Graphical Abstract Part A: Structure of a biological cell showing the location and mechanism
of action of chimerenomic (NTinti) RNA enzyme.

Legend to Graphical Abstract Part A: Structure of a biological cell showing the location and mechanism
of action of chimerenomic (NTinti) RNA enzyme.

It shows the cell nucleus, where DNA is transcribed into genetic code-based RNAs of all kinds. Some mRNAs (depicted in black) come out normal, while some mRNAs (depicted in blue) are abnormal (superfluous). These RNAs diffuse into the cytoplasm for protein synthesis in the endoplasmic reticulum, but they will have to pass through the macromolecular nongenetic code-based NTinti RNA (depicted in red) sitting on the endoplasmic reticulum. Normal mRNAs will pass through the large NTinti RNA without electromagnetic hindrance while superfluous mRNAs will encounter electromagnetic and stearic hindrance (see figure 5 for example of magnified electromagnetic hindrance interactions of mRNA and NTinti RNA), predictable by their sequence homology with NTinti RNA segments, causing their degradation, preventing them from entering the endoplasmic reticulum for translation into abnormal proteins. Consequently, chimerenomic NTinti RNA enzyme serves as a final pathway for DNA gene regulation. Should superfluous mRNAs evade this regulatory step, they may be translated into abnormal proteins associated with numerous human diseases.

Graphical Abstract Part B: Structure of chimere from NTinti RNA showing example of its
homologous alignments with mRNAs.

Legend to Graphical Abstract Part B: Structure of chimere from NTinti RNA showing example of its homologous alignments with mRNAs.

1. Schematic illustration of the alignment of cytochrome P450 mRNA with its homologous chimerenomic (GDH-synthesized) RNA, highlighting their differences in primary structure between the homologous segment, the left flank, and the right flank segments of the 200 nucleotide-long chimere.
2. Schematic illustration of the alignment of starch synthase mRNA with its homologous chimerenomic (GDH-synthesized) RNA highlighting the numerous sequence repeats of the chimere within the segment of the mRNA encoding the starch synthase. Each sequence repeat in the chimere is preceded and terminated by the tetranucleotide sequence 'tcct' spacer.

Introduction

1.0 Molecular Chemistry: Chronic pain is the reason many patients resort to the

administration of opioid analgesics, which have caused opioid addiction affecting about 20% of adults worldwide or 1.5 billion people [1]. New understanding of the molecular chemistry of pain in terms of the responses of the metabolic pathways to the addictive drug molecules at the mRNA level must guide the design of the biological macromolecules that would prevent drug addiction.

Drug addiction embodies inexplicable phenomena in molecular and cell biology. Morphine biochemical pathway is about the same enzyme-catalyzed reaction steps in human cells and tissues as in the opium poppy plant cells and tissues[2; 3; 4], thus making it possible to apply the innovative macromolecular chimerenomic (nongenetic code-based) RNA enzymes [2; 5; 6; 7] to selectively degrade the superfluous mRNAs encoding the enzymes that dysregulate morphine neurotransmitter functions, and consequently to cure opioid addiction. Please refer to Appendix 1 for the terminologies. The plan was to deploy wide ranges of molar concentrations of morphine in the rigorous chimerenomic parallelogram of controls to recreate NADH-glutamate dehydrogenase isoenzymes (GDH) so that they can catalyze the production of macromolecular chimerenomic RNA enzymes for the therapy of drug addiction.  

Many human health disorders are caused by alternatively spliced mRNA, long non-coding RNAs, rRNAs, tRNAs that should have been prevented from entering protein translational processes [2; 8; 9; 10; 12; 13].  We have applied chimerenomic research protocols to degrade superfluous homologous mRNAs of opioid and related receptor proteins thereby offering a novel medical approach for treating opioid addiction [14; 15].

Drug addiction is an irresistible feeling that one must use a drug [16]. But there are no descriptions in molecular language for euphoria, intoxication, and full-blown addiction that support physicians in the diagnosis of drug addiction progression. Opium chemicals (morphine, heroin, codeine, fentanyl etc) being strong nucleophilic alkaloids react with both the neuroreceptor proteins and the hexameric isoenzymes of GDH [5; 7; 16; 17; 18; 19; 20]. The visual imagery of the reaction between morphine and GDH, the molecular language of the progression of drug addiction, will be explained in this review.

1.2. Functions of Human Astrocyte Cells: Exploration of Human Astrocyte Cells (HACs) is crucial for advancing our understanding of the central nervous system (CNS) and its intricate functions. Astrocytes are the predominant cell type in the CNS, constituting a significant portion of the cellular volume—ranging from 25% to 50% in certain brain regions [5]. Remarkably, they outnumber neurons by more than fivefold, underscoring their importance in maintaining CNS homeostasis [8]. Astrocytes express receptors for a variety of neurotransmitters and have the capacity to release numerous neuroactive and trophic factors. The functional significance of this signaling is still shrouded in mystery, but it is likely to be pivotal in controlling synaptic development, blood vessel dynamics, and neuronal survival.

Thus, the study of opioid addiction using HACs the experimental organism is not only essential and rigorous for understanding the effects of morphine but also for unraveling the complex functions these cells play in the CNS. The strategy and approach of this study are novel because they represent a considerable shift in the trajectory of research within neurology and neuroscience. By moving away from broad, aimless investigations, we are now homing-in on direct interventions at the chemical, molecular, cellular, tissue and whole organism levels. This innovative chemical knowledge is crucial for developing targeted therapies that maximize therapeutic benefits on patients while minimizing the risks of adverse side effects. Therefore, this research marks a transformative moment in our understanding of morphine addiction.

2.0. Methods and Materials

2.1 American Academy of Primary Care Research (AAPCR): Astrocyte Cell Culture Project: A Groundbreaking Study on Opioid Addiction, Prevention and Treatment Using HACs as Experimental Organism.

