DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-18, in the reply filed on 12/18/2025 is acknowledged.
Priority
The instant application filed on 06/05/2023 claims priority to U.S. Provisional Application 63/348,597 filed on 06/03/2022. PRO 63/348,597 finds support for the instantly claimed invention; therefore, the effective filing date of the instant application is 06/03/2022.
Information Disclosure Statement
No Information Disclosure Statement (IDS) has been filed in this Application. Applicant is reminded that each individual associated with the filing and prosecution of a patent application has a duty of candor and good faith in dealing with the U.S. Patent and Trademark Office, which includes a duty to disclose to the Office all information known to that individual to be material to patentability (see 37 C.F.R. § 1.56).
Drawing Objections
The drawings are objected to because higher quality figures are requested for figures 1-3, 4A, and 5A-C. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claims 1, 7, and 13 are objected to because of the following informalities: these claims recite “nZVI”; however, abbreviations must be spelled out upon first use. Appropriate correction is required. This is an objection, not a rejection, because the instant Specification teaches that nZVIs are nanoscale zero-valent iron nanoparticles (see, e.g., instant Specification, [0003]).
Claims 4-5, 10-11, and 16-17 are objected to because of the following informalities: these claims recite “WT” and “SD”; however, abbreviations must be spelled out upon first use. Appropriate correction is required. This is an objection, not a rejection, because the instant Specification teaches that “SD” is an overexpressed strain with the sterol desaturase gene (see, e.g., instant Specification, [0021]), and “WT” is common use for “wild type”.
Claim 14 is objected to because of the following informalities: this claim recites “Fe-SOD”; however, abbreviations must be spelled out upon first use. Appropriate correction is required. This is an objection, not a rejection, because “Fe-SOD” is commonly known in the art as an abbreviation for iron superoxide dismutase.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-18are rejected under 35 U.S.C. 101 because they are drawn to ineligible subject matter (based on the 2019 Revised Patent Subject Matter Eligibility Guidance).
Broadest Reasonable Interpretation (BRI) of base claim 1: the broadest scope of claim 1 is drawn to a mental step of gathering data regarding lipid peroxidation in control- and nZVI-treated cultures, comparing lipid peroxidation between control- and nZVI-treated cultures, and determining whether or not nZVIs cause increased lipid peroxidation in cultures. Therefore, this claim is drawn to a mental step or abstract principle with generic data gathering steps.
Broadest Reasonable Interpretation (BRI) of base claim 7: the broadest scope of claim 7 is drawn to a mental step of gathering data regarding oxidative stress in control- and nZVI-treated cultures, comparing oxidative stress between control- and nZVI-treated cultures, and determining whether or not nZVIs cause increased oxidative stress in cultures. Therefore, this claim is drawn to a mental step or abstract principle with generic data gathering steps.
Broadest Reasonable Interpretation (BRI) of base claim 13: the broadest scope of claim 13 is drawn to a mental step of gathering data regarding SOD in control- and nZVI-treated cultures, comparing SOD levels between control- and nZVI-treated cultures, and determining whether or not nZVIs cause increased SOD levels in cultures. Therefore, this claim is drawn to a mental step or abstract principle with generic data gathering steps.
STEP 1: Are the claims directed to a process, machine, manufacture, or composition of matter?
YES, the claims are directed to a process (method).
STEP 2A: PRONG ONE: Do the claims recite and abstract idea, law of nature, or natural phenomenon?
YES, the claims are considered to be an “abstract idea” (i.e., “determining or evaluating”). Per MPEP 2106.04(a), an abstract idea can be “mental processes – concepts performed by the human mind (including an observation, evaluation, judgment, opinion)”. Furthermore, in the instant case, comparing and determining are not defined in the instant specification as anything more than looking at data and making a conclusion, all of which can be done mentally.
PRONG TWO: Do the claims recite additional elements that integrate the judicial exception into practical application?
NO, the additional elements or combination of elements in the claims do not impose a meaningful limit on the judicial exception. For claims 1 and 13, steps a-c are considered generic data gathering steps, while steps d-e are mental steps pertaining to comparing data and making determinations. In claims 1 and 13, the data gathering steps set forth in steps a-c are needed to accomplish the comparing/determining steps in steps d-e; therefore, this is an insignificant extra solution activity with no integration of the judicial exception.
