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Global E-Services Bi-Weekly newsletter

May 30, 2021 | Issue 21

Coming feature stories:ont

In this discussion we include anti-pollination mechanisms with PNA-conjugated or carriered (viz. with auxins) agents that can disrupt the pollen mother sac's pollinating activity requiring genetic engineering and as genetically modified organisms (GMO) such as with the first technological developments with Seagrass Tech, originally a marine, hydroponics-based technology of growing marine grasses used for pharma purposes to be later 'downstream" processed, for e. g. with: enzymes, hormones, monoclonals, vaccines, orthopharma (steroidals), plant bioactives including novel nutritional food bioactives (NFB) that we are proposing at 'SkyeBlue'. We believe this technology will precede 'farming' for biomass as feedstock, e. g. for grass-fed dairy cows and beef, including conjugated PNA-auxin driven non-GMO XL-growth varieties for improved economies of scale in a race for time to scale-up the industrial process. This will also be congruent (from both pharma and feedstock for dairy and beef) with the use for renewables for bioenergy like ethanol (etOH) and isobutanol (isoButOH).

The feature story deals with an 'old' problem the author has dealt with in the past involving his introduction into applications of rec-DNA techniques to lignocellulose degradation in the rumen stomachs with bacteria (yeast and fungi are also considered viable options at this time) such as Fibrobacter succinogenes and Ruminococcus albus and flavefaciens using low-quality feeds resources for livestock during his Masteral program where he introduced a tri-innoculant approach using both fibrolytic spp. including ligninolytics and a 'piggy-backed' approach with surrogate proteins that are recombinant in nature that are relatively impervious to proteolytic attack within the rumen. There will be a need to continue work identifying gene 'targets' and characertizing operon sequencing and structure between regulatory operator/promoter (O/P) regions and structural genes and, if possible, TFs and their promoter binding sites. Also reported is any non-GMO protoplast fusion studies as an alternative approach to the above for new phenotypes. PNA-B12, a popularized approach at 'SkyeBlue' will also be touched upon as a non-GMO option for boosting fribrolytics, ligninolytics (e. g. with ferulic acid esterase activity, etherase activity) and native protein synthesis analogous to boosting surrogates introduced into hosts in rumen microbes. Any queries for proposed Ph.D. studies are welcomed by the author regards this featured story.

In this story functional feed components in animal feeding for health or well-being with new biopharma discovery will be discussed. With respect to health, immuno responsiveness is of interest covering two areas: (1) immuno suppression regarding energy status and metabolites and the phenomenon of immunosuppression vs. immuno responsiveness in periparturient cows, and 2) the chronic inflammatory response, e. g. s. of which are: a) omega-3 fatty acids deactivating NF-kappaB which down regulates pro-inflammatory cytokines, b) lactoferrin (LF) from dairy milk also deactivating NF-kappaB from binding DNA at its promoter (P) region and then down-regulating pro-inflammatory cytokines, and c) fructans interacting with the immune system and its role with chronic inflammation. Functional nutritional components for feeding livestock includes: 1) oilseeds (e. g. canola, soybean) and their omega-3 fatty acid content in DHA and EPA and other free fatty acids, 2) grains and forages (grasses and legumes) for their complex carbohydrates and sugars, e. g. s. fructans and glucose, 3) vitamin and mineral supplements, e. g. VitD, and 4) protein supplements, e. g from oilseed meals and soybean meals and protected amino acids (also known as functional amino acids or FAA) such as arginine (ARG), methionine (MET), phenylalanine (PHE), lysine (LYS) and histidine (HIS). Transcription factor engineering or TFE is our version of using peptide nucleic acid (PNA) carriered (e. g. conjugated with Vit B12) technology carrying gene silencing nucleic acid sequences to mammalian target cells, in vivo, through their cell membrane or barrier involving transcription factors (TF) invovled with metabolism for such functional nutritional components in animal feeding. Still more advanced approaches are using protein structural-functional design aided with X-ray crystallographic structure proofing to supposedly manipulate by enhancing binding of TF in their promoter region on the operon involved with metabolism. These are just being speculated at this time at 'SkyeBlue'. Components of the response system such as those participating in the cascade phenomenon can be engineered to boost or decrease their metabolic effect with activity.

Protocol Sequence Derivation of VitaminD-Agonist (VitD-A) from Lanosteral via the 'Flores Synthesis'.

D. A. Flores (1980) earlier arrived at the synthesis to derivatize lanosterol, the beginning molecule that eventually is converted by animals and fungi to cholesterol and then to VitD's two active forms (1, 25 dihydroxy-VitD and 25-hydroxy-VitD), to lanosteral or its end-of-chain aldehydic derivative, to converted sequentially in the yeast model system using its ~30 or so enzymic steps to the agonist forms of VitD active forms acting to compete with FBI and sustain VitD's activation at this site supporting better titres for the vitamin in support of managing the chronic inflammatory condition and its aetiologies that are beginning to be uncovered for several conditions including that for certain Cancers and with the epidemiological studies describing a relationship bet-ween latitude, exposure to activating UV-B radiation from the sun, skin pigmentation or tone, intake of enriched bread and milk as a source of VitD, and incidence of such Cancers.

In essence the 7-step chemical 'priming' 'Flores Sythesis' process involves severing the side-chain in the seco-steroidal molecule of lanosterol at its double bond, and reattachment with the analogue moiety containing a carboxyllic acid derivative end-group and then recovering the trans-configurated double bonded of the seco-steroidal derivative molecule, followed by successive reduction of the carboxyllic acid end group to its endproduct of the aldehydic derivative of Lanosteral. The endproduct represents a drug analogue where one of the methyl side group at the side-chain end has been substituted for an aldehydic group analogous to the methyl form that competes with the conventional form with FBI during curtailment once activation has been initiated to the activated form of VitD, 2 for either the liver and kidney, in the process, increasing physiological titre compared to without it.