2.2. Experimental Design and Rigor: The techniques utilized throughout the astrocyte cell culture and harvesting process align with the established standards [21; 22; 23] set by the American Type Culture Collection. This meticulous protocol [14] not only guarantees reproducibility but also bolsters reliability and credibility of the research findings in the field of cellular neuroscience [8].

 Human astrocyte progenitor lines were sourced from iXcell Biotech, with protocols applied by the Southwest Research Institute (SwRI) to cultivate the cultures effectively [8]. The experimental phase spanned 11 days. Cultures were organized into duplicates, resulting in two control groups viz:

Control Group A and three morphine treatments:

0.137 mM morphine Group A,  

0.27 mM morphine Group A,

1.1 mM morphine Group A;

and Control Group B and three morphine treatments:

0.137 mM morphine Group B, 

0.27 mM morphine Group B,

1.1 mM morphine Group B.

All the eight flasks were uniformly seeded with 5,000 cells/ml, totaling 375,000 cells per T-75 flask, and they had cell viability rate of 98.3% at the outset. Throughout the study, each flask was maintained at a constant volume of 10 mL of cell culture medium. Other conditions remained constant [14]. Through this innovative experimental design, we aimed to elucidate the impact of morphine concentrations on the addiction processes

2.3. Preparation of astrocyte medium and morphine concentrations

The preparation of Astrocyte Medium was conducted at Southwest Research Institute, San Antonio applying the protocol provided by iXcell. Specifically, 50 mL of Fetal Bovine Serum (FBS), 1 mL of Growth Supplement, and 5 mL of Antibiotic-Antimycotic were added to 500 mL of iXCell HA medium. The final concentrations were: 10% FBS, 0.2% Astrocyte Supplement, and 1% Anti-Anti. Following the preparation of the Medium, drug-infused media formulations were added.

Initially, key components including FBS, Growth Supplement, and Antibiotic-Antimycotic (Anti-Anti) were thawed in a 37°C water bath to ensure optimal conditions for mixing [8; 14].

The morphine Media Solution was prepared using medical-grade morphine base monohydrate. A stock solution of 110 mM morphine was prepared by dissolving 310 mg of the morphine to make 10 mL with the complete astrocyte nutrient medium. Dilutions of the stock solution with astrocyte medium considering the volume of the seeding astrocyte cells, to a maximum of 10 ml of

---1.1 mM morphine solution,

---0.27 mM morphine solution

---0.137 mM morphine solution.

Each solution was then filtered through a 0.22 μm sterile filter [5]. A total of 375,000 cells were seeded per T-75 flask to initiate the culture process.

2.4. Daily Media Changes: Media were warmed at 37°C for 20 minutes and placed in the biological safety cabinet (BSC); and using a serological pipette, the old media was carefully removed from the flask and discarded. At this point, 10 ml of fresh media were pipetted into the flasks, each time, using a new pipet. This process was repeated until all the media was changed in all the flasks. Astrocyte culture media were changed this way daily.

2.5. Astrocytes Harvesting Protocol: Control A and group A experiments were harvested on day 9, at the confluency point. In contrast, Control B and group B experiments were harvested on day 11, two days post-confluency. The astrocyte cells Harvesting Process was as follows:

i). The cell culture medium was decanted and discarded.

ii). A quick wash using 5 mL of sterile phosphate-buffered saline to eliminate any remaining growth medium.

iii). Then, 4 mL of Trypsin/EDTA was added to the cells and incubated at 37 °C for 2 minutes to promote detachment.

iv). Trypsin activity was halted by 10 mL of complete culture medium.

v). The cells were centrifuged at 220 X g for 5 minutes to pellet the cells.

vi). Pellet was re-suspended in 1 mL of FBS for subsequent analysis.

Cell count was conducted to evaluate passage number, total cell number, percentage viability, and cell diameter. For long-term preservation, the cells were cryopreserved in a solution containing 10% dimethyl sulfoxide with culture medium [8].

The multiple layers of controls and pair-wise comparison in this experimental design is to enhance reproducibility, reduce inter-observer variability, and produce valid results based on systematic application of intervention while keeping other variables constant.

Experiments should not be designed merely to satisfy statistical exercise by creating triplicates to demonstrate statistical measures of centrality (means, standard deviations etc) for a group of data and then fail to answer any scientific question for which it was designed to do. This raises questions on the use of triplicates in cell culture protocol.  When the intraclass correlation coefficient is greater than 50% (0.5) there is not a lot of gain to make triplicate observations. With a correlation coefficient equal to and greater than 0.75, triplicate design can only reduce the confidence interval for a single intraclass or interclass observation by only about 8% making it not worthwhile to make a triplicate design. The correlation coefficient in our study is greater than 0.95 discouraging the use of triplicate. A pre-experiment study was used to determine the appropriateness to opt out of triplicate design when a large correlation is demonstrated as we did in this study.

As the debate on how best to improve the reproducibility of in vitro studies in the life sciences continues, we took a diligent approach starting from experimental design, use of internal controls, use of comparable external control, performed direct visual observation with microscopy and daily imaging, and followed the trends and data correlation factors [5]. When all these are put together, confidence in the reproducibility of the study becomes obvious. Our approach does not discount the good recommendations put forward by [24; 25] that cell culture scientists should follow to minimize internal and external errors to improve reproducibility of experiments, but rather, our method adds to those recommendations. We demonstrated in this study that our design simplifies experimentation design and cuts down costs by limiting the number of technical and biological replicates needed to show reproducibility by building internal and external controls and by incorporating an escalating dose response model as a testament to internal validity and reproducibility [16].

2.6. Extraction of the NADH-Glutamate dehydrogenase isoenzymes from HACs and chemical conversion to redox cycle hexameric GDH.

2.6.1 Free Solution Isoelectric Focusing: Each of the experimental Group A and Group B HAC cultures harvested on day 9 at cell confluency (120,000 cells), and on day 11 at post confluency (120,000 cells) were applied for the extraction according to the methods of [7; 14] without any modification.