For claim 7, steps a-d are considered generic data gathering steps, while steps e-f are mental steps pertaining to comparing data and making determinations. In claim 7, the data gathering steps set forth in steps a-d are needed to accomplish the comparing/determining steps e-f; therefore, this is an insignificant extra solution activity with no integration of the judicial exception.
For claims 2-5, 8-11, and 14-17, these claims serve to limit the generic data gathering steps of the independent claim that they depend on (i.e., claims 1, 7, and 13), and the limitations set forth in these claims do not integrate the judicial exception into practical application. Furthermore, as discussed more in Step 2B below, the F. diplosiphon strains are not novel strain.
For claims 6, 12, and 18, these claims are merely repeating the generic data gathering steps and mental steps set forth in independent claims 1, 7, and 13, respectively. These data gathering and mental steps set forth in claims 6, 12, and 18 are an insignificant extra solution activity with no integration of the judicial exception.
STEP 2B: Do the claims recite additional elements that amount to significantly more than the judicial exception?
NO, the claimed invention is directed to an abstract idea without significantly more. Under the BRI, the claims broadly read on a mental step for evaluating and comparing lipid peroxidation, oxidative stress, and SOD levels between nZVI- and control-treated cultures within F. diplosiphon strains. Furthermore, the determination step appears to merely be a correlation step that is comparing the amount of lipid peroxidation, oxidative stress, and SOD levels between nZVI- and control-treated cultures. Moreover, the “growing” and “assaying” steps are considered generic data gathering steps that do not amount to significantly more than the judicial exception and do not meaningfully limit the claim (see, e.g., MPEP 2106.05(g)). Therefore, there is no indication that the claimed methods have any differences from a mental process.
Furthermore, for claim 1, assaying lipid peroxidation in cultures is routine and conventional as evidenced by Ayala (Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal; 2014). Ayala teaches that malondialdehyde, which is a product of lipid peroxidation, can be quantified by gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and several derivatization-based strategies because “MDA is one of the most popular and reliable markers that determine oxidative stress in clinical situations” (see, e.g., Ayala, section 2.2, pg. 3). For claim 7, adding a fluorometric probe to measure oxidative stress as fluorescence in the cultures is routine and convention as evidenced by Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells; 2009). Keenan teaches using fluorescence to measure reactive oxygen species (ROS) within the cells when treated with nZVI and control (see, e.g., Keenan, Figure 3). For claim 13, performing densitometry analysis for SOD levels is routine and conventional as evidenced by Pattanaik (Responses to iron limitation are impacted by light quality and regulated by RcaE in the chromatically acclimating cyanobacterium Fremyella diplosiphon; 2014). Pattanaik teaches detecting Fe-SOD by non-denaturing PAGE and immunoblotting (see, e.g., Pattanaik, Materials and Methods, “Detection of SOD by non-denaturing PAGE and immunoblot analyses”, pg. 160). For claims 3-5, 9-11, and 15-17, the B481-WT F. diplosiphon and B481-SD F. diplosiphon strains are not novel strains, as evidenced by Sitther (US 2020/0131469; Date of Publication: April 30, 2020) and Sitther 2019 (US 2019/0071699; Date of Publication: March 7, 2019). Sitther teaches a wild-type F. diplosiphon B481 (see, e.g., Sitther, [0049]-[0050]), and Sitther 2019 teaches a recombinant strain of F. diplosiphon that overexpresses the sterol desaturase gene to enhance lipid production, wherein this recombinant strain is called B481-SD F. diplosiphon (see, e.g., Sitther 2019, abstract).
Therefore, the claims are interpreted under the BRI standard, wherein the claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims do not recite any additional elements. The assaying steps are considered routine and conventional, as evidenced by the art cited above. The F. diplosiphon strains are not novel, as evidenced by the art cited above.
Therefore, the claims are considered an abstract idea (i.e., mental process) which are directed to judicially recognized exceptions without amounting to significantly more than what can practically be performed in the human mind, and are not eligible under 35 U.S.C. 101.
