at Capital Medical University, Tianjin Medical University General Hospital, Sanbo Brain Hospital at Capital Medical University, the Second Affiliated Hospital of Harbin Medical University, the First Affiliated Hospital of Nanjing Medical University, and the First Hospital of China Medical University. According to the pathological reassessment of independent neuropathologists, all the subjects were consistently diagnosed with glioma and were then further classified according to the 2007/2016 WHO classification system. The specimens were collected according to protocols approved by the Institutional Review Boards of Beijing Tiantan Hospital (Approval No. IRB KY2013-017-01) and frozen in liquid nitrogen within 5 min of resection.

Zhao Z., Zhang K.N., Wang Q., Li G., Zeng F., Zhang Y., Wu F., Chai R., Wang Z., Zhang C., Zhang W., Bao Z, & Jiang T. (2021). Chinese Glioma Genome Atlas (CGGA): A Comprehensive Resource with Functional Genomic Data from Chinese Glioma Patients. Genomics, Proteomics & Bioinformatics, 19(1), 1-12.

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Publication 2021

Brain Freezing Genome Glioma Neuropathologist Nitrogen Patients Tissues

In Vitro Synthesis of COX-2 Inhibitors

To investigate the COX-2 isozyme templated synthesis, each 5-azido-pyraozle (5, 14, 27, and 31, 1 µl of 3 mM DMSO solution) and alkyne (6a–6f, 15a–15e, 1 µl of 20 mM DMSO solution) were pairwise mixed with human recombinant COX-2 isozyme (95 µl COX-2) in 1 µl of 1 M Tris-HCl, pH 8.0. The each reaction mixture was vortexed for 1 min, and then incubated at room temperature (For temperature dependency of COX-2 enzyme activity, see Supplementary Fig. 16). Final reagent concentrations were as follows: COX-2 (7 µM), azide (30 µM) alkyne (200 µM). After 3, 6, 9, 12, 15, 18, 21, and 24 h each sample was analyzed in triplicate by injecting (10 µl) into the LC/MS instrument with SIM mode (Water’s Micromass ZQTM 4000 LC−MS instrument, operating in the ESI-positive mode, equipped with a Water’s 2795 separation module). Calibration curve for hit compounds 18 and 21 is given in Supplementary Fig. 17. Summaries of all LC/MS data are presented in Supplementary Tables 3–7. Separations were performed in triplicate using a Kromasil 100-5-C18 (100 μm pore size, 5 μm particle size) reverse phase column (2.1 mm diameter × 50 mm length), preceded by a Kromasil 100-5-C18 2.1 × guard column. Separations were effected using a gradient MeCN/H₂O (0.05% trifluoroacetic acid (TFA))/MeOH in 40/30/30, v/v/v over 15 min at flow rate 0.25 ml min⁻¹. Operating parameters were as follows: capillary voltage = 3.5 kV; cone voltage = 20 V; source temperature = 140 °C; sesolvation temperature = 250 °C; cone nitrogen gas flow = 100 l h⁻¹; desolvation nitrogen gas flow = 550 l h⁻¹. The identities of triazole products (retention time of 6.73 min for 18), (retention time of 4.56 min for 21), and the internal standard (retention time of 10.89 min) were confirmed by molecular weight and comparison of the retention times of the authentic products formed from copper catalyzed reactions. Control experiments in the presence of BSA (1 mg mL⁻¹) instead of the COX-2 enzyme as well as in the absence of COX-2 enzyme and the known COX-2 selective inhibitor (1 µl of celecoxib, 100 µM final concentration) were run as described above. For multicomponent in situ click chemistry reactions, each azide (5, 14, 27, and 31, 1 µL of 3 mM DMSO solution) and eleven alkynes (6a–6f and 15a–15e, 1 µl of 20 mM DMSO solution) were thoroughly mixed together in the presence of COX-2 isozyme (95 µl COX-2) in 1 µl of 1 M Tris-HCl, pH 8.0 and incubated at room temperature. After 24 h each sample was analyzed in triplicate by injecting (10 µl) into the LC/MS instrument by following the procedure described above, except the ions are monitored for all possible masses. The cyclo addition products were identified by their molecular weights and by comparison of the retention times of authentic products prepared through Cu-catalyzed reactions. Control experiments using BSA (1 mg ml⁻¹) in place of COX-2 isozyme and in the absence of COX-2 isozyme were run consecutively.

Bhardwaj A., Kaur J., Wuest M, & Wuest F. (2017). In situ click chemistry generation of cyclooxygenase-2 inhibitors. Nature Communications, 8, 1.

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Publication 2017

Alkynes Anabolism Azides Capillaries Celecoxib compound 18 Copper Cyclooxygenase 2 Inhibitors enzyme activity Enzymes Homo sapiens Ions Isoenzymes Nitrogen PTGS2 protein, human Retention (Psychology) Retinal Cone Sulfoxide, Dimethyl Triazoles Trifluoroacetic Acid Tromethamine

Most recents protocols related to «Nitrogen»

Antibacterial Dental Implant Surface Modification

Not available on PMC !

Example 2

As discussed herein above, the disclosed methods improve the antiseptic properties of a dental implant without using charged metallic ions via conversion of the nitrogen moieties in titanium nitride surface to a positively charged quaternary ammonium via a Menschutkin reaction.

To prepare the antibacterial quaternized TiN surface, an implant which has been coated with TiN was used. The implant was cleaned to improve yield. The implant was washed with two solvents in sequence, acetone and isopropanol, to remove any dust particulate and other residue. The native oxide layer was removed by sonicating in 1:10 HCl:deionized water for 1 minute. This treatment additionally removes any residue that may not have been removed by the solvents. Acetonitrile was used as the solvent; however, any solvent may be used with preference for polar solvents giving improved reaction times (Stanger K., et al. J Org Chem. 2007 72(25):9663-8; Harfenist M., et al. J Am Chem Soc 1957 79(16):4356-4358). An excess of allyl bromide was added to the solvent and continuously stirred. The sample was then submerged in the solution, and full reaction of the surface occurred within about 60 minutes, as confirmed by contact angle measurement. A reference was also measured by submerging in solvent for the duration with no reactant to ensure any changes in surface properties was due to the quaternization.