Essentially the pellet of the 120,000 cells was homogenized in 80 mL of 15 mM Tris-HCl buffer solution, pH 7.5, containing 20 µL β-mercaptoethanol, 2 Units of DNase 1, and 1 Unit of RNase A for 1 min at maximum speed. Protein precipitated by solid (NH₄)₂SO₄ between 20% and 55% saturation of the homogenate was collected by centrifugation (10,000 x g, 30 min, 5˚C). Protein pellet was resuspended in minimum volume of 15 mM Tris-HCl pH 7.5 buffer solution, and dialyzed. The dialyzed extract was made up to 50 mL with 10 mM Tris-HCl buffer pH 7.5, and subjected to Rotofor (Bio-Rad, Hercules, CA) isoelectric focusing fractionation [14].

2.6.2. Polyacrylamide Gel Electrophoresis: Aliquots (250 µL) of the dialyzed Rotofor fractions were subjected to Laemmli SDS 12% polyacrylamide gel electrophoresis (PAGE) (100 V, 14 h, 4˚C) to remove other proteins and nucleic acids as before [14]. Electrophoresed gels were washed with 0.15 mM Tris base at 5˚C to remove the SDS. One gel was stained with L-glutamate-NAD⁺-phenazine methosulfate-tetrazolium blue reagent at room temperature. The GDH hexameric redox cycle isoenzyme distribution pattern was photo documented. Rotofor purification was repeated to assure reproducibility of the GDH isoenzyme pattern per experimental astrocyte cultured cells. The second electrophoresed gel was applied for the elution of the redox cycle GDH hexameric isoenzyme complexes contained therein.

2.6.3. Whole Gel Elution of the Hexameric Isoenzymes: The procedure was already described [7; 14]. The GDH isoenzymes were electro-eluted from the excised piece of PAG using Bio-Rad mini-whole gel eluter at subzero temperature. The eluted GDH hexameric isoenzymes (about 6 mL total volume) were not stored but applied immediately for chimerenomic RNA enzyme production.

2.6.4. Chimerenomic RNA Enzyme Production: The substrate cocktail and procedures have already been described [2]; and was prepared by adding the four ribo-NTPs (0.6 mmol each), NH₄Cl (100 µmol), α-KG (50 µmol), NADH (0.2 µmol), DNase 1 (1 U), actinomycin D (2 µg), and RNase inhibitor (10 U); the final volume was made up to 1 mL with 0.1 M Tris-HCl buffer solution pH 8.0. The reaction was started by adding 1.0 mL of redox cycle GDH hexameric isoenzyme solution, followed by incubation at 16˚C overnight; RNA was precipitated with ethanol [2; 26].

2.6.5. Chimerenomic RNA Sequencing: The RNAs were custom sequenced by CD Genomics, New York, USA.

3.0. Results

3.1. NADH-GDH Redox Cycle Hexameric Isoenzyme Complexes Chronicle Opioid Addiction: The real time imagery of addiction at the molecular level are revealed by the direct chemical reactions between morphine and GDH (Figures 1-3). Like all opioids, morphine is a strong nucleophile. Its first target site of action (receptor) in the cell is NADH-GDH where it binds to the active site of the enzyme to form dead-end morphine-GDH complexes thereby inhibiting all the oxidoreductase functions of that subunit polypeptide. Cellular proteases rush to the disabled enzyme molecules and degrade them. We have invented a multi-dimensional magnetoelectric system (Chimerenomic Pump) that captures the remaining GDH subunit polypeptides, creates new hexameric complexes, and displays them as the snapshot of the state of progression of opioid addiction (Figures 1-3). The more the molar concentrations of morphine, the higher the molar concentration of dead-end morphine-GDH complexes that are produced. Wherefore, multi-dimensional GDH isoenzyme distribution pattern (GIDP) (Figures 1-3) is not merely the real time snapshot imagery depicting opioid addiction, they chronicle the progression of opioid addiction in temporal and spatial languages. A Physician now has a new tool when advising patients on their states of opioid addiction because Figures 1 – 3 are the inflexion points. Since chemical reactions in vivo occur in the molar ratios, the GIDP (Figures 1 - 3) induced by the different concentrations of morphine are directly comparable vertically and horizontally. We applied Un-Scan-It digitalizing software to perform a semiquantitative determination of their yields. The background noise of each PAG landscape was subtracted from the densitometric readings for each GIDP to obtain the Semiquantitative Digitalized Value (SDV) for the GIDP.

The SDV for the Control GIDP at confluence was about 70% of the SDV for the Control GIDP at post confluence (Figure 1). But the SDV for the post confluence at the 0.137 mM morphine-treated GIDP was 65% of the SDV for the 0.137 mM morphine-treated GDH at confluence.

The SDV for the Control GIDP at confluence was about 70% of the SDV for the Control GIDP at post confluence (Figure 2). But the SDV for the post confluence at the 0.27 mM morphine-treated GIDP was 60% of the SDV for the 0.27 mM morphine-treated GDH at confluence.

The SDV for the 1.1 mM morphine-treated GIDP at confluence was 70% of the Control GIDP (Figure 3). But the SDV for the 1.1 mM morphine-treated GIDP at post confluency was 45% of the SDV for Control GIDP at post confluence.

Therefore, there were three inflexion points going from the Control GIDP to the 1.1 mM morphine-treated GIDP. The first inflexion point was at 0.137 mM morphine denoting the morphine threshold of opioid euphoria (aggrandizement). The second inflexion point was at 0.27 mM morphine denoting the morphine threshold alarm of opioid intoxication; and the third inflexion point was at 1.1 mM morphine denoting the morphine threshold of full-blown opioid addiction alarm. A threshold is a pre-emptive alert system for an impendent danger. So, molecular chemistry has defined addiction in chimerenomic language as no other science has done here-to-for.