Claim Rejections - 35 USC § 112(a), Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 4-5, 10-11, and 16-17 are rejected under 35 U.S.C. 112(a) as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The invention appears to employ novel biological materials, specifically B481-WT F. diplosiphon and B481-SD F. diplosiphon. Since the biological materials are essential to the claimed invention they must be obtainable by a repeatable method set forth in the specification or otherwise readily available to the public. If the biological materials are not so obtainable or available, the requirements of 35 U.S.C. §112 may be satisfied by a deposit of the biological materials.
If the deposit is made under the Budapest Treaty, then an affidavit or declaration by Applicant, or a statement by an attorney of record over his or her signature and registration number, stating that the specific biological materials have been deposited under the Budapest Treaty and that the biological materials will be irrevocably and without restriction or condition released to the public upon the issuance of a patent, would satisfy the deposit requirement made herein. If the deposit has not been made under the Budapest Treaty, then in order to certify that the deposit meets the criteria set forth in 37 C.F.R. § 1.801-1.809, Applicant may provide assurance of compliance by an affidavit or declaration, or by a statement by an attorney of record over his or her signature and registration number, showing that:
during the pendency of this application, access to the invention will be afforded to the Commissioner upon request;
all restrictions upon availability to the public will be irrevocably removed upon granting of the patent;
the deposit will be maintained in a public depository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer;
a test of the viability of the biological material at the time of deposit will be made (see 37 C.F.R. § 1.807); and
the deposit will be replaced if it should ever become inviable.
Applicant's attention is directed to M.P.E.P. § 2400 in general, and specifically to MPEP § 2411.05, as well as to 37 C.F.R. § 1.809(d), wherein it is set forth that "the specification shall contain the accession number for the deposit, the date of the deposit, the name and address of the depository, and a description of the deposited material sufficient to specifically identify it and to permit examination.” The specification should be amended to include this information; however, Applicant is cautioned to avoid the entry of new matter into the specification by adding any other information.
Claim Rejections - 35 USC § 103, Obviousness
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-4 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Sitther (US 2020/0131469; Date of Publication: April 30, 2020) in view of Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells; 2009) and Ayala (Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal; 2014).
Sitther’s general disclosure relates to zero-valent iron nanoparticles (nZVIs) in solution with photosynthetic bioproduct producing microorganisms, wherein the nZVIs cause increased growth, lipid production, and fatty acid production (see, e.g., Sitther, abstract). Moreover, Sitther discloses Fremyella diplosiphon can be used as a model organism for these experiments because this microorganism can absorb light and grow in a range of environmental conditions through a process called complementary chromatic adaptation (see, e.g., Sitther, [0006]). Furthermore, Sitther discloses that nZVIs can be used to “increase microorganism/cyanobacterial growth, total lipid content, as well as oleic and linoleic acids, which are primary fatty acid components in biofuel production. Enhancing F. diplosiphon lipid productivity will enhance harvest efficiency of the resultant biofuel. In addition, enhancement of unsaturated FA profile benefits F. diplosiphon-derived biodiesel by reducing the cloud and pour points, thus increasing the percentage that could be used in commercial blends” (see, e.g., Sitther, [0060]).
Regarding claims 1 and 3-4 growth and detection of lipid peroxidation in cyanobacterium, Sitther teaches growing F. diplosiphon B481 in “BG11/HEPES medium with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mgL-1 Nanofer 25s and untreated control over a period 15 days” (see, e.g., Sitther, [0036], [0046], and Figures 6-7). Moreover, Sitther teaches methods of assaying cultures of F. diplosiphon B481 that are treated with and without nZVIs (see, e.g., Sitther, [0049]-[0050]). Furthermore, Sitther teaches “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0057]).
However, Sitther does not teach: assaying said first culture and control culture for lipid peroxidation (claim 1c); or comparing a level of lipid peroxidation in said first culture and a level of lipid peroxidation in said control culture (claim 1d); or making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of lipid peroxidation in said first culture of said cyanobacterium is statistically higher than said level of lipid peroxidation in said control culture of said cyanobacterium (claim 1e).