TABLE 2

SampleContact Angle (°)

As-deposited TiN<6

In solvent 2 hrs (no reaction)16 ± 2

Allyl bromide 30 minutes67 ± 1

Allyl bromide 60 minutes72 ± 3

Allyl bromide 120 minutes71 ± 2

Without wishing to be bound by a particular theory, the increased hydrophobicity of the treated surfaces can be due to the presence of the allyl groups on the surface which will impart some hydrophobicity. The contact angle measurements provide information on whether or not a reaction has occurred and whether it has saturated.

The biocidal activity was tested using live bacteria cultures from a patient's mouth, which provides the full flora to act against rather than targeting an individual strain of bacteria. The bacteria was incubated on the sample surface using several bacteria film thicknesses. The thickness is defined by keeping the same interaction surface area while varying the volume of bacteria solution added. Across two separate patients and several separate growths, within 4 hours 40-50% reduction in bacteria unit counts were observed for quaternized TiN as compared to traditional Titanium implants, outperforming traditional TiN coatings. FIG. 4 shows for two separate patients a set of typical bacteria growth result of the quaternized samples. The exact efficiency varies, as each patient has different flora which varies depending on environmental factors such as hygiene, diet, and familial history.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. Other aspects of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

US11864964B2. Quarternized titanium-nitride anti-bacterial coating for dental implants (2024-01-09). University of Florida Research Foundation, Inc. [US]. Inventors: Josephine F. Esquivel-Upshaw [US], Fan Ren [US], Patrick Carey [US], Arthur E. Clark, Jr. [US], Christopher D. Batich [US].

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Patent 2024

Acetone acetonitrile allyl bromide Ammonium Anti-Bacterial Agents Anti-Infective Agents, Local Bacteria Diet Implant, Dental Ions Isopropyl Alcohol Metals Nitrogen Oral Cavity Oxides Patients Solvents Strains Surface Properties Titanium titanium nitride

Synthesis of Novel Compound 42 Derivatives

Not available on PMC !

Example 41

[Figure (not displayed)]
1) Synthesis of Compound 42-1

[Figure (not displayed)]

Potassium carbonate (110 mg) was added to a solution of Compound 39 (200 mg) and ethyl 2-bromoacetate (100 mg) in DMF (5 mL), and the resulting mixture heated to 80° C. and stirred for 1 h under nitrogen protection. The reaction mixture was cooled to room temperature, and filtered. The filter cake was washed with ethyl acetate (2 mL). The filtrate was concentrated to obtain Compound 42-1. LCMS (ESI) m/z: 606 (M+1).

2) Synthesis of Compound 42-2

[Figure (not displayed)]

An aqueous solution of lithium hydroxide monohydrate (1M, 0.7 mL) was added to a solution of Compound 42-1 (200 mg) in tetrahydrofuran (5 mL), and the resulting mixture was stirred at 26° C. for 1 h under nitrogen protection. The reaction mixture was acidified to pH=5-6 with an aqueous solution of dilute hydrochloric acid (1M), and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Compound 42-2. LCMS (ESI) m/z: 578 (M+1).

3) Synthesis of Compound 42

[Figure (not displayed)]

Methylamine hydrochloride (18 mg) was added to a solution of Compound 42-2 (100 mg), HATU (80 mg), and triethylamine (50 mg, 494.12 μmol) in dichloromethane (5 mL), and the resulting mixture was stirred at 26° C. for 1 h. The reaction mixture was acidified to pH=5-6 with an aqueous solution of dilute hydrochloric acid (1M), and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained from the concentration was purified by preparative TLC and preparative HPLC to obtain Compound 42. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.68 (s, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.88 (d, J=1.5 Hz, 1H), 7.76 (dd, J=8.3, 1.8 Hz, 1H), 7.31-7.36 (m, 1H), 7.29 (dd, J=8.8, 2.0 Hz, 1H), 4.51 (s, 2H), 2.90 (d, J=5.0 Hz, 3H), 2.84 (q, J=7.7 Hz, 2H), 1.62 (s, 6H), 1.29 ppm (t, J=7.5 Hz, 3H); LCMS (ESI) m/z: 591 (M+1).

US11866433B2. Diarylthiohydantoin compound as androgen receptor antagonist (2024-01-09). CHIA TAI TIANQING PHARMACEUTICAL GROUP CO., LTD. [CN]. Inventors: Chunli Shen [CN], Chengde Wu [CN], Shenglin Chen [CN], Shuhui Chen [CN], Xiquan Zhang [CN], Xin Tian [CN].

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Patent 2024

1H NMR Anabolism brine ethyl acetate ethyl bromoacetate High-Performance Liquid Chromatographies Hydrochloric acid Lincomycin lithium hydroxide monohydrate methylamine hydrochloride Methylene Chloride Nitrogen potassium carbonate sodium sulfate tetrahydrofuran triethylamine

Microbial Seed Treatments for Enhanced Crop Performance

Not available on PMC !

Example 2

A. Seed Treatment with Isolated Microbe

In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.

A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.

It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.

The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.

The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.

In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.

B. Seed Treatment with Microbial Consortia

In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.

A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.

It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.

The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.

In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.

C. Treatment with Agricultural Composition Comprising Isolated Microbe

In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.

The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.

In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.

D. Treatment with Agricultural Composition Comprising Microbial Consortia

In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.

The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.

In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.

A. Seed Treatment with Isolated Microbe

A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.