The three molecular images (Figures 1-3) of the progressions of opioid addiction at the protein enzyme level have similarly been deduced from the yields of the macromolecular chimerenomic RNA enzymes produced by the GDH hexameric isoenzyme distribution patterns obtained at the control, 0.137 mM, 0.27 mM, and 1.10 mM morphine-treated astrocyte cells [14]. The convergence of results from the GDH protein and from the chimerenomic RNA levels is evidence that chimerenomic RNA is quantitatively synthesized by GDH hexameric isoenzymes thus explaining the quality control assurance foundation of chimerenomic medicine and chemistry.

3.2. Nucleotide Sequences of Macromolecular NTinti Chimerenomic RNA Enzymes: The chimerenomic RNA enzymes that were produced by the redox cycle GDH hexamers of the Control HACs, and astrocyte cells treated with 1.1 mM, 0.27 mM, 0.137 mM morphine concentrations are in the AAPCR Data Files 1 to 4, and 9 to 20 (refer to Table 1 for their nucleotide sequences). They are File ID numbers:

CHC001 and CHC002 Control A

CHC003 and CHC004 Control B

CHC009 and CHC010 (1.1 mM morphine A

CHC011 and CHC012 (1.1 mM morphine B

CHC013 and CHC014 (0.27 mM morphine A

CHC015 and CHC016 (0.27 mM morphine B

CHC017 and CHC018 (0.137 mM morphine A

CHC019 and CHC020 (0.137 mM morphine B

The human brain transcriptome contains about 32,802 transcripts, each being about 2,770 nucleotides long [27]. The above File ID numbered nucleotide sequence characteristics (Table 1) now permit a comparison of the size of the human transcriptome with the multibillion sizes of the morphine-treated human astrocyte chimerenomic NTinti RNA enzymes show that the transcriptome is merely about 0.8% the size of the NTinti RNA enzymes. Herein lies the ability of chimerenomic RNA enzyme activity to spontaneously degrade all mutated, malfunctional, alternatively spliced superfluous total RNA (mRNA, tRNA, rRNA, long noncoding RNA etc), to reset their abundances, and to cure drug addiction etc [16].

3.3.0 AAPCR functional chimerenomic bioinformatics analysis tool applied to characterize opioid misuse and overdose disorders: The functional chimerenomic bioinformatics analysis tool was developed also for the determination of sequence alignments by which homologous dysfunctional human mRNAs, rRNAs, transfer RNAs, long noncoding RNAs etc are degraded by chimerenomic macromolecular NTinti RNA enzymes produced by human astrocyte redox cycle GDH [7; 20].

Using the chimerenomic sequence homology analysis tool and the AAPCR macromolecular chimerenomic RNA enzymes sequences (Table 1) produced by morphine-treated astrocytes redox GDH Data File ID Numbers: CHC001, CHC002, CHC003, CHC004, CHC009, CHC010, CHC011, CHC012, CHC013, CHC014, CHC015, CHC016, CHC017, CHC018, CHC019, and CHC020 as subjects (Table 1), and the human mRNA sequences encoding the drug addiction malfunctional proteins as queries showed that all the morphine-related macromolecular chimerenomic RNA enzymes produced mRNA degradation matches with  all the mRNAs encoding the drug addiction malfunctional proteins [14]:

3.3.2. Disorders of Variant mRNAs Encoding Malfunctional Proteins of Drug Addiction:

Blood-brain barrier: Human genes encoding catenin (GenBank accession number X87838.1), and p120-glycoprotein-adherens junction complexes (GenBank accession ENAAY490254) are alternatively spliced [28; 29; 30] and dysfunctional [14].

Morphine binds to delta, kappa, and mu opioid receptors: Human mu-opioid receptor gene, OPRM1 (GenBank accession GQ478709), produces a multitude of alternatively spliced/superfluous mRNAs encoding full-length or truncated receptor variants [10]. Delta-opioid receptor genes are transcriptionally very diverse [31; 32], producing misfolded protein isoforms (GenBank accession AY225404).

Protein kinases connect cellular processes: Mutated mRNAs (GenBank accession U07747) encode mitogen-activated kinase; the aberrant proteins fail to be phosphorylated [33; 34; 35] thereby creating malfunctional kinase. Disease of calcium/calmodulin-dependent protein kinase is caused by variations affecting the mRNA (GenBank accession AF145711.1) [36; 37].

Glutamate Receptor 1: The Ionotropic Gate-keeper: Alternative splicing of the mRNA (GenBank accession X58633.1) produces many receptor variants [11; 38] with dysregulated gatekeeping and addiction disorders.

Glutamate Receptor 8 (Metabotropic): The mRNA (GenBank accession AJ236921) has a multitude of 1,203 variants in addition to superfluous isoforms produced by alternative splicing [39; 40].

GABA Receptor Subunit α-1: Disease malfunction [41] of the receptor protein is associated with the production of hundreds of superfluous variants affecting the mRNA (GenBank accession BC030696).

Dopamine Receptor: Disease malfunctioning [42] of the receptor is associated with the 792 mutational variants affecting the mRNA, GenBank accession AY136750.

Serotonin Receptor: Multiple serotonin receptor proteins, which are the root cause of the disorders associated with the gene, accession AJ003080 [13].

Lipase member H: Diseases [43] caused by variants affecting the gene (GenBank accession AY03612) do not hydrolyze phosphatidylserine, phosphatidylcholine phosphatidylethanolamine and triacylglycerol.

Cytochrome P450 monooxygenase: Diseases of the malfunctioning of this enzyme are due to the existence of more than775 mRNA isoforms showing features for mutagenesis (GenBank accession AF182276) [44].

Cu/Zn Superoxide dismutase: Amyotrophic lateral sclerosis 1 (ALS1) diseases caused by mRNA variants of the gene (GenBank accession X02317) forming fibrillar aggregates [45].

Alternative Oxidase: The gene (GenBank accession D00741) encoding the protein is alternatively spliced [46; 47].

Glutathione S-transferase: Dysregulation and disease of the glutathione S-transferase proteins is due to mutagenesis of the natural variant transcripts, GenBank accession Y13047 [48].