Keenan’s general disclosure relates to exposing human bronchial epithelial cells to nZVI to determine if nZVI damages cells (see, e.g., Keenan, abstract). Moreover, Keenan discloses that exposure of human bronchial epithelial cells to nZVI leads to the same level of reactive oxygen species production in the cells compared to when cells are exposed of the same concentration of dissolved Fe(II) (see, e.g., Keenan, abstract). Furthermore, Keenan discloses that “Iron is believed to cause oxidative stress via redox cycling and ROS generation, resulting in lipid peroxidation and DNA damage” (see, e.g., Keenan, Introduction, pg. 4555).
Regarding claim 1 pertaining to detection of lipid peroxidation in the cultures, Keenan teaches that “Iron is believed to cause oxidative stress via redox cycling and ROS generation, resulting in lipid peroxidation and DNA damage” (see, e.g., Keenan, Introduction, pg. 4555).
Ayala’s general disclosure relates to “biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE)” (see, e.g., Ayala, abstract). Moreover, Ayala discloses “MDA appears to be the most mutagenic product of lipid peroxidation, whereas 4-HNE is the most toxic” (see, e.g., Ayala, Section 2.2, pg. 3). Furthermore, Ayala teaches “The hydroperoxyl radical (HO∙2) plays an important role in the chemistry of lipid peroxidation. This protonated form of superoxide yields H2O2 which can react with redox active metals including iron or copper to further generate
HO∙ through Fenton or Haber-Weiss reactions. The HO∙2 is a much stronger oxidant than superoxide anion-radical and could initiate the chain oxidation of polyunsaturated phospholipids, thus leading to impairment of membrane function [28–30] (see, e.g., Ayala, Section 2, pg. 2).
Regarding claims 1 and 6 pertaining to detecting lipid peroxidation in cultures, Ayala teaches that malondialdehyde, which is a product of lipid peroxidation, can be quantified by gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and several derivatization-based strategies because “MDA is one of the most popular and reliable markers that determine oxidative stress in clinical situations” (see, e.g., Ayala, section 2.2, pg. 3). Ayala teaches that iron is a redox active metal that can interact with hydrogen peroxide to further generate superoxide radicals, wherein these radicals can cause lipid peroxidation (see, e.g., Ayala, section 2, pg. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to grow Sitther’s F. diplosiphon cultures in the presence and absence of nZVIs, followed by assaying the cultures for lipid peroxidation, as taught by Keenan and Ayala. One would have been motivated to do so because Keenan teaches that “Iron is believed to cause oxidative stress via redox cycling and ROS generation, resulting in lipid peroxidation and DNA damage” (see, e.g., Keenan, Introduction, pg. 4555). Furthermore, Ayala teaches that “MDA is one of the most popular and reliable markers that determine oxidative stress in clinical situations” (see, e.g., Ayala, section 2.2, pg. 3). Moreover, Sitther teaches that cultures of F. diplosiphon B481 can be assayed for lipid content, wherein the cultures are treated with and without nZVIs (see, e.g., Sitther, [0049]-[0050] & Figure 8). Therefore, based on the teachings of Sitther, Keenan, and Ayala, it would have been obvious to assay F. diplosiphon for lipid peroxidation, wherein the cultures are treated with and without nZVI, because this would allow for one to determine if nZVIs cause increased lipid peroxidation. One would have expected success because Sitther, Keenan, and Ayala all teach oxidative stress that can be caused by iron or iron nanoparticles.
Regarding claim 6 pertaining to carrying out steps a and c-e with a second culture, the Examiner has interpreted this to merely be repetition of the same method steps recited in independent claim 1. The simple repetition of a known step to achieve and art-recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc. v. InfoUSA, Inc. 587 F.3d 1324 (Fed. Cir. Dec. 2, 2009)). In the instant case, an artisan would understand that repeating steps a and c-e with a second concentration of zero-valent iron nanoparticles would predictably lead to verification of increased or decreased oxidative stress depending on the concentration of the zero-valent iron nanoparticles, and therefore, one of ordinary skill in the art would readily appreciate that such steps could be repeated logically to achieve a desired outcome.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Sitther, Ayala, and Keenan as applied to claims 1-4 and 6 above, and further in view of Sitther (US 2019/0071699; Date of Publication: March 7, 2019 – herein referred to as “Sitther 2019”).
