Medicine in motion: Opportunities, challenges and data analytics-based solutions for traditional medicine integration into western medical practice.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional pharmacopeias have been developed by multiple cultures and evaluated for efficacy and safety through both historical/empirical iteration and more recently through controlled studies using Western scientific paradigms and an increasing emphasis on data science methodologies for network pharmacology. Traditional medicines represent likely sources of relatively inexpensive drugs for symptomatic management as well as potential libraries of new therapeutic approaches. Leveraging this potential requires hard evidence for efficacy that separates science from pseudoscience. MATERIALS AND METHODS: We performed a review of non-Western medical systems and developed case studies that illustrate the epistemological and practical translative barriers that hamper their transition to integration with Western approaches. We developed a new data analytics approach, in silico convergence analysis, to deconvolve modes of action, and potentially predict desirable components of TM-derived formulations based on computational consensus analysis across cultures and medical systems. RESULTS: Abstraction, simplification and altered dose and delivery modalities were identified as factors that influence actual and perceived efficacy once a medicine is moved from a non-Western to Western setting. Case studies on these factors highlighted issues with translation between non-Western and Western epistemologies, including those where epistemological and medicinal systems drive markets that can be epicenters for zoonoses such as the novel Coronavirus. The proposed novel data science approach demonstrated the ability to identify and predict desirable medicinal components for a test indication, pain. CONCLUSIONS: Relegation of traditional therapies to the relatively unregulated nutraceutical industry may lead healthcare providers and patients to underestimate the therapeutic potential of these medicines. We suggest three areas of emphasis for this field: First, vertical integration and embedding of traditional medicines into healthcare systems would subject them to appropriate regulation and evidence-based practice, as viable integrative implementation mode. Second, we offer a new Bradford-Hill-like framework for setting research priorities and evaluating efficacy, with the goal of rescuing potentially valuable therapies from the nutraceutical market and discrediting those that are pseudoscience. Third, data analytics pipelines offer new capacity to generate new types of TMS-inspired medicines that are rationally-designed based on integrated knowledge across cultures, and also provide an evaluative framework against which to test claims of fidelity and efficacy to TMS made for nutraceuticals.

X chromosome reactivation in mouse embryonal carcinoma cells.

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon.

In both humans and mice, two genes encode phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The pgk-1 gene is expressed in all somatic cells, is located on the X chromosome, and contains 10 introns. The pgk-2 gene is expressed only in sperm cells, is located on an autosome, and has no introns. The nucleotide sequence of the pgk-2 gene suggests that it arose from pgk-1 more than 100 million years ago by RNA-mediated gene duplication. The pgk-2 gene may, then, be a transcribed retroposon. Thus, gene duplication by retroposition may have been used as a mechanism for evolutionary diversification.

Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter.

We report the cloning of the mouse pgk-1 gene encoding the somatic cell isoform of the enzyme phosphoglycerate kinase. The gene is contained within a 16-kb region of the X chromosome and is interrupted by at least ten introns. The promoter region of the pgk-1 gene is rich in G and C nucleotides and contains five copies of the hexadeoxynucleotide, GGGCGG, the potential binding site for the Sp 1 transcription factor, a CCAAT sequence, but no TATA box. This promoter functions following DNA-mediated transfection into mammalian cells. The promoter of the mouse pgk-1 gene is homologous to the human pgk-1 promoter. A number of conserved motifs in the promoter may indicate a significant role for these sequences in expression of the pgk-1 gene.

Timing of paternal Pgk-1 expression in embryos of transgenic mice.

In mouse development, the paternal allele of the X-linked gene Pgk-1 initiates expression on day 6, two days later than the maternal allele, which is activated on day 4. The different timing of expression of the maternal and paternal alleles may be determined by (i) imprinting of the chromosome region in which the gene resides, but not aimed specifically at the Pgk-1 gene; (ii) gene specific imprinting, acting on Pgk-1 irrespective of the chromosomal localization of the gene; (iii) an interplay between embryo cell differentiation, timing of X-inactivation and Pgk-1 expression, without the involvement of imprinting at the Pgk-1 locus itself (Fundele R., Illmensee, K., Jagerbauer, E. M., Fehlau, M. and Krietsch, W. K. (1987) Differentiation 35, 31-36). Our findings in transgenic mouse lines, carrying Pgk-1 on autosomes, indicate the importance of the X chromosomal location for the delayed expression of the paternal Pgk-1 allele, and are in agreement with the first of the explanations listed above. We propose that the late activation of the paternal Pgk-1 locus is a consequence of imprinting targeted at, and centered around, the X chromosome controlling element.

The family of mouse phosphoglycerate kinase genes and pseudogenes.

The mammalian genome contains two genes encoding phosphoglycerate kinase; the pgk-1 gene is X-linked and is expressed in all cells except sperm, while the pgk-2 gene is expressed exclusively in sperm cells. The mouse genome contains no pseudogenes derived from pgk-2. On the other hand, the genomes of Balb/c and C3H/He strain mice contain six other regions with sequences homologous to those of pgk-1 cDNA. These pgk-related sequences are likely derived from the pgk-1 gene by retroposition because all are located on autosomal chromosomes and because none appear to be interrupted by introns. Two of the presumed pseudogenes contain sequences homologous to all regions of the pgk-1 cDNA while the other four genomic regions were truncated at the 5', 3', or both ends. One of the truncated pseudogenes was sequenced. Its pgk-related sequence was not flanked by direct repeats, suggesting that loss of the 5' and/or 3' ends of this retrogene may have occurred following its integration into the genome. Our evidence suggests that pgk-1-derived retroposons arose initially more than 100 million years ago and have continued to arise until so recently that some are unique to different mouse strains.