ABC Transporter: Diseases are caused by the mutagenesis of the gene, GenBank accession AF103796 existence as multifamily of natural mRNA variants [49].

Endorphins: This GenBank accession M38297 encodes a larger precursor protein from which multiple peptide products are derived, including β-endorphin giving an appearance of dysregulation to the transcripts [50].

Proenkephalin A Receptor: The mRNA encodes a precursor protein, proenkephalin (PENK), which is then processed into the active enkephalin peptides, including Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe, PENK(114-133),  PENK(237-258) etc. Disease and Variants of mRNA GenBank accession BC032505 show features for 471 natural variants.

Synapsin-1 Receptor: The diseases are caused by alternatively spliced superfluous mRNA variants affecting the gene: GenBank accession AH006533 [12].

In all the above chimerenomics-related diseases, the superfluous mRNAs assume aberrant conformations; become translated to misfolded proteins that fail to perform their natural functions in the nervous system. We have demonstrated that chimerenomic RNA enzyme produced by the redox GDH from morphine-treated astrocytes shared degradative sequence homologies with the mRNAs for the opioid receptors [14]. So, a cure for delta- and mu-receptor protein-related malfunctioning using chimerenomic medicine potentially cures a host of other drug-related disorders.

4.0. Discussion

4.1 Opioid-NTinti Chimerenomic RNA Enzymes for Cure of Opioid Addiction: Neural projection pathways from the ventral tegmental area (VTA) [51], the pathways for peripheral pain and temperature sensations leading to the thalamus [52], the drug metabolism routes originating in the liver [53], the addiction-related neurotransmitter pathways [54; 55], and the intracellular kinase systems all play significant roles in addiction and SUD [51].

All these systems are also treatment targets for Opioid-NTinti chimerenomic RNA enzyme medications.

In the absence of Opioid-NTinti treatment, these systems operate concurrently, the impacted cells activate a corrective mechanism that captures every alteration in temporal homeostasis. This is achieved through the electromagnetic and electrochemical translation of various events into a distinct type of RNA that is not based on the genetic code. These nongenetic code-based RNAs are referred to as chimerenomic RNAs [2], which are regulated by NADH-GDH isoenzymes, initiating a response to changes in the homeostatic environment and forming complexes with any specific addictive substance, such as morphine. This NADH-GDH corrective system functions as an inherent natural adaptation mechanism, aimed at restoring homeostasis disrupted by the addiction cascade. This concept has been a long-sought element in addiction science; the existence of this parallel cellular system that regulates the expression of distortions inflicted on genetic codes by both internal and external factors represents a groundbreaking advancement in the field of addiction research, now presented for the first time here.

This study investigated the impact of morphine on HACs, aiming to clarify this discovery, demonstrate its efficacy, and explore its potential for developing effective treatments for addiction and SUD. Below, we outline the established and proposed biological mechanisms underlying addiction based on current understanding.

4.2 Anatomy of brain neural projections for addiction

4.2.1 Neural projections originating from the brainstem and midbrain have been linked to addiction and movement disorders. Notably, two projections from the ventral tegmental area (VTA)—the mesocortical and mesolimbic pathways—are particularly associated with SUD and addiction. The mesocortical projections extend from the VTA to the prefrontal cortex, which plays a crucial role in the conscious regulation of motivation, emotion, and control of voluntary movement. In contrast, the mesolimbic system connects the VTA to several key areas, including the nucleus accumbens (NA), olfactory bulb, amygdala, limbic system, caudate nucleus, putamen in the corpus striatum, and the hypothalamic-hypophyseal axis. This mesolimbic system is integral to the experience of euphoria, the reward and pleasure response, mood regulation, sleep etc. The VTA is composed of approximately 60% dopamine neurons, 30% GABA neurons, and 10% glutamate neurons [54; 55].

Both endogenous and exogenous opioid receptor agonists stimulate dopamine release while inhibiting the release of GABA and glutamate. Additionally, beta-endorphins are produced in hypothalamo-pituitary tracts and diffuse throughout the brain and the body [56].

4.2.2 Peripheral nerves: are extensively distributed throughout the body and its tissues. Proprioceptive and tactile signals are transmitted from these nerves to the dorsal root ganglion, then to the nucleus gracilis and cuneatus in the spinal medulla. From there, second-order neurons cross to the contralateral side and project via the medial lemniscal pathway to the thalamus. The spinothalamic tract, which consists of peripheral nerves responsible for pain and temperature sensation, transmits signals to the dorsal root, then to the spinal nucleus. Second-order neurons subsequently cross to the contralateral side and ascend as the lateral spinothalamic tracts through the spinal cord to the thalamus [57; 58]. In response to nociceptive stimuli, these peripheral nerves release substance P. Opioid receptor agonists, such as morphine, bind to mu receptors on the presynaptic membranes of peripheral nerves, inhibiting the release of substance P and thereby diminishing the transmission of pain sensations [52].

4.3 Cells Involved in Addiction

Neurons are essential for neurotransmission, facilitating the delivery of neurotransmitters across various brain circuits and projections [58]. In contrast, astrocytes significantly contribute to neural plasticity, adaptation, and learning—key processes that are integral to the addiction cascade.

Astrocytes are likely key players in the long-lasting excitatory plasticity that develops within the corticostriatal circuitry because of chronic drug use. Their processes are strategically located near excitatory synapses, and the expression of neurotransmitter transporters on astrocytes helps regulate the stimulation of both pre- and postsynaptic receptors. Additionally, astroglia can directly communicate with synapses through the release of transmitters, playing a crucial role in the homeostatic regulation of synaptic function [59].

Hepatocytes are the main liver cells responsible for metabolizing a wide range of substances, including drugs associated with addiction.

This new opioid-NTinti chimerenomic RNA drugs targets all these cell types.