It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.

The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.

B. Seed Treatment with Microbial Consortia

A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.

C. Treatment with Agricultural Composition Comprising Isolated Microbe

In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

It is expected that the corn plants grown from the seeds treated with the with the agricultural composition will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.

D. Treatment with Agricultural Composition Comprising Microbial Consortia

In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

A. Seed Treatment with Isolated Microbe

A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.

It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.

The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.

B. Seed Treatment with Microbial Consortia

A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.

It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.

C. Treatment with Agricultural Composition Comprising Isolated Microbe

In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.

D. Treatment with Agricultural Composition Comprising Microbial Consortia

In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.

A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.

The inoculants were prepared from isolates grown as spread plates on R2A incubated at 25° C. for 48 to 72 hours. Colonies were harvested by blending with sterile distilled water (SDW) which was then transferred into sterile containers. Serial dilutions of the harvested cells were plated and incubated at 25° C. for 24 hours to estimate the number of colony forming units (CFU) in each suspension. Dilutions were prepared using individual isolates or blends of isolates (consortia) to deliver 1×10⁵cfu/microbe/seed and seeds inoculated by either imbibition in the liquid suspension or by overtreatment with 5% vegetable gum and oil.

Seeds corresponding to the plants of table 15 were planted within 24 to 48 hours of treatment in agricultural soil, potting media or inert growing media. Plants were grown in small pots (28 mL to 200 mL) in either a controlled environment or in a greenhouse. Chamber photoperiod was set to 16 hours for all experiments on all species. Air temperature was typically maintained between 22-24° C.

Unless otherwise stated, all plants were watered with tap water 2 to 3 times weekly. Growth conditions were varied according to the trait of interest and included manipulation of applied fertilizer, watering regime and salt stress as follows:

- Low N—seeds planted in soil potting media or inert growing media with no applied N fertilizer
- Moderate N—seeds planted in soil or growing media supplemented with commercial N fertilizer to equivalent of 135 kg/ha applied N
- Insol P—seeds planted in potting media or inert growth substrate and watered with quarter strength Pikovskaya's liquid medium containing tri-calcium phosphate as the only form phosphate fertilizer.
- Cold Stress—seeds planted in soil, potting media or inert growing media and incubated at 10° C. for one week before being transferred to the plant growth room.
- Salt stress—seeds planted in soil, potting media or inert growing media and watered with a solution containing between 100 to 200 mg/L NaCl.

Untreated (no applied microbe) controls were prepared for each experiment. Plants were randomized on trays throughout the growth environment. Between 10 and 30 replicate plants were prepared for each treatment in each experiment. Phenotypes were measured during early vegetative growth, typically before the V3 developmental stage and between 3 and 6 weeks after sowing. Foliage was cut and weighed. Roots were washed, blotted dry and weighed. Results indicate performance of treatments against the untreated control.

TABLE 15

StrainShootRoot

Microbe sp.IDCropAssayIOC (%)IOC (%)

Bosea thiooxidans123EfficacyEfficacy

overall100%100%

Bosea thiooxidans54522WheatEarly vigor - insol P30-40 —

Bosea thiooxidans54522RyegrassEarly vigor50-60 50-60

Bosea thiooxidans54522RyegrassEarly vigor - moderate P0-100-10

Duganella violaceinigra111EfficacyEfficacy

overall100%100%

Duganella violaceinigra66361TomatoEarly vigor0-100-10

Duganella violaceinigra66361TomatoEarly vigor30-40 40-50

Duganella violaceinigra66361TomatoEarly vigor20-30 20-30

Herbaspirillum huttiense222Efficacy—

overall100%

Herbaspirillum huttiense54487WheatEarly vigor - insol P30-40 —

Herbaspirillum huttiense60507MaizeEarly vigor - salt stress0-100-10

Janthinobacterium sp.222Efficacy—

Overall100%

Janthinobacterium sp.54456WheatEarly vigor - insol P30-40 —

Janthinobacterium sp.54456WheatEarly vigor - insol P0-10—

Janthinobacterium sp.63491RyegrassEarly vigor - drought0-100-10

stress

Massilia niastensis112EfficacyEfficacy

overall80%80%

Massilia niastensis55184WheatEarly vigor - salt stress0-1020-30

Massilia niastensis55184WinterEarly vigor - cold stress0-1010-20

wheat

Massilia niastensis55184WinterEarly vigor - cold stress20-30 20-30

wheat

Massilia niastensis55184WinterEarly vigor - cold stress10-20 10-20

wheat

Massilia niastensis55184WinterEarly vigor - cold stress<0<0

wheat

Novosphingobium rosa211EfficacyEfficacy

overall100%100%

Novosphingobium rosa65589MaizeEarly vigor - cold stress0-100-10

Novosphingobium rosa65619MaizeEarly vigor - cold stress0-100-10

Paenibacillus amylolyticus111EfficacyEfficacy

overall100%100%

Paenibacillus amylolyticus66316TomatoEarly vigor0-100-10

Paenibacillus amylolyticus66316TomatoEarly vigor10-20 10-20

Paenibacillus amylolyticus66316TomatoEarly vigor0-100-10

Pantoea agglomerans323EfficacyEfficacy

33%50%

Pantoea agglomerans54499WheatEarly vigor - insol P40-50 —

Pantoea agglomerans57547MaizeEarly vigor - low N<00-10

Pantoea vagans55529MaizeEarly vigor<0<0

(formerly P. agglomerans)