The prior art of Sitther, Ayala, and Keenan, herein referred to as modified-Sitther-Ayala-Keenan, is discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, modified-Sitther-Ayala-Keenan does not teach: wherein said cyanobacterium is B481-SD F. diplosiphon (claim 5).
Sitther 2019’s general disclosure relates to “A recombinant strain of F. diplosiphon was made by transforming wild type F. diplosiphon with a pGEM-7Zf (+) plasmid containing sterol desaturase gene (SD) via electroporation. The recombinant strain was designated B481-SD and overexpressed the sterol desaturase gene to result in enhanced lipid production” (see, e.g., Sitther 2019, abstract). Moreover, Sitther 2019 discloses that “Gas chromatography-mass spectrometry(GC-MS) revealed a 23% increase in desirable unsaturated fatty acid methyl esters (FAMEs) in B481-SD transesterified lipids, with methyl octadecenoate (C18:1) and methyl octadecadienoate (C18:2) as the most abundant desaturated components. Two-dimensional gas chromatography with time-of-flight mass spectrometry of transesterified lipids identified C12:0, C15:0, C18:3, and C18:4 components which were not previously detected in 1D GC-MS. Results of the study indicated that overexpression of the SD gene increased total lipid content as well as essential unsaturated fatty acids that maximize the potential of F. diplosiphon as a large-scale biofuel agent” (see, e.g., Sitther 2019, [0015]).
Regarding claim 5 pertaining to B481-SD F. diplosiphon, Sitther 2019 teaches a recombinant strain of F. diplosiphon that overexpresses the sterol desaturase gene to enhance lipid production, wherein this recombinant strain is called B481-SD F. diplosiphon (see, e.g., Sitther 2019, abstract). Sitther 2019 teaches that the B481-SD F. diplosiphon strain has increased total lipid content compared to the B481-WT F. diplosiphon strain (see, e.g., Sitther 2019, Figure 8).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to assay lipid peroxidation in F. diplosiphon cultures treated with and without nZVIs, as taught by modified-Sitther-Ayala-Keenan, wherein the F. diplosiphon is B481-SD F. diplosiphon, as taught by Sitther 2019. One would have been motivated to do so because Sitther 2019 teaches that the B481-SD F. diplosiphon strain is a recombinant strain that overexpresses a sterol desaturase gene in order to enhance lipid production (see, e.g., Sitther 2019, abstract). Additionally, Sitther teaches that the B481-SD F. diplosiphon strain has increased total lipid content compared to the B481-WT F. diplosiphon strain (see, e.g., Sitther 2019, Figure 8). Furthermore, modified-Sitther-Ayala-Keenan teaches that treatment of F. diplosiphon cultures with nZVI causes increased lipid peroxidation (see, e.g., Keenan, Introduction, pg. 4555) (see, e.g., Sitther, [0057]) (see, e.g., Keenan, section 2, pg. 2). Therefore, based on the teachings of modified-Sitther-Ayala-Keenan and Sitther 2019, it would have been obvious to measure lipid peroxidation in the B481-SD F. diplosiphon strain because this would allow for one of ordinary skill in the art to assay and assess lipid peroxidation by nZVIs within a culture that overexpresses lipids. One would have expected success because modified-Sitther-Ayala-Keenan and Sitther 2019 both teach assaying lipid levels in F. diplosiphon strains.
Claims 7-10 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Sitther (US 2020/0131469; Date of Publication: April 30, 2020) in view of Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells; 2009).
Sitther’s general disclosure is discussed above.
Regarding claims 7 and 9-10 pertaining to growth of a cyanobacterium, Sitter teaches growing F. diplosiphon B481 in “BG11/HEPES medium with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mgL-1 Nanofer 25s and untreated control over a period 15 days” (see, e.g., Sitther, [0036], [0046], and Figures 6-7). Furthermore, Sitther teaches “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0057]).
However, Sitther does not teach: adding a fluorometric probe to each of said first culture and said control culture (claim 7c); or measuring a fluorescence of said first culture and said control culture (claim 7d); or comparing a level of fluorescence of said first culture and said control culture (claim 7e); or making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of fluorescence of said first culture of said cyanobacterium is statistically higher than said level of fluorescence in said control culture of said cyanobacterium (claim 7f); or wherein said fluorometric probe is 2′,7′-dichlorodihydrofluorescein diacetate fluorometric probe (claim 8).