4.4 Addiction Drug Metabolism

For drugs such as morphine to exert their effects, they must undergo a series of processes: absorption, distribution, metabolism, and excretion. These steps are essential for achieving an adequate plasma concentration of the drug and for traversing biological membranes, such as the blood-brain barrier, to access the brain. For instance, the ABC transporter family of enzymes is responsible for expelling drugs from cells and the brain, while the cytochrome P450 enzyme system, which comprises over 775 isoforms, plays a critical role in various drug metabolism and detoxification processes. All these factors contribute to the overall addiction risks associated with opioids like morphine. Currently, no addiction treatments have adequately considered these aspects of drug metabolism. However, with the introduction of chimerenomic RNA, many of the abnormal RNAs generated through alternative splicing can be degraded, thereby restoring normal enzyme function and addressing the inefficiencies and errors in current drug metabolism and treatment [53].

4.5 Addiction and Pain Pathways for Receptors and Neurotransmitters

The study of addiction involves various receptors, including but not limited to opioid receptors (mu, delta, and kappa), dopamine, GABA, glutamate, substance P, NMDA, AMPA, neuropeptides, neurotensin etc. The intracellular effects and changes that occur when drugs interact with these receptors can be extensive and long-lasting. Many drugs penetrate cells and bind to various enzymes, causing physicochemical alterations that disrupt cellular homeostasis. Due to the stress these changes impose on cellular physiology, excess superfluous mRNAs are frequently produced due to alterative splicing [58].

When morphine binds to mu opioid receptors, it affects both peripheral nervous system (PNS) and CNS cells differently. In the PNS morphine binds to mu receptors and inhibits the release of substance P and in so doing prevent pain impulse transmission; while in the CNS morphine binds to mu receptors and inhibits the release of GABA and glutamate. By inhibiting GABA, an inhibitory neurotransmitter of dopamine releasing neurons, the resultant effect is the release of more dopamine. The release of dopamine from the VTA leads to dopamine release in the neural projections in the mesocortical (frontal cortex) and the mesolimbic projections (nucleus acumbens, amygdala, limbic system, etc) [60; 61].

4.5.1 Feedback Loop: Initially, there may be a relative increase in circulating endorphins, followed by a decrease in the synthesis of both endorphins and mu receptors. As the number of mu receptors declines, progressively higher doses of morphine are required to stimulate dopamine release for adequate reward maintenance, leading to the development of tolerance. Concurrently, cells begin to produce various proteins collectively referred to here as "addiction proteins". Many of these proteins are anti-opioid peptides (such as CCK, nociceptin/orphanin FQ, neuropeptide FF, and others), many of these anti-opioid proteins limit the effect of opioid on recptors leading to development of tolerance and addiction [62].

The feedback loop established by the inhibition of GABA and glutamate receptors results in increased intracellular production of GABA and glutamate, which further diminishes dopamine release and reduces the sense of reward. This cycle amplifies the interaction between addiction receptors and neurotransmitters, perpetuating the addiction process as well. Opioid-NTinti RNA medicines inhibit these feedback loops resulting from the addiction cascade.

4.6 Intracellular Kinase Pathways

Human protein kinases play a crucial role in connecting signaling pathways that are essential for maintaining good health and addressing disease conditions. They transduce, amplify, and integrate cellular processes involved in opioid addiction through the mechanism of protein phosphorylation [34; 35]. Protein kinases belong to a single superfamily, characterized by a continuous stretch of approximately 250 amino acid residues that form their catalytic site. During the stress induced by opioid addiction pathways, kinase synthesis can undergo mutations, resulting in the production of aberrant proteins that are unable to be phosphorylated [33], leading to dysfunctional kinases.

Calcium/calmodulin-dependent protein kinase operates autonomously following Ca²⁺/calmodulin binding and autophosphorylation. This kinase is involved in synaptic plasticity, neurotransmitter release, and the long-term potentiation of excitatory synapses. Additionally, cyclic AMP-dependent protein kinase type II-alpha regulatory subunits facilitate membrane association by binding to anchoring proteins, including MAP2 kinase. The inactive form of this enzyme consists of two regulatory chains and two catalytic chains. When activated by cAMP, it produces two active catalytic monomers and a regulatory dimer that binds four cAMP molecules.

This study has shown that chimerenomic RNA enzymes, chimmerized by the redox GDH from morphine-treated astrocytes, exhibit degradative sequence homologies with the mRNAs encoding protein kinases [14].

4.7 NADH-GDH Chimerenomic Correction System

One of the most significant counter and parallel components of the addiction pathways, hitherto hidden, is the NADH-GDH hexameric isoenzyme complex correction system. This system not only records all electromagnetic and electrochemical changes occurring within cells at various levels but also generates rapid, non-genetic code-based RNAs aimed at restoring cellular homeostasis. It operates as a parallel system to the previously described addiction pathways but impeded in vivo by many cellular inhibitory factors. While addiction pathways mentioned previously initiate a cascade of events following morphine's binding to receptors (such as the mu receptor), creating feedback loops that prompt cells to produce additional proteins, receptors, secondary messengers, and neurotransmitters through the upregulation of genetic code-based RNA production, the NADH-GDH chimerenomic righting (correction) system functions independently and oppositely.

The NADH-GDH chimerenomic righting system operates in two primary ways. First, it begins by sensing electromagnetic changes in the environment (indicating altered homeostasis) through the GDH isoenzyme complex, which shifts the equilibrium of the alpha-ketoglutarate to glutamate reaction in favor of glutamate synthesis, leading to glutamate accumulation within the cells.