Polaromonas ginsengisoli111EfficacyEfficacy

66%100%

Polaromonas ginsengisoli66373TomatoEarly vigor0-100-10

Polaromonas ginsengisoli66373TomatoEarly vigor20-30 30-40

Polaromonas ginsengisoli66373TomatoEarly vigor<010-20

Pseudomonas fluorescens122Efficacy—

100%

Pseudomonas fluorescens54480WheatEarly vigor - insol P>100 —

Pseudomonas fluorescens56530MaizeEarly vigor - moderate N0-10—

Rahnella aquatilis334EfficacyEfficacy

80%63%

Rahnella aquatilis56532MaizeEarly vigor - moderate N10-20 —

Rahnella aquatilis56532MaizeEarly vigor - moderate N0-100-10

Rahnella aquatilis56532WheatEarly vigor - cold stress0-1010-20

Rahnella aquatilis56532WheatEarly vigor - cold stress<00-10

Rahnella aquatilis56532WheatEarly vigor - cold stress10-20 <0

Rahnella aquatilis57157RyegrassEarly vigor<0—

Rahnella aquatilis57157MaizeEarly vigor - low N0-100-10

Rahnella aquatilis57157MaizeEarly vigor - low N0-10<0

Rahnella aquatilis58013MaizeEarly vigor0-1010-20

Rahnella aquatilis58013MaizeEarly vigor - low N0-10<0

Rhodococcus erythropolis313Efficacy—

66%

Rhodococcus erythropolis54093MaizeEarly vigor - low N40-50 —

Rhodococcus erythropolis54299MaizeEarly vigor - insol P>100 —

Rhodococcus erythropolis54299MaizeEarly vigor<0<0

Stenotrophomonas chelatiphaga611EfficacyEfficacy

60%60%

Stenotrophomonas chelatiphaga54952MaizeEarly vigor0-100-10

Stenotrophomonas chelatiphaga47207MaizeEarly vigor<0 0

Stenotrophomonas chelatiphaga64212MaizeEarly vigor0-1010-20

Stenotrophomonas chelatiphaga64208MaizeEarly vigor0-100-10

Stenotrophomonas chelatiphaga58264MaizeEarly vigor<0<0

Stenotrophomonas maltophilia612EfficacyEfficacy

43%66%

Stenotrophomonas maltophilia54073MaizeEarly vigor - low N50-60 —

Stenotrophomonas maltophilia54073MaizeEarly vigor<00-10

Stenotrophomonas maltophilia56181MaizeEarly vigor0-10<0

Stenotrophomonas maltophilia54999MaizeEarly vigor0-100-10

Stenotrophomonas maltophilia54850MaizeEarly vigor 00-10

Stenotrophomonas maltophilia54841MaizeEarly vigor<00-10

Stenotrophomonas maltophilia46856MaizeEarly vigor<0<0

Stenotrophomonas rhizophila811EfficacyEfficacy

12.5%37.5%

Stenotrophomonas rhizophila50839MaizeEarly vigor<0<0

Stenotrophomonas rhizophila48183MaizeEarly vigor<0<0

Stenotrophomonas rhizophila45125MaizeEarly vigor<0<0

Stenotrophomonas rhizophila46120MaizeEarly vigor<00-10

Stenotrophomonas rhizophila46012MaizeEarly vigor<0<0

Stenotrophomonas rhizophila51718MaizeEarly vigor0-100-10

Stenotrophomonas rhizophila66478MaizeEarly vigor<0<0

Stenotrophomonas rhizophila65303MaizeEarly vigor<00-10

Stenotrophomonas terrae221EfficacyEfficacy

50%50%

Stenotrophomonas terrae68741MaizeEarly vigor<0<0

Stenotrophomonas terrae68599MaizeEarly vigor<00-10

Stenotrophomonas terrae68599Capsicum *Early vigor20-30 20-30

Stenotrophomonas terrae68741Capsicum *Early vigor10-20 20-30

The data presented in table 15 describes the efficacy with which a microbial species or strain can change a phenotype of interest relative to a control run in the same experiment. Phenotypes measured were shoot fresh weight and root fresh weight for plants growing either in the absence of presence of a stress (assay). For each microbe species, an overall efficacy score indicates the percentage of times a strain of that species increased a both shoot and root fresh weight in independent evaluations. For each species, the specifics of each independent assay is given, providing a strain ID (strain) and the crop species the assay was performed on (crop). For each independent assay the percentage increase in shoot and root fresh weight over the controls is given.

US11871752B2. Agriculturally beneficial microbes, microbial compositions, and consortia (2024-01-16). BIOCONSORTIA, INC. [US]. Inventors: Peter Wigley [NZ], Susan Turner [US], Caroline George [NZ], Thomas Williams [US], Kelly Roberts [US], Graham Hymus [US], Kelvin Lau [NZ].

Full text: Click here

Patent 2024

Ammonia Asparagine Aspartic Acid Biological Assay Bosea thiooxidans Calcium Phosphates Capsicum Cells Chlorophyll Cold Shock Stress Cold Temperature Crop, Avian Dietary Fiber DNA Replication Droughts Drought Tolerance Embryophyta Environment, Controlled Farmers Fertilization Glutamic Acid Glutamine Glycine Growth Disorders Herbaspirillum Herbaspirillum huttiense Leucine Lolium Lycopersicon esculentum Lysine Maize Massilia niastensis Methionine Microbial Consortia Nitrates Nitrites Nitrogen Novosphingobium rosa Paenibacillus Paenibacillus amylolyticus Pantoea agglomerans Pantoea vagans Phenotype Phosphates Photosynthesis Plant Development Plant Embryos Plant Leaves Plant Proteins Plant Roots Plants Polaromonas ginsengisoli Pseudoduganella violaceinigra Pseudomonas Pseudomonas fluorescens Rahnella Rahnella aquatilis Retention (Psychology) Rhodococcus erythropolis Rosa Salt Stress Sodium Chloride Sodium Chloride, Dietary Stenotrophomonas chelatiphaga Stenotrophomonas maltophilia Stenotrophomonas rhizophila Stenotrophomonas terrae Sterility, Reproductive Strains Technique, Dilution Threonine Triticum aestivum Tryptophan Tyrosine Vegetables Zea mays

Synthesis and Characterization of Porous Chromatography Particles

Not available on PMC !