Keenan’s general disclosure is discussed above.
Regarding claim 7 pertaining to measuring oxidative stress by fluorescence, Keenan teaches using fluorescence to measure reactive oxygen species (ROS) within the cells when treated with nZVI and control (see, e.g., Keenan, Figure 3). Furthermore, Keenan compared ROS production between the nZVI- and control-treated cells and determined that ROS production was increased in the cells that were treated with nZVI compared to control (see, e.g., Keenan, Results, “Cytotoxicity”, and Figure 3).
Regarding claim 8 pertaining to the fluorometric probe, Keenan teaches “The 2′7′-dichlorodihyrdofluorescein diacetate (HDCF-DA) assay (27-29) was used to measure ROS production inside the lung cells.” (see, e.g., Keenan, Material and Methods, “Cytotoxicity Experiments”, pg. 4556).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to grow Sitther’s F. diphosiphon cultures in the presence and absence of nZVIs, wherein the cultures are further assayed for oxidative stress by fluorescence, as taught by Keenan. One would have been motivated to do so because Keenan teaches that ROS production between the nZVI- and control-treated cells and determined that ROS production was increased in the cells that were treated with nZVI compared to control (see, e.g., Keenan, Results, “Cytotoxicity”, and Figure 3). Furthermore, ROS production in Keenan’s method can be obtained by measuring fluorescence through the 2′7′-dichlorodihyrdofluorescein diacetate (HDCF-DA) assay because this allows for measurement of ROS within cells (see, e.g., Keenan, Material and Methods, “Cytotoxicity Experiments”, pg. 4556). Moreover, Sitther teaches “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0057]). Therefore, based on the teachings of Sitther and Keenan, it would have been obvious to measure ROS production within F. diplosiphon cultures treated with and without nZVIs because this would allow for one of ordinary skill in the art to assess if nZVI treatment alters ROS production. One would have expected success because Sitther and Keenan both teach treatment of cells with nZVI.
Regarding claim 12 pertaining to carrying out steps a and c-e with a second culture, the Examiner has interpreted this to merely be repetition of the same method steps recited in independent claim 1. The simple repetition of a known step to achieve and art-recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc. v. InfoUSA, Inc. 587 F.3d 1324 (Fed. Cir. Dec. 2, 2009)). In the instant case, an artisan would understand that repeating steps a and c-e with a second concentration of zero-valent iron nanoparticles would predictably lead to verification of increased or decreased oxidative stress depending on the concentration of the zero-valent iron nanoparticles, and therefore, one of ordinary skill in the art would readily appreciate that such steps could be repeated logically to achieve a desired outcome.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Sitther and Keenan as applied to claims 7-10 and 12 above, and further in view of Sitther (US 2019/0071699; Date of Publication: March 7, 2019 – herein referred to as “Sitther 2019”).
The prior art of Sitther and Keenan, herein referred to as modified-Sitther-Keenan, is discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, modified-Sitther-Keenan does not teach: wherein said cyanobacterium is B481-SD F. diplosiphon (claim 11).
Sitther 2019’s general disclosure is discussed above.
Regarding claim 11 pertaining to B481-SD F. diplosiphon, Sitther 2019 teaches a recombinant strain of F. diplosiphon that overexpresses the sterol desaturase gene to enhance lipid production, wherein this recombinant strain is called B481-SD F. diplosiphon (see, e.g., Sitther 2019, abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to assay lipid peroxidation in F. diplosiphon cultures treated with and without nZVIs, as taught by modified-Sitther-Keenan, wherein the F. diplosiphon is B481-SD F. diplosiphon, as taught by Sitther 2019. One would have been motivated to do so because Sitther 2019 teaches that the B481-SD F. diplosiphon strain is a recombinant strain that overexpresses a sterol desaturase gene in order to enhance lipid production (see, e.g., Sitther 2019, abstract). Additionally, Sitther 2019 teaches that the B481-SD F. diplosiphon strain has increased total lipid content compared to the B481-WT F. diplosiphon strain (see, e.g., Sitther 2019, Figure 8). Modified-Sitther-Keenan teaches “high levels of free radicals or reactive oxygen species (ROS) can inflict direct damage to lipids” and ROS can cause lipid peroxidation (see, e.g., Keenan, section 2, pg. 2). Furthermore, modified-Sitther-Keenan teaches that treatment of F. diplosiphon cultures with nZVI causes increased lipid peroxidation (see, e.g., Keenan, Introduction, pg. 4555) (see, e.g., Sitther, [0057]) (see, e.g., Keenan, section 2, pg. 2). Therefore, based on the teachings of modified-Sitther-Keenan and Sitther 2019, it would have been obvious to measure lipid peroxidation in the B481-SD F. diplosiphon strain because this would allow for one of ordinary skill in the art to assay and assess ROS production by nZVIs within a culture that overexpresses lipids, wherein lipid peroxidation is correlated to ROS production. One would have expected success because modified-Sitther-Keenan and Sitther 2019 both teach assaying lipids in F. diplosiphon strains.