The process of deriving enzymes that have been 'point-mutated' that can do the same reaction but with analogue substrates down the ~30 or so sequential steps would involve, as stated, lanosteral as starting material, taking yeast culture that is plated out, then replica plating onto a lawn of lanosteral to thousands of colonies as first precursor and then using low-level X-ray mutagenesis to the hundreds of plates, examination of individual colonies for clearing of the lawns for positive colonies with the appropriate 'adapted' enzyme(s), with the positive colonies then collected, grown again to bulk in a batch fermentor or reactor and the enzyme, using the original gene 'library' of the enzyme to compared sequencing of the amino acids residues and that which was substituted by mutation and further X-ray crystallographic analysis to elucidate the candidate enzyme's 2-D and 3-D structural analysis and function at the active site. The last chosen host source colony from the sequence is then replica replated on lawns of substrate from the previous bulk fermentation of lanosteral in the presence of the first 'adapted' yeast culture, and then again exposed to low-levels of X-ray to undergo mutagenesis and then examined for clearing colonies on plates that will again determine the cloned colonies to be studied as previously determined for successful point mutations followed by cloning the second 'adapted' enzyme in our growing one-enzyme libraries in sequence.

Management of libraries will be in E. coli and their step-wise obtainment in order to generate more intermediates in bulk along the ~30 or so individual steps required for substrate maintenance in kilogram (kg) amounts.

After mutating microbial cultures for their "adapted" enzymes they will be eventually introduced to plant hosts for "plant pharming" using hydroponics culture to process lanosteral for transformation along the ~30 or so steps to the desired drug agent or derivative of VitD, recovered "downstream" later for purification.

It has been mentioned that this type of research is ideal for medical research scholars who desire to achieve innovation with this significant breakthrough for the biotech or pharma industry.

The project has already been presented for consideration at the author's alma mater located in the United States, at Kalamazoo College, an authors copy made for reference that is now available at their Upjohn Library commons for possible consideration at the Community Global Health (CGHL) Now program in conjunction with their Health Sciences Division.

No designation has been made as to a candidate site or location for carrying out the preliminary microbial mutational experiments with organic synthesis preliminaries using the author's 'Flores Synthesis' or proprietary synthetic methodology as has been documented earlier in his senior individualized project (SIP) project (1980) performed at Simon Fraser University, Burnaby campus, British Columbia Canada at their former laboratory of A. C. Oehlschlager in Organic Chemistry where he received NSERC funding as an undegraduate student at the time and achieved Honours standing for it.

Candidates expressing an interest in this type of research should refer to the said references and also conduct a surveyed search for centres of excellence (COE) in seco-steroidal organic chemistry research for drug discovery around the country of the United States of America.

How Vit D-Agonists (also cf. to as antagonists at their site of action) support health or even ameliorate disease involving the family of chronic inflammatory-related aetiologies beginning to be implicated with this condition.

Thus far, chronic inflammation has been implicated with asthma, cancers, diabetes, cardiovascular disease (CVD) and arthritis. And recently with mutliple sclerosis - and even Alzheimers and Parkinsons. We will only touch on a few of these for our story here, including vascular disease or atherosclerosis and Cancers.

Research by Elena Goleva and colleagues of National Jewish Health (NJH) have discovered molecular and signaling events by which inflammation is inhibited by VitD. Low levels of Vit D found in millions of people failed to inhibit the inflammatory cascade while adequate levels did. The study outlines a clear chain of cellular events, from the binding of DNA, through specific signaling, to a reduction of proteins known to trigger inflammation.

Incubated white blood cells (WBC) with varying levels of VitD, then exposed to lipopolysaccharide (LPS), a molecule associated with bacterial cell walls and are known to promote a strong inflamma- tory response, demonstrated that cells incubated with no VitD in solution containing 15 ng/ml produced high levels of cytokines IL-6 and TNF-alpha, major actors in the inflammatory response. Cells incubated at 30 ng/ml VitD and above showed significantly reduced response to LPS, the highest levels of inhibition occurring at 50 ng/ml (Cf. Y. Zhang et al., 2012, J. Immunol. 188(5): 2127-2135).

VitD protects against vascular disease via several mechanisms, including reducing the chronic inflammatory response that contributes to the pathology of the disease and it has been shown that low VitD levels are associated with increased cardiovascular mortality with recommended levels at 50-80 ng/ml. In the same study low serum levels of 25-hydroxy-Vit D are associated with high prevalence of cardiovascular risk factors and disease (Cf. H. Dobnig et al., 2008, Archives of Internal Medicine 168(12): 1340-1349).

With Cancers, VitD response elements in hundreds of genes serve to input cell processes of proliferation, differentiation and apoptosis. It has been suggested that the latitudinal exposure of UV-B radiation and VitD activation underneath the skin may account for the geographical pattern of cancer mortality. An increase of 25-hydroxy VitD in ng/ml was associated a risk reduction of 59% for rectal cancer and 22% for colon cancer. In the study stratification by anatomical site did not reach statistical significance. In a larger pooled study, higher 25-hydroxy-VitD were statistically significantly associated with decreased risks of colorectal cancer (CRC) without any predilection for proximal or distal location of tumour (Cf. M. Raman et al., 2011, Therapeutic Adv. in Gastroenterology 4(1): 49-62).

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