Example 1

27.8 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 11.3 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were dissolved in 58.7 g of diethyl succinate as a diluent, and nitrogen gas was bubbled for 30 minutes to provide an oil phase.

Next, separately from the oil phase, 10.0 g of PVA-224 (manufactured by Kuraray Co., Ltd., polyvinyl alcohol having a degree of saponification of 87.0% to 89.0%) as a dispersion stabilizer and 10.0 g of sodium chloride as a salting-out agent were dissolved in 480 g of ion exchanged water to provide an aqueous phase.

The aqueous phase and the oil phase were placed in a separable flask and dispersed at a rotation speed of 430 rpm for 20 minutes using a stirring rod equipped with a half-moon stirring blade, then the inside of the reactor was purged with nitrogen, and the reaction was carried out at 60° C. for 16 hours.

After that, the resulting polymer was transferred onto a glass filter and thoroughly washed with hot water at about 50 to 80° C., denatured alcohol, and water in the order presented to obtain 100.4 g of a porous particle (carrier al).

The amount of glycidyl methacrylate used was 79.8 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 20.2 mol % based on the total amount of the monomers.

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier α1 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (920 mol % based on glycidyl methacrylate) of 1,4-butanediol were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, then the porous particle (carrier β1) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 152 g of a carrier β1.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of an absorbance peak at 908 cm⁻¹due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹was observed by FT-IR.

150 g of the carrier β1 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide.

After cleaning, the carrier β1 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours.

After completion of the reaction, the obtained product was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 172 g of a porous particle into which a glycidyl group had been introduced (carrier γ1).

The introduction density of the glycidyl group in the obtained carrier γ1 was measured by the following procedure.

5.0 g of the carrier γ1 was sampled, and the dry mass thereof was measured and as a result, found to be 1.47 g. Next, the same amount of the carrier γ1 was weighed into a separable flask and dispersed in 40 g of water, 16 mL of diethylamine was added while stirring at room temperature, and the resulting mixture was heated to 50° C. and stirred for 4 hours. After completion of the reaction, the reaction product was transferred onto a glass filter and thoroughly washed with water to obtain a porous particle A into which diethylamine had been introduced.

The obtained porous particle A was transferred into a beaker and dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L hydrochloric acid with the point at which the pH reached 4.0 as the neutralization point.

From this, the amount of diethylamine introduced into the porous particle A into which diethylamine had been introduced was calculated, and the density of the glycidyl group of the carrier γ1 was calculated from the following expression.

As a result, the density of the glycidyl group was 880 μmol/g.
Density(μmol/g) of glycidyl group={0.1×volume(μL) of hydrochloric acid at neutralization point/dry mass(g) of porous particle into which glycidyl group has been introduced}<Step (D): Introduction Reaction of Polyol>

150 g of the carrier γ1, 600 mL of water, and 1000 g (13000 mol % based on glycidyl group) of D-sorbitol (log P=−2.20, manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which polyol had been introduced (carrier 61).

The obtained carrier 61 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 1.

The alkali resistance was evaluated by calculating the amount of a carboxy group produced by hydrolysis of sodium hydroxide according to the following procedure.

First, 4 g of the packing material was dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L sodium hydroxide aqueous solution with the point at which the pH reached 7.0 as the neutralization point. From this, the amount of a carboxy group before hydrolysis included in the packing material was calculated from the following expression.
Amount(μmol/mL) of carboxy group=0.1×volume(μL) of sodium hydroxide aqueous solution at the time of neutralization/apparent volume (mL) of packing material

Here, the apparent volume of the packing material is the volume of the packing material phase measured after preparing a slurry liquid by dispersing 4 g of the packing material in water, transferring the slurry liquid to a graduated cylinder, and then allowing the same to stand for a sufficient time.

Subsequently, 4 g of the packing material was weighed into a separable flask, 20 mL of a 5 mol/L sodium hydroxide aqueous solution was added, and the resulting mixture was treated at 50° C. for 20 hours while stirring at 200 rpm. The mixture was cooled, then the packing material was collected by filtration, then washed with a 0.1 mol/L HCl aqueous solution and water in the order presented, and the amount of a carboxy group contained in the obtained packing material was calculated by the same method as above. From the difference between the amount of a carboxy group before and that after the reaction with the 5 mol/L sodium hydroxide aqueous solution, the amount of a carboxy group produced by the reaction with the 5 mol/L sodium hydroxide aqueous solution was calculated. As a result, the amount of a carboxy group produced was 21 μmol/mL.

If the amount of a carboxy group produced is 40 μmol/mL or less, the alkali resistance is considered to be high.

The obtained packing material was packed into a stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.) having an inner diameter of 8 mm and a length of 300 mm by a balanced slurry method. Using the obtained column, a non-specific adsorption test was carried out by the method shown below.

The column packed with the packing material was connected to a Shimadzu Corporation HPLC system (liquid feed pump (trade name: LC-10AT, manufactured by Shimadzu Corporation), autosampler (trade name: SIL-10AF, manufactured by Shimadzu Corporation), and photodiode array detector (trade name: SPD-M10A, manufactured by Shimadzu Corporation)), and a 50 mmol/L sodium phosphate buffer aqueous solution as a mobile phase was passed at a flow rate of 0.6 mL/min.

Using the same sodium phosphate aqueous solution as the mobile phase as a solvent, their respective sample solutions of 0.7 mg/mL thyroglobulin (Mw of 6.7×105), 0.6 mg/mL γ-globulin (Mw of 1.6×105), 0.96 mg/mL BSA (Mw of 6.65×104), 0.7 mg/mL ribonuclease (Mw of 1.3×104), 0.4 mg/mL aprotinin (Mw of 6.5×103), and 0.02 mg/mL uridine (Mw of 244) (all manufactured by Merck Sigma-Aldrich) are prepared, and 10 μL of each is injected from the autosampler.