Claims 13-16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Sitther (US 2020/0131469; Date of Publication: April 30, 2020) in view of Pattanaik (Responses to iron limitation are impacted by light quality and regulated by RcaE in the chromatically acclimating cyanobacterium Fremyella diplosiphon; 2014) and Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells (2009).
Sitther’s general disclosure is discussed above.
Regarding claims 13 and 15-16 pertaining to growth of a cyanobacterium, Sitter teaches growing F. diplosiphon B481 in “BG11/HEPES medium with 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mgL-1 Nanofer 25s and untreated control over a period 15 days” (see, e.g., Sitther, [0036], [0046], and Figures 6-7). Furthermore, Sitther teaches “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0057]).
However, Sitther does not teach: carrying out a densitometric analysis of superoxide dismutase (“SOD”) in said first culture and said control culture (claim 13c); or comparing a level of SOD in said first culture and said control culture (claim 13d); or making a determination that said concentration of zero-valent iron nanoparticles in said first culture of said cyanobacterium is causing oxidative stress in said first culture of said cyanobacterium when said level of SOD in said first culture of said cyanobacterium is statistically higher than said level of SOD in said control culture of said cyanobacterium (claim 13e); or wherein said SOD is Fe-SOD (claim 14).
Pattanaik’s general disclosure relates to the impact of iron limitation and light quality on Fremyella diplosiphon (see, e.g., Pattanaik, abstract). Moreover, Pattanaik discloses “light-regulated responses to iron limitation in F. diplosiphon. RL-grown cells exhibited significant reductions in growth and pigment levels, and alterations in iron-associated proteins, which impact the accumulation of reactive oxygen species under iron-limiting conditions, whereas GL-grown cells exhibited partial resistance to iron limitation” (see, e.g., Pattanaik, abstract). Furthermore, Pattanaik discloses that iron levels can affect oxidative stress responses within F. diplosiphon, which can in response lead to alterations in the levels of catalases, peroxidases, and some superoxide dismutases (see, e.g., Pattanaik, Introduction, pg. 993).
Regarding claim 13-14 pertaining to measuring SOD via densitometric analysis, Pattanaik teaches detecting Fe-SOD by non-denaturing PAGE and immunoblotting (see, e.g., Pattanaik, Materials and Methods, “Detection of SOD by non-denaturing PAGE and immunoblot analyses”, pg. 160).
Keenan’s general disclosure is discussed above.
Regarding claim 13 pertaining to comparing a first culture and control culture of cyanobacteria, Keenan teaches treating human bronchial epithelial cells with and without nZVI and measuring oxidative stress (see, e.g., Keenan, Figures 1C-D). Furthermore, Keenan teaches that “Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, Results, “Oxidant Production”, pg. 4556).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to grow Sitther’s F. diplosiphon B481 strain with and without nZVIs, wherein the F. diplosiphon strain is assess for SOD and oxidative stress levels, as taught by Pattanaik and Keenan, respectively. One would have been motivated to do so because Pattanaik teaches that iron levels can affect oxidative stress responses within F. diplosiphon, which can in response lead to alterations in the levels of catalases, peroxidases, and some superoxide dismutases (see, e.g., Pattanaik, Introduction, pg. 993). Furthermore, Keenan teaches “Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, Results, “Oxidant Production”, pg. 4556). Moreover, Sitther teaches that “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0057]). Therefore, based on the teachings of Sitther, Pattanaik, and Keenan, it would have been obvious to assess ROS production in F. diplosiphon treated with and without nZVIs in order to further assess oxidative stress. One would have expected success because Sitther, Pattanaik, and Keenan all teach measuring oxidative stress in cells treated with iron.
