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 .
Withdrawal of Rejections
The response and amendments filed on 07/20/2026 are acknowledged. Any previously applied minor objections and/or minor rejections (i.e., formal matters), not explicitly restated here for brevity, have been withdrawn necessitated by Applicant’s formality correction and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/or maintaining, if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner’s Response to Arguments section.
Briefly, the previous claim rejections under 35 U.S.C. 112(a), 112(b), and 101 have been withdrawn necessitated by Applicant’s amendments.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
New Grounds of Rejection Necessitated by Amendments
Claim Rejections - 35 USC § 112(a), New Matter
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-18 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1, 7, and 13 have been amended to recite “f. adjusting at least one of said concentration of zero-valent nanoparticles or a culture condition based on said determination to modulate oxidative stress in said cyanobacterium”; however, there is no support for adjusting concentration(s) of zero-valent nanoparticles or culture conditions based on a previous determination to modulate oxidative stress in the cyanobacterium. Therefore, this is new matter and should be removed from the instant claims (see, e.g., MPEP 608.04).
Claims 2-6, 8-12, and 14-18 are included in this rejection for depending on a rejected independent claim and failing to rectify the noted deficiency.
New and/or Maintained Grounds of Rejection Necessitated by Amendments
Claim Rejections - 35 USC § 103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 – previously cited) in view of Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells; 2009 – previously cited) and Ayala (Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal; 2014 – previously cited).
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); or adjusting at least one of said concentration of zero-valent nanoparticles or a culture condition based on said determination to modulate oxidative stress in said cyanobacterium (claim 1f); or wherein said assaying of said first culture and said control culture of lipid peroxidation comprises a determination of malondialdehyde content in said first culture and said control culture (claim 2).
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 and adjusting conditions based on oxidative stress, 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). Keenan teaches that “Fe(II) released from nZVI or added to PBS was oxidized to Fe(III) within 60 min and Fe(III) slowly precipitated out of solution (Figure 1b). Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, “Oxidant Production”, pg. 4556); therefore, the nZVI cause increased oxidative stress, which would result in increased lipid peroxidation, compared to a control culture. Furthermore, Keenan teaches that “The choice of solution conditions affected oxidant production by nZVI. Experiments with PBS and Hank’s balanced salt solution (HBSS), which has been used in previous nanoparticle toxicity studies (14, 29), demonstrated that oxidant production (i.e., HCHO) was approximately three times higher in PBS than it was in HBSS (SI Figure S2). Oxidant production by nZVI in PBS at pH7 also was 2-3 times higher than it was in 1mMpiperazine-N,N′-bis(ethanesulfonic acid) (PIPES), which was used in previous studies because the buffer does not complex iron (25), or in 1 mM PIPES with 155mMNaCl. The addition of bicarbonate to PBS decreased oxidant production, with HCHO production decreasing from 11.2 to 5.6 μM with the addition of 10 mM HCO3- (SI Figure S3) (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Additionally, Keenan teaches “the presence of 155 mM chloride decreased oxidant production by 29.8±2.7% in 2mM PIPES” (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches adjustment of culture conditions based on oxidative stress produced by nZVI.
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 1-2 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 firstly 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). Keenan teaches that “Fe(II) released from nZVI or added to PBS was oxidized to Fe(III) within 60 min and Fe(III) slowly precipitated out of solution (Figure 1b). Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, “Oxidant Production”, pg. 4556); therefore, the nZVI causes increased oxidative stress, which would result in increased lipid peroxidation, compared to a control culture. 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). Therefore, based on these teachings, 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.
It would have been secondly 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 of nZVIs, wherein the culturing conditions are adjusted based on oxidative stress, as taught by Keenan. One would have been motivated to do so because Keenan teaches that different solution conditions affects oxidant production by nZVI, wherein oxidant production is approximately three times higher in PBS than in HBSS (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches that modulating culture or solution conditions will influence the amount of oxidant production. Additionally, Keenan teaches that nZVI generate reactive oxygen species (see, e.g., Keenan, Introduction, pg. 4555 & Figure 1) and 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, modulating the concentration of nZVI will modulate the amount of reactive oxygen species and oxidative stress. Therefore, based on the teachings of Sitther and Keenan, it would be obvious to modulate the concentration of nZVI or a culture condition to modulate oxidative stress since both the nZVI concentration and culture conditions influence the amount of oxidative stress. One would have expected success because both Sitther and Keenan both teach treatment of cells with nZVI.
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” – previously cited).
The references of Sitther, Ayala, and Keenan are discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, the references do 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 Sitther, Ayala, and 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). Furthermore, Sitther and Keenan teach that treatment of F. diplosiphon cultures with nZVI causes increased lipid peroxidation and oxidative stress (see, e.g., Keenan, Introduction, pg. 4555) (see, e.g., Sitther, [0057]) (see, e.g., Keenan, section 2, pg. 2). Therefore, based on these teachings, it would have been obvious to measure lipid peroxidation and oxidative stress 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 and oxidative stress by nZVIs within a cyanobacteria culture, wherein the cyanobacteria overexpresses lipids. One would have expected success because Sitther, Ayala, Keenan, and Sitther 2019 all teach lipids and/or lipid peroxidation in cell cultures. Additionally, Sitther and Sitther 2019 both teach 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 – previously cited).