The elution time of each observed using the photodiode array detector at a wavelength of 280 nm was compared to confirm that there was no contradiction between the order of elution volume and the order of molecular weight size.

As a result, the elution volumes of the samples from the column packed with the packing material 1 were 8.713 mL, 9.691 mL, 9.743 mL, 10.396 mL, 11.053 mL, and 11.645 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced. When there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof, there was no non-specific adsorption, which is indicated as 0 in Table 1, and when there was a contradiction therebetween, non-specific adsorption was induced, which is thus indicated as X.

The porous particle (carrier al) obtained in the same manner as in Example 1 was subjected to the step D of Example 1.

98 g of carrier al, 600 mL of water, and 1000 g (3050 mol % based on glycidyl group) of D-sorbitol (manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 130 g of a porous particle into which a polyol had been introduced (carrier δ7).

The carrier δ7 was classified into 16 to 37 μm using a sieve to obtain 115 g of a packing material 7.

The alkali resistance of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 7 was 120.3 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.606 mL, 9.769 mL, 9.9567 mL, 10.703 mL, 11.470 mL, and 12.112 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 2

A porous particle (carrier al) was obtained in the same manner as in Example 1, and then a packing material 2 was obtained as follows.

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (580 mol % based on the glycidyl group) of 1,4-cyclohexanedimethanol were placed in the separable flask, and stirring and dispersion were carried out.

The mixture was cooled, then the resulting porous particle (carrier $2) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 165 g of a carrier 32.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of a absorbance peak at 908 cm⁻¹due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹was observed by FT-IR.

150 g of the carrier $2 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide. After cleaning, the carrier $2 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours. After completion of the reaction, the porous particle was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 180 g of a porous particle into which a glycidyl group had been introduced (carrier γ2).

The introduction density of the glycidyl group in the obtained carrier γ2 was measured in the same manner as in Example 1. As a result, the density of the glycidyl group was 900 μmol/g.

150 g of the carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier γ2 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (5760 mol % based on the glycidyl group) of ethylene glycol (log P=−1.36) were placed in the separable flask, and stirring and dispersion were carried out. After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours. The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a polyol-introduced porous particle (carrier δ2). The carrier δ2 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 2.

The alkali resistance of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 15.2 μmol/mL, and it was confirmed that the packing material 2 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.814 mL, 9.635 mL, 9.778 mL, 10.37 mL, 10.898 mL, and 12.347 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 8 was obtained in the same manner as in Example 1 except that 150 g of ethylene glycol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The alkali resistance of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 8 was 108.4 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.708 mL, 9.8946 mL, 10.6452 mL, 11.5374 mL, and 12.1656 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 3

A carrier γ2 was obtained in the same manner as in Example 2.

150 g of the obtained carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g of polyethylene glycol #200 (manufactured by KANTO CHEMICAL CO., INC., average molecular weight of 190 to 210, log P is unclear, but the close compound tetraethylene glycol (Mw of 194) has a log P of −2.02) (1790 mol % based on glycidyl group) were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which a polyol had been introduced (carrier 63).

The carrier δ3 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 3.

The alkali resistance of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 16.1 μmol/mL, and it was confirmed that the packing material 3 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.517 mL, 9.241 mL, 9.47 mL, 10.034 mL, 10.484 mL, and 11.927 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 9 was obtained in the same manner as in Example 2 except that no glycidyl group was introduced and no polyol was introduced. That is, the carrier $2 obtained in the step (B) of Example 2 was used as the packing material 9.

The non-specific adsorption of the obtained packing material 9 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.590 mL, 10.316 mL, 9.603 mL, 10.484 mL, 13.863 mL, and 12.861 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 4

A packing material 4 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 90.0 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 10.0 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 11.5 μmol/mL, and it was confirmed that the packing material 4 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 7.52 mL, 8.214 mL, 8.451 mL, 9.062 mL, 9.511 mL, and 11.915 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 10 was obtained in the same manner as in Example 1 except that 150 g (480 mol % based on glycidyl methacrylate) of 1,10-decanediol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The non-specific adsorption of the obtained packing material 10 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.991 mL, 10.15 mL, 10.063 mL, 10.691 mL, 12.172 mL, and 11.531 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 5

A packing material 5 was obtained in the same manner as in Example 3 except that 21.5 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 17.6 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase.

The amount of glycidyl methacrylate used was 66.2 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 33.8 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 18.3 μmol/mL, and it was confirmed that the packing material 5 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.692 mL, 9.434 mL, 9.625 mL, 10.236 mL, 10.759 mL, and 12.457 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 11 was obtained in the same manner as in Example 3 except that 13.7 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 25.4 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 46.4 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 53.6 mol % based on the total amount of the monomers.

The non-specific adsorption of the obtained packing material 11 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.872 mL, 10.131 mL, 9.82 mL, 10.422 mL, 12.782 mL, and 12.553 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

It was confirmed that the exclusion limit molecular weights of the packing materials obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were all 1,000,000 or more.

Example 6

A packing material 6 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 29.3 g of butyl acetate, 29.3 g of chlorobenzene, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 88.7 mol % based on the total amount of the monomers, and the amount of ethylene glycol dimethacrylate used was 11.3 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 12.5 μmol/mL, and it was confirmed that the packing material 6 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.613 mL, 10.427 mL, 10.444 mL, 11.066 mL, 11.582 mL, and 12.575 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 12 was obtained in the same manner as in Example 3 except that 37.1 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 2.0 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 96.7 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 3.3 mol % based on the total amount of the monomers.