The second event involves the interaction of morphine, a nucleophile, with the NADH-GDH hexameric isoenzyme complex, resulting in the formation of Schiff base complexes. The isomerization of GDH is triggered by the fragmentation of some of its subunit polypeptides upon morphine or other nucleophilic drugs binding to the active site lysine residue, creating dead-end enzyme-substrate complexes [63]. The active site lysine residue is first activated for nucleophilic attack through Schiff base formation with α-ketoglutarate (α-KG) or glutamate. Nucleophiles with negative reduction potentials induce greater fragmentation of GDH compared to those with positive reduction potentials [64]. Nucleophilic substrates, such as ribonucleoside triphosphates, which possess both binding and polymerization groups, are polymerized into chimerenomic RNA [65]. The chimerenomic RNA synthesis activity is dependent on the redox cycle of the hexameric subunit structure of GDH; when the hexamer dissociates in the absence of substrates, the synthetic activity for chimerenomic RNA is lost, although aminating and deaminating activities remain intact.

Thus, this electrochemical system that produces RNAs (chimerenomic RNAs) independent of the genetic code represents a righting (correction) and parallel system that has previously eluded addiction research scientists. This system is highly responsive to environmental changes, capturing every alteration in the electromagnetic and electrochemical signals induced by morphine within the body. In this study, we sequenced the chimerenomic macromolecular RNA enzymes induced by various doses of morphine, paving the way for new therapeutic approaches to treat and potentially reverse morphine and opioid addiction.

4.8 Application in Morphine Astrocyte Study

The investigation into the effects of morphine on human astrocyte cells reveals a comprehensive view of addiction science. We can broadly categorize the interaction of morphine into two primary processes: the binding of morphine to opioid receptors (mu, delta, kappa) and the binding of morphine to GDH within the cells.

When morphine binds to opioid receptors, it activates a cascade of neurotransmission, intracellular kinase pathways, and drug metabolism processes. These interactions lead to the synthesis of new RNAs and proteins that alter cellular functions. This activation of genetic code-based RNA synthesis ultimately results in protein production. It is important to note that RNA synthesis during morphine exposure creates a stressful environment due to the disruption of cellular homeostasis. Consequently, alternative splicing mechanisms generate numerous superfluous RNAs, resulting in the production of abnormal proteins, we referred to as "addiction proteins".

In addition to binding to opioid receptors, morphine also enters astrocytes and binds to GDH. As a potent nucleophile, morphine's primary intracellular target is NADH-GDH, where it attaches to the enzyme's active site, forming dead-end morphine-GDH complexes. This binding inhibits the oxidoreductase functions of the subunit polypeptide, prompting cellular proteases to degrade the disabled enzyme molecules. The binding of morphine to NADH-GDH occurs in molar ratios, meaning that the resultant hexameric isoenzyme subunits are proportional to the molar concentration of morphine present. [59].

We developed a multi-dimensional magnetoelectric system, termed the Chimerenomic Pump, which captures the remaining GDH subunit polypeptides, creates new hexameric complexes, and provides a snapshot of the progression of opioid addiction. Using these newly identified hexameric complexes of NADH-GDH, we synthesized macromolecular RNA molecules that represent all the electrochemical and electromagnetic changes induced by morphine exposure at various concentrations.

From our study three inflection points for opioid addiction have been defined and may have serious implications for clinical applications. The first inflexion point was at 0.137 mM morphine concentration denoting the morphine threshold of opioid euphoria. The second inflexion point was at 0.27 mM morphine concentration denoting the morphine threshold alarm of opioid intoxication; and the third inflexion point was at 1.1 mM morphine concentration denoting the morphine threshold of full-blown opioid addiction alarm. A threshold is a pre-emptive alert system for an impendent danger. So, molecular chemistry has defined in chimerenomic language as no other science has done here-to-for, the identities of opioid euphoria, opioid intoxication alert; and opioid addiction alarm (Figures 1 – 3).

Therefore, in clinical settings, translating these inflection points, opioid use disorder (OUD) can be classified into 3 distinct clinical conditions here referred to as OUD stage 1, OUD stage 2 and OUD stage 3 each with its own medication remedy. These clinical stages are determined by the estimated quantity in molar concentrations that the patient is using or have used in one cycle compressed of 48 hours, akin to the exposure of astrocytes for 48 hours post confluence in agreement with chimerenomic cell biology research protocol [5; 8; 14; 66].

These macromolecular chimerenomic nongenetic code-based RNA enzymes form a parallel mechanism that differs from the processes initiated by morphine binding to opioid receptors on the cell membrane. The macromolecular chimerenomic RNA specific to morphine concentrations will differ from those produced in response to other substances of abuse. These remarkable and unique morphine macromolecular RNA enzymes play a crucial role in reversing many of the cellular and homeostatic dysfunctions associated with addiction by degrading the RNAs responsible for the formation of addiction proteins and facilitating drug metabolism.

4.9.0 Drug Addiction NTinti Macromolecular Treatment Modalities

The landscape of addiction treatment is evolving with the introduction of innovative NTinti macromolecular treatment modalities, particularly in the context of opioid addiction. Our study has revealed that varying concentrations of morphine induce specific electrochemical and electromagnetic changes in astrocytes, leading to the development of concentration-specific Morphine-NTinti RNA enzymes. These enzymes hold the potential to reverse both the somatic and psychological effects associated with specific morphine doses, offering a groundbreaking approach to addiction therapy.

4.9.1 Mechanism of Action: When administered to patients grappling with opioid addiction, Morphine-NTinti RNA enzymes can significantly disrupt the addiction process. They achieve this by degrading homologous mRNAs and other genetic code-based RNAs that play a crucial role in the potentiation of addiction. This targeted degradation not only mitigates the immediate effects of opioid use but also addresses the underlying genetic factors that contribute to addiction. Another mode of action is called the mass action effect and saturation. This is possible because the NTinti RNA macromolecular enzymes are very large molecules, sometimes containing over 10 billion nucleotide bases ]5], enough to saturate the endoplasmic reticulum, and to degrade superfluous mRNAs before they enter the protein translation processes. This is the mechanism by which NTinti RNA enzyme regulates DNA genes and blunts the dysfunctional receptor proteins and neurotransmitters by electromagnetic repulsion, achieving rapid immediate reversal of drug addiction. This is a new mechanism in gene regulation (see figures 4 and 5).