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 adjusting at least one of said concentration of zero-valent nanoparticles or a culture condition based on said determination to modulate oxidative stress in said cyanobacterium (claim 7g); 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). Keenan teaches that “Fe(II) released from nZVI or added to PBS was oxidized to Fe(III) within 60 min and Fe(III) slowly precipitated out of solution (Figure 1b). Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, “Oxidant Production”, pg. 4556); therefore, the nZVI cause increased oxidative stress, which would result in increased lipid peroxidation, compared to a control culture. Furthermore, Keenan teaches that “The choice of solution conditions affected oxidant production by nZVI. Experiments with PBS and Hank’s balanced salt solution (HBSS), which has been used in previous nanoparticle toxicity studies (14, 29), demonstrated that oxidant production (i.e., HCHO) was approximately three times higher in PBS than it was in HBSS (SI Figure S2). Oxidant production by nZVI in PBS at pH7 also was 2-3 times higher than it was in 1mMpiperazine-N,N′-bis(ethanesulfonic acid) (PIPES), which was used in previous studies because the buffer does not complex iron (25), or in 1 mM PIPES with 155mMNaCl. The addition of bicarbonate to PBS decreased oxidant production, with HCHO production decreasing from 11.2 to 5.6 μM with the addition of 10 mM HCO3- (SI Figure S3) (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Additionally, Keenan teaches “the presence of 155 mM chloride decreased oxidant production by 29.8(2.7% in 2mM PIPES” (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches adjustment of culture conditions (i.e., culture media) based on oxidative stress produced by nZVI.
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 firstly 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 measurement of 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 measure if nZVI treatment alters ROS production. One would have expected success because Sitther and Keenan both teach treatment of cells with nZVI.
It would have been secondly 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 of nZVIs, wherein the culturing conditions are adjusted based on oxidative stress, as taught by Keenan. One would have been motivated to do so because Keenan teaches that different solution conditions affects oxidant production by nZVI, wherein oxidant production is approximately three times higher in PBS than in HBSS (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches that modulating culture or solution conditions will influence the amount of oxidant production. Additionally, Keenan teaches that nZVI generate reactive oxygen species (see, e.g., Keenan, Introduction, pg. 4555 & Figure 1) and 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, modulating the concentration of nZVI will modulate the amount of reactive oxygen species and oxidative stress. Therefore, based on the teachings of Sitther and Keenan, it would be obvious to modulate the concentration of nZVI or a culture condition to modulate oxidative stress since both the nZVI concentration and culture conditions influence the amount of oxidative stress. One would have expected success because both 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” – previously cited).
The references of Sitther and Keenan is discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, Sitther and Keenan do 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 Sitther and 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). Furthermore, Sitther and Keenan teach that treatment of F. diplosiphon cultures with nZVI causes increased lipid peroxidation and oxidative stress (see, e.g., Keenan, Introduction, pg. 4555) (see, e.g., Sitther, [0057]) (see, e.g., Keenan, section 2, pg. 2). Therefore, based on these teachings, it would have been obvious to measure lipid peroxidation and oxidative stress 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 and oxidative stress by nZVIs within a cyanobacteria culture, wherein the cyanobacteria overexpresses lipids. One would have expected success because Sitther, Keenan, and Sitther 2019 all teach lipids peroxidation and/or lipid peroxidation in cell cultures. Additionally, Sitther and Sitther 2019 both teach 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 – previously cited) 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 – previously cited) and Keenan (Oxidative Stress Induced by Zero-Valent Iron Nanoparticles and Fe(II) in Human Bronchial Epithelial Cells; 2009 – previously cited).
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 adjusting at least one of said concentration of zero-valent nanoparticles or a culture condition based on said determination to modulate oxidative stress in said cyanobacterium (claim 13f); 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 in protein extracts obtained from non-denaturing lysis of F. diplosiphon cells 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). Keenan teaches that “Fe(II) released from nZVI or added to PBS was oxidized to Fe(III) within 60 min and Fe(III) slowly precipitated out of solution (Figure 1b). Superoxide and hydrogen peroxide were produced as nZVI was oxidized (Figure 1c-d)” (see, e.g., Keenan, “Oxidant Production”, pg. 4556); therefore, the nZVI cause increased oxidative stress, which would result in increased lipid peroxidation, compared to a control culture. Furthermore, Keenan teaches that “The choice of solution conditions affected oxidant production by nZVI. Experiments with PBS and Hank’s balanced salt solution (HBSS), which has been used in previous nanoparticle toxicity studies (14, 29), demonstrated that oxidant production (i.e., HCHO) was approximately three
times higher in PBS than it was in HBSS (SI Figure S2). Oxidant production by nZVI in PBS at pH7 also was 2-3 times higher than it was in 1mMpiperazine-N,N′-bis(ethanesulfonic acid)
(PIPES), which was used in previous studies because the buffer does not complex iron (25), or in 1 mM PIPES with 155mMNaCl. The addition of bicarbonate to PBS decreased oxidant production, with HCHO production decreasing from 11.2 to 5.6 μM with the addition of 10 mM HCO3- (SI Figure S3) (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Additionally, Keenan teaches “the presence of 155 mM chloride decreased oxidant production by 29.8(2.7% in 2mM PIPES” (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches adjustment of culture conditions (i.e., culture media) based on oxidative stress produced by nZVI.