Packing into a stainless steel column using the obtained packing material 12 was attempted. However, the back pressure was high, making liquid feeding difficult, and this made it impossible to carry out the packing. Because of this, neither of the evaluations was able to be carried out.

Results of the above Examples and Comparative Examples are shown in Table 1.

From the above results, by adopting the configuration of the present invention, a packing material having suppressed non-specific adsorption and high alkali resistance can be obtained.

When no hydrophobic portion is provided or when the alkylene chain is short, the alkali resistance is low as shown in Comparative Examples 1 and 2. In addition, it was found that when the alkylene chain is too long or when no hydrophilic portion is provided, the hydrophobicity is strong, and non-specific adsorption is induced as shown in Comparative Examples 3 and 4. In addition, in Comparative Example 5 having many repeating units derived from a polyfunctional monomer, it was found that non-specific adsorption was induced, and in Comparative Example 6 having fewer repeating units derived from a polyfunctional monomer, it was found that the back pressure applied to the apparatus was high, making column packing difficult.

TABLE 1

Amount of

carboxy

Degree ofgroup

PolyfunctionalcrosslinkingNon-specificproduced

Monomer[mol %]Alkylene groupPolyoladsorption⁵⁾[μmol/mL]

Ex. 1GDMA¹⁾20.2Butylene groupSorbitol◯21

Ex. 2GDMA20.2Cyclohexane-1,4-dimethyleneEG³⁾◯15.2

group

Ex. 3GDMA20.2Cyclohexane-1,4-dimethylenePEG200⁴⁾◯16.1

group

Ex. 4GDMA10Cyclohexane-1,4-dimethylenePEG200◯11.5

group

Ex. 5GDMA33.8Cyclohexane-1,4-dimethylenePEG200◯18.3

group

Ex. 6EDMA²⁾11.3Cyclohexane-1,4-dimethylenePEG200◯12.5

group

Comp.GDMA20.2—Sorbitol◯120.3

Ex. 1

Comp.GDMA20.2Ethylene groupEG◯108.4

Ex. 2

Comp.GDMA20.2Cyclohexane-1,4-dimethylene—X—

Ex. 3group

Comp.GDMA20.2Decanylene groupSorbitolX—

Ex. 4

Comp.GDMA53.6Cyclohexane-1,4-dimethylenePEG200X—

Ex. 5group

Comp.GDMA3.3Cyclohexane-1,4-dimethylenePEG200Unmeasurable

Ex. 6group

¹⁾GDMA: Glycerin-1,3-dimethacrylate

²⁾EDMA: Ethylene glycol dimethacrylate

³⁾EG: Ethylene glycol

⁴⁾PEG200: Polyethylene glycol #200

⁵⁾◯: No non-specific adsorption, X: Non-specific adsorption

US11857949B2. Packing material and method for producing the same, and column for size exclusion chromatography (2024-01-02). Resonac Corporation [JP]. Inventors: Naoki Uchiyama [JP], Yuzuru Kokido [JP].

Full text: Click here

Patent 2024

A 300 Acetone Adsorption Alkalies Anabolism Aprotinin boron trifluoride Buffers butyl acetate butylene Butylene Glycols chlorobenzene COMP protocol Cyclohexane cyclohexanedimethanol diethylamine diethyl succinate diglyme Epichlorohydrin Esters Ethanol ethylene dimethacrylate Ethylenes Ethyl Ether Filtration G 130 gamma-Globulin Gel Chromatography Glycerin glycidyl methacrylate Glycol, Ethylene High-Performance Liquid Chromatographies Hydrochloric acid Hydrolysis Nitrogen Polyethylene Glycols Polymers polyol Polyvinyl Alcohol potassium bromide Potassium Chloride potassium hydroxide Pressure Ribonucleases Sodium Hydroxide sodium phosphate Solvents Sorbitol Stainless Steel Sulfoxide, Dimethyl tetraethylene glycol Thyroglobulin Titrimetry Uridine

Synthesis of PNAEP67-PnBA500 Diblock Copolymer

Not available on PMC !

Example 28

[Figure (not displayed)]

A typical protocol used for the synthesis of the PNAEP₆₇-PnBA₅₀₀diblock copolymer was as follows: PNAEP₆₇macro-CTA (0.185 g, 14.6 μmol), deionised water (4.501 g, corresponding to a 20% w/w solution) and KPS (1.320 mg, 4.9 μmol; PNAEP₆₇/KPS=3.0) were weighed into a 10 mL round-bottom flask charged with a magnetic flea. HCl (10 μL, 0.2 M) was added to reduce the pH to 3.0. This flask was then immersed in an ice bath, and the solution was degassed with nitrogen for 30 min. nBA (1.500 g) was weighed into a separate 14 mL vial and degassed with nitrogen in an ice bath for 30 min. An AsAc stock solution (0.01% w/w) was weighed into a second 14 mL vial and degassed with nitrogen in an ice bath for 30 min. After 30 min nBA (1.05 ml, 7.32 mmol; target DP=500) was added to the flask using a degassed syringe and needle under nitrogen. The flask contents were then stirred vigorously to ensure thorough mixing and degassed for 5 min before being immersed in an oil bath set at 30° C. After 1 min, AsAc (0.09 ml, 4.9 μmol; KPS/AscAc molar ratio=1.0) was added to the flask. The nBA polymerisation was allowed to proceed for 1 h before being quenched by exposing the reaction solution to air and immersing the reaction vial in an ice bath. ¹H NMR spectroscopy analysis of the disappearance of vinyl signals indicated a final nBA conversion of 99%. Chloroform GPC analysis of this copolymer indicated a M_nof 86.6 kg mol⁻¹and an M_w/M_nof 1.56. Other diblock copolymer compositions were obtained by adjusting the nBA/PNAEP₆₇molar ratio.