Regarding claim 18 pertaining to carrying out steps a and c-e with a second culture, the Examiner has interpreted this to merely be repetition of the same method steps recited in independent claim 1. The simple repetition of a known step to achieve and art-recognized outcome is “merely the logical result of common sense application to the maxim ‘try, try again’ (see, e.g., Perfect Web Technologies, Inc. v. InfoUSA, Inc. 587 F.3d 1324 (Fed. Cir. Dec. 2, 2009)). In the instant case, an artisan would understand that repeating steps a and c-e with a second concentration of zero-valent iron nanoparticles would predictably lead to verification of increased or decreased oxidative stress depending on the concentration of the zero-valent iron nanoparticles, and therefore, one of ordinary skill in the art would readily appreciate that such steps could be repeated logically to achieve a desired outcome.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Sitther, Pattanaik, and Keenan as applied to claims 13-16 and 18 above, and further in view of Sitther (US 2019/0071699; Date of Publication: March 7, 2019 – herein referred to as “Sitther 2019”).
The prior art of Sitther, Pattanaik, and Keenan, herein referred to as modified-Sitther-Pattanaik-Keenan, is discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, modified-Sitther-Pattanaik-Keenan does not teach: wherein said cyanobacterium is B481-SD F. diplosiphon (claim 17).
Sitther 2019’s general disclosure is discussed above.
Regarding claim 17 pertaining to B481-SD F. diplosiphon, Sitther 2019 teaches a recombinant strain of F. diplosiphon that overexpresses the sterol desaturase gene to enhance lipid production, wherein this recombinant strain is called B481-SD F. diplosiphon (see, e.g., Sitther 2019, abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to assay SOD levels in F. diplosiphon cultures treated with and without nZVIs, as taught by modified-Sitther-Pattanaik-Keenan, wherein the F. diplosiphon is B481-SD F. diplosiphon, as taught by Sitther 2019. One would have been motivated to do so because Sitther 2019 teaches that the B481-SD F. diplosiphon strain is a recombinant strain that overexpresses a sterol desaturase gene in order to enhance lipid production (see, e.g., Sitther 2019, abstract). Additionally, Sitther 2019 teaches that the B481-SD F. diplosiphon strain has increased total lipid content compared to the B481-WT F. diplosiphon strain (see, e.g., Sitther 2019, Figure 8). Modified-Sitther-Pattanaik-Keenan teaches “high levels of free radicals or reactive oxygen species (ROS) can inflict direct damage to lipids” and ROS can cause lipid peroxidation (see, e.g., Keenan, section 2, pg. 2). Furthermore, modified-Sitther-Pattanaik-Keenan teaches that treatment of F. diplosiphon cultures with nZVI causes increased lipid peroxidation (see, e.g., Keenan, Introduction, pg. 4555) (see, e.g., Sitther, [0057]) (see, e.g., Keenan, section 2, pg. 2). Additionally, modified-Sitther-Pattanaik-Keenan teaches iron levels can affect oxidative stress responses within F. diplosiphon, which can in response lead to alterations in the levels of catalases, peroxidases, and some superoxide dismutases (see, e.g., Pattanaik, Introduction, pg. 993). Therefore, based on the teachings of modified-Sitther-Pattanaik-Keenan and Sitther 2019, it would have been obvious to measure lipid peroxidation in the B481-SD F. diplosiphon strain because this would allow for one of ordinary skill in the art to assay and assess SOD production by nZVIs within a culture that overexpresses lipids, wherein lipid peroxidation is correlated to ROS production and wherein ROS levels are correlated to SOD levels. One would have expected success because modified-Sitther-Pattanaik-Keenan and Sitther 2019 all teach assaying lipids in F. diplosiphon strains.
Conclusion
Claims 1-18 are rejected.
No claims are allowed.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653