It would have been first 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 SOD 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.
It would have been secondly 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 of nZVIs, wherein the culturing conditions are adjusted based on oxidative stress, as taught by Keenan. One would have been motivated to do so because Keenan teaches that different solution conditions affects oxidant production by nZVI, wherein oxidant production is approximately three times higher in PBS than in HBSS (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Therefore, Keenan teaches that modulating culture or solution conditions will influence the amount of oxidant production. Additionally, Keenan teaches that nZVI generate reactive oxygen species (see, e.g., Keenan, Introduction, pg. 4555) and 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, modulating the concentration of nZVI will modulate the amount of reactive oxygen species and oxidative stress. Therefore, based on the teachings of Sitther and Keenan, it would be obvious to modulate the concentration of nZVI or a culture condition to modulate oxidative stress since both the nZVI concentration and culture conditions influence the amount of oxidative stress. One would have expected success because both Sitther and Keenan both teach treatment of cells with nZVI.
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” – previously cited).
The references of Sitther, Pattanaik, and Keenan are discussed above as it pertains to detecting oxidative stress in F. diplosiphon in the presence and absence of nZVIs.
However, the references do 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 Sitther, Pattanaik, and 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). 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, 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, 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 these teachings, it would have been obvious to measure lipid peroxidation in the B481-SD F. diplosiphon strain because this would allow for one 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 Sitther, Pattanaik, Keenan and Sitther 2019 all teach assessment of oxidative stress in cell cultures following nZVI or iron treatment.
Examiner’s Response to Arguments
Applicant's arguments filed 07/20/2026 have been fully considered but they are not persuasive.
Regarding Applicant’s arguments that the amended claims are not taught by any of the cited references (remarks, pages 12-14), this argument is not persuasive because, as discussed above, Keenan teaches the amended claim limitations. Therefore, Applicant’s arguments are moot.
Regarding Applicant’s arguments that the specification demonstrates unexpected results (remarks, page 12), this argument is not persuasive because the results are not commensurate in scope with the claimed invention. Applicant is relying upon specific concentrations of nZVIs, specific treatment duration times, specific culturing conditions for the F. diplosiphon strains, and methods of centrifuging the cells and incubation with trichloroacetic acid at specific volumes and concentrations in order to measure peroxidation of lipids (see, e.g., instant specification, [0011], [0021], [0022]). Therefore, based on these conditions, Applicant’s results are not commensurate in scope with the claimed invention.
Regarding Applicant’s arguments pertaining to impermissible hindsight reconstruction (remarks, pages 14-15), this argument is not persuasive because it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). The references used within the above presented rejection are not unrelated and pertain to the same field(s) of endeavor. Sitther’s general disclosure pertains to F. diplosiphon treatment with nZVIs, wherein “nZVIs in green technologies can be toxic to microorganisms due to damage caused by ROS activity (Sevcu et al. 2011)” (see, e.g., Sitther, [0050]-[0051], [0057]). Keenan’s general disclosure pertains to ROS production and lipid peroxidation following treatment with nZVIs (see, e.g., Keenan, “Oxidant Production”, pg. 4556). Ayala’s general disclosure pertains to ROS and lipid peroxidation following iron treatment in cell cultures (see, e.g., Ayala, section 2, pgs. 2-3). Sitther 2019’s general disclosure pertains to a mutant F. diplosiphon called B481-SD F. diplosiphon, wherein this strain is a recombinant strain that overexpresses a sterol desaturase gene in order to enhance lipid production (see, e.g., Sitther 2019, abstract). Pattanaik’s general disclosure pertains to the influence of iron levels on ROS in F. diplosiphon (see, e.g., Pattanaik, abstract). Therefore, these references all pertain to F. diplosiphon strains treated with nZVI, nZVI treatment in cell culture causing oxidative stress, and/or ROS and lipid peroxidation following iron treatment in cell culture. All of these references are analogous because they pertain to the same general concept pertaining to iron or nZVI causing oxidative stress in cell culture; therefore, these references were combined based on knowledge in the art at the time the claimed invention was made and not based on knowledge from Applicant’s disclosure.
Conclusion
Claims 1-18 are rejected.
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