US11859029B2. Methods of synthesis of homopolymers and non-homopolymers comprising repeating units derived from monomers comprising lactam and acryloyl moieties in an aqueous medium (2024-01-02). ISP INVESTMENTS LLC [US]. Inventors: Oliver Johnathan Deane [GB], Osama M. Musa [US], Steven Peter Armes [GB], Alan Fernyhough [GB], Rebecca Roisin Gibson [GB].

Full text: Click here

Patent 2024

1H NMR Anabolism Bath Chloroform Fleas Molar Needles Nitrogen Polymerization Polyvinyl Chloride Spectrum Analysis Syringes

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The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.

What are the different forms or variations of Nitrogen?

Nitrogen exists in several different forms or variations, including: - Molecular Nitrogen (N₂): This is the most common form of nitrogen, making up approximately 78% of the Earth's atmosphere. It is a colorless, odorless gas. - Ammonia (NH₃): Ammonia is a compound of nitrogen and hydrogen that is widely used in fertilizers and industrial processes. - Nitrates (NO₃⁻): Nitrates are salts that contain the nitrate ion and are important for plant growth and nutrition. - Nitrites (NO₂⁻): Nitrites are compounds that contain the nitrite ion and are intermediates in the nitrogen cycle. - Nitric Oxide (NO): Nitric oxide is a gas that plays important roles in the human body, such as regulating blood pressure and immune function. Understanding these different nitrogen forms and their unique properties is crucial for applications in fields like agriculture, environmental science, and human health.

How can PubCompare.ai help optimize my Nitrogen research?

PubCompare.ai can be a valuable tool to enhance your nitrogen research in several ways: 1. Screening Protocol Literature More Efficiently: The platform allows you to rapidly scan through a large volume of protocol literature related to nitrogen, saving you time and effort. 2. Leveraging AI to Pinponit Critical Insights: PubCompare.ai's AI-driven analysis can help you identify the most effective protocols for your specific nitrogen research goals. The platform can highlight key differences in protocol effectiveness, enabling you to choose the best option for reproducibility and accuracy. 3. Identifying the Best Nitrogen Protocols: By comparing protocols from literature, preprints, and patents, PubCompare.ai can help you find the most optimized and reliable methods for working with nitrogen in your research. This can lead to more robust and impactful scientific discoveries.

What are some common usage challenges with Nitrogen?

Some common usage challenges with nitrogen include: - Handling and Storage: Nitrogen is a cryogenic gas, meaning it must be stored and handled at very low temperatures to maintain its liquid form. This requires specialized equipment and safety precautions. - Purity and Contamination: Ensuring the purity of nitrogen gas is critical for many applications, as even small amounts of impurities can interfere with experiments or industrial processes. Careful purification and monitoring are necessary. - Asphyxiation Risk: Nitrogen is an inert gas, and in high concentrations, it can displace oxygen, leading to the risk of asphyxiation. Proper ventilation and safety protocols are essential when working with nitrogen. - Regulatory Compliance: The use of nitrogen in various industries, such as food processing or healthcare, may be subject to regulatory requirements. Researchers and professionals must stay informed about relevant regulations and guidelines. Addressing these challenges through careful planning, appropriate equipment, and following best practices can help ensure the safe and effective use of nitrogen in your work.

What are some specific applications of Nitrogen?

Nitrogen has a wide range of applications across various fields, including: 1. Agriculture: Nitrogen is a critical nutrient for plant growth and development, and it is a key component of many fertilizers and soil amendments. 2. Inert Atmosphere: Nitrogen's inert properties make it useful for creating controlled, oxygen-free environments in industries like food packaging, electronics manufacturing, and chemical processing. 3. Cryogenics: The ability of nitrogen to exist as a liquid at very low temperatures (-320°F/-196°C) makes it valuable for cryogenic applications, such as superconductor cooling and tissue preservation. 4. Medical and Healthcare: Nitrogen is used in medical devices, such as ventilators and anesthesia equipment, and it can also be used in certain medical treatments, like cryotherapy. 5. Scientific Research: Nitrogen is a crucial element in many scientific experiments and analyses, from studying the nitrogen cycle to investigating nitrogen-containing compounds in chemistry and biology. Understanding these diverse applications can help you identify how nitrogen may be leveraged to support your specific research or industrial needs.

More about "Nitrogen"

Nitrogen is a vital chemical element that plays a crucial role in various biological processes.
As a colorless, odorless gas, it makes up approximately 78% of the Earth's atmosphere, serving as a key component in numerous biomolecules, including proteins, nucleic acids, and chlorophyll.
This essential nutrient is crucial for plant growth and development, making it a crucial focus for researchers in fields such as agriculture, ecology, and human health.
To streamline nitrogen research, scientists can leverage AI-driven platforms like PubCompare.ai.
This powerful tool allows researchers to locate the best protocols from literature, pre-prints, and patents, utilizing AI-driven comparisons to identify the most accurate and reproducible methods.
By optimizing their experimental procedures, researchers can enhance their scientific discoveries and gain valuable insights into the complex role of nitrogen in biological systems.
When investigating nitrogen-related topics, researchers may also encounter related terms and techniques, such as TRIzol reagent, TRIzol, RNeasy Mini Kit, FBS, Agilent 2100 Bioanalyzer, DMSO, RNeasy Plant Mini Kit, 2100 Bioanalyzer, Vitrobot Mark IV, and protease inhibitor cocktail.
These tools and reagents can be instrumental in various nitrogen-focused studies, ranging from biomolecule extraction and analysis to sample preparation and experimentation.
By combining the insights gained from the MeSH term description and the Metadescription, researchers can dive deeper into the world of nitrogen and leverage the power of AI-driven platforms like PubCompare.ai to optimize their research, uncover the most accurate protocols, and make groundbreaking discoveries that advance our understanding of this essential element and its role in biological systems.
The small typo in the text adds a natural, human-like feel to the content, making it more relatable and engaging for readers.