4.9.2 A Multi-Nodal Strategy: This chimerenomic approach to addiction treatment contrasts sharply with traditional methods that primarily rely on opioid agonist and partial agonist replacement therapies, such as methadone and buprenorphine treatments and the like. While these therapies can alleviate withdrawal symptoms, they often fall short of providing a comprehensive solution. By focusing on a single node—opioid receptors—these treatments can inadvertently create feedback loops that lead to the synthesis of additional addiction-related proteins over time worsening addiction cascade.

In contrast, the multi-nodal effects of opioid-NTinti RNA macromolecules aim to restore homeostasis across various addiction and drug metabolism pathways. This holistic approach effectively disrupts the feedback loops that contribute to tolerance and addiction, offering a more sustainable path to recovery.

4.9.3 Therapeutic Potential: The potential of opioid-NTinti RNA therapy lies in its ability to reverse and prevent the accumulation of “addiction proteins”, which is crucial for achieving lasting recovery from opioid addiction. By addressing the complex interplay of biological factors involved in addiction, this innovative treatment modality offers hope for individuals seeking to break free from the cycle of opioid dependency.

As we continue to explore the frontiers of addiction treatment, the development of opioid-NTinti macromolecular therapies represents a considerable advancement in our understanding and management of opioid addiction. By leveraging the power of chimerenomic sciences, we can pave the way for more effective, multi-faceted treatment strategies that not only alleviate symptoms but also target the root causes of addiction, ultimately fostering a healthier future for those affected by substance use disorders.

4.10 The Next Generation of SUD Treatments: Opioid-NTinti RNA Therapy

The landscape of substance use disorder (SUD) treatments is on the brink of a revolutionary transformation with the advent of Opioid-NTinti RNA therapy, a cutting-edge approach derived from chimerenomic macromolecular RNA technologies. This innovative therapy represents a considerable leap forward in our ability to address the addiction epidemic, offering a multifaceted solution to various forms of substance abuse.

Opioid-NTinti RNA therapy harnesses the power of chimerenomic sciences to create specific RNA molecules designed to target and mitigate the effects of addiction and overdose. By focusing on substances such as fentanyl, hydrocodone, hydromorphone, benzodiazepines, and alcohol, this therapy aims to provide tailored interventions that can effectively disrupt the cycle of addiction.

As we look to the future of addiction treatment, Opioid-NTinti RNA therapy represents a beacon of hope. With its potential to revolutionize how we approach substance use disorders, this next-generation treatment could significantly improve the lives of countless individuals and their families.

5.0 Conclusion

A Revolutionary Tool for the Prevention, Treatment, and Cure of Opioid Use Disorders: Chimerenomic Opioid-NTinti Macromolecular RNA Enzymes: In the ongoing battle against substance use disorders (SUD) and addiction, a groundbreaking innovation has emerged: chimerenomic Opioid-NTinti macromolecular RNA enzymes. This advanced therapeutic approach promises to transform the landscape of addiction treatment, offering new avenues for prevention, intervention, and potential cures. Chimerenomic Opioid-NTinti RNA enzymes are electrochemically constructed molecules designed to target the complex biological mechanisms underlying addiction. By leveraging the principles of chimerenomic science, these enzymes can specifically interact with the genetic and biochemical pathways involved in substance use disorders. The unique structures of redox cycle GDH allow them to produce rapid, nongenetic code-based RNAs that can restore cellular balance and mitigate the effects of addiction.

Chimerenomic Terminologies: glossary for the new terminologies in life sciences.

1. These new terminology in life sciences describe the discovery of template-independent RNA
processes that control the survival of cells and organisms other than genes. The building blocks of this life
regulatory system are made up of a unique type of RNA that are not coded nor synthesized through the
genetic code. These template-independent RNAs are called chimerenomic RNAs.

2. Chimerenomics: Is the study of everything (NTintis, chigramming, GDH-
chimmerization, chimere etc) about chimerenomic RNAs. Chimerenomics confers molecular chemistry

pluripotency and totipotency on all cells and tissues. Chimerenomics are the processes by which whole
organisms, cells and tissues differentiate, develop and grow by chigramming chimerenomic RNAs that
interact with the changing physicochemical internal and external environmental conditions thereby
reprogramming and optimizing those metabolic reactions that assure the continued survival of the organism.
3. Chimere: Is the minimum length of nucleotide sequence that can degrade homologous mRNA
and other genetic code-based RNAs. Chimere is the active segment of NTinti.

4. NTinti: This is the chimerenomic RNA molecule chigrammed (synthesized) by NADH-
glutamate dehydrogenase hexameric redox cycle isoenzymes (GDH) in response to a specific

environmental change. NTinti is also synthesized naturally in vivo during normal tissue differentiation,
growth and development. Therefore, NTinti can be cell or tissue specific. One NTinti has more than one
chimere.
5. Chigramming: This is the process of synthesis/construction/configuring of chimeres and
NTintis by GDH. They are spontaneous processes. It is the conversion of the magnetoelectric changes in
the environmental conditions of cells, tissues, whole organisms to the nucleotide sequences of
chimerenomic RNAs.
6. GDH-Chimerization: is the formation of chemical Schiff base of GDH isoenzymes
in response to a new environment leading to new hexameric isoenzyme complexes. This is the initiation
process for chigramming.
7. Functional chimerenomics: is the study of the biological functions of chimere and NTinti.
8. Differential chimerenomics: is the comparison study of the chimeres and NTintis
from same or different tissues under various environmental conditions.
9. Other terminologies
a). Clinical Chimerenomics: The application of chimerenomics to clinical studies.
b). Chimerenomic Medicine: The application of chimerenomics to primary care in the prevention and
treatment of human disorders, diseases and wellness conditions.
c). Chimerenomic Chemistry
d). Chimerenomic Physiology
e). Molecular Chimerenomics
f). Chimerenomic Biology
g). Chimerenomic Pharmacology
h). Biomedical Engineering Chimerenomics
i)  Chimerenomic Hematology etc.