Reactivation of hprt on the inactive X chromosome with DNA demethylating agents.

The C86 line of female embryonal carcinoma cells contains one active and one inactive X chromosome. Following methylnitrosourea mutagenesis, a clone called C86AGM2 was isolated that carries a mutated hprt gene on the active X chromosome. This hprtm allele encodes an HPRT enzyme that has less than 1% normal enzyme activity, is thermolabile, and has an altered isoelectric point. Following treatment with drugs that demethylate DNA, the hprt+ gene from the inactive X chromosome in C86AGM2 cells became active as determined by the appearance of HPRT activity with the thermodenaturation and electrofocusing characteristics of the normal enzyme. No expression of this hprt+ gene occurred if C86AGM2 cells were induced to differentiate prior to DNA demethylation. Stable lines of C86AGM2 cells expressing both the hprtm and hprt+ genes did not inactivate either gene following differentiation.

Inhibition of hematopoietic development from embryonic stem cells by antisense vav RNA.

The vav proto-oncogene is universally and specifically expressed in hematopoietic cells. vav contains a unique array of motifs allowing the protein to function as a signal transducer and possibly as a transcription factor. Under certain in vitro culture conditions murine embryonic stem cells develop into colonies containing multiple hematopoietic lineages. In embryonic stem cell lines, constitutively expressing high levels of antisense vav transcripts through a stably integrated transgene, differentiation into hematopoietic cells is disrupted. This observation presents the first evidence that vav has a critical role in the development of hematopoietic cells from totipotent cells.

DNA methylation of two X chromosome genes in female somatic and embryonal carcinoma cells.

The extent of methylation of DNA sequences upstream and within the two X-linked genes, Pgk-1 and Hprt, was analyzed in male and female somatic cells and in female embryonal carcinoma cells carrying either two active X chromosomes (Xa) or one active and one inactive X chromosome (Xi). Sites upstream and within the first intron of both Pgk-1 and Hprt were heavily methylated on the Xi in somatic cells and in embryonal carcinoma cells with an Xi. Reactivation of this Xi was accompanied by extensive demethylation of these sites. In female embryonal carcinoma cells with two active X chromosomes, one X inactivates during differentiation in culture; however, methylation did not occur during differentiation, consistent with the idea that DNA methylation does not play a role in the initiation of X inactivation but may be involved in maintaining inactivation of those genes on the Xi.

cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins.

We have identified the mRNA for a human gene, denoted D4, which is expressed at very high levels in hematopoietic cell lines and in normal cells of lymphoid and myeloid origin. The 1.5-kb transcript is absent or detectable only at low levels in nonhematopoietic tissues. D4 encodes a 201-amino acid protein with homology to rhoGDI, an inhibitor of GDP dissociation for the ras-homologous protein rho. D4 might function also as a regulator of guanine nucleotide exchange for small GTP-binding proteins. A homologous transcript of similar size is also preferentially expressed in murine hematopoietic tissues. When totipotent murine embryonic stem cells develop in vitro into hematopoietic cells, the gene is activated with the onset of hematopoiesis. When hematopoietic cell lines are induced to differentiate, the expression of D4 is modulated. Thus, D4 appears to be a developmentally regulated gene. Its preferential expression in hematopoietic cells indicates that D4 likely plays some significant role in the growth and differentiation processes of hematopoietic cells. This significance is underscored by increasing evidence for the involvement of regulators of G proteins in clinical diseases.

The mouse Pgk-1 gene promoter contains an upstream activator sequence.

The Pgk-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed. This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells. The Pgk-1 promoter is in a region rich in nucleotides G and C. This promoter can efficiently drive high levels of expression of reporter genes such as E. coli lacZ and neo. We have determined that the 120 bp upstream of the transcription start site functions as a core promoter. Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion. This 320 bp region does not enhance transcription from the core promoter of the SV40 early region. Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity.

A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins for the Rho- and Rab-subtype GTP-binding proteins. As a first step toward identifying the key functional domain(s) on the Rho-subtype GDI, truncations of the amino and carboxyl termini were performed. Deletion of the final four amino acids from the carboxyl terminus of Rho GDI or the removal of 25 amino acids from the amino terminus had no significant effect on the ability of the GDI to inhibit GDP dissociation from the Rho-like protein Cdc42Hs or on its ability to release Cdc42Hs from membrane bilayers. However, the deletion of 8 amino acids from the carboxyl terminus of Rho GDI eliminated both activities. To further test the importance of the carboxyl-terminal domain of the Rho GDI molecule, chimeras were constructed between this GDI and a related protein designated LD4, which is 67% identical to Rho GDI but is less potent by a factor of 10-20 than Rho GDI in functional assays with the Cdc42Hs protein. Two sets of chimeras were constructed that together indicated that as few as 6 amino acids near the carboxyl terminus of Rho GDI could impart full GDP dissociation inhibition and membrane dissociation activities on the LD4 molecule. Further analysis of this region by site-directed mutagenesis showed that a single change at residue 174 of LD4 to the corresponding residue of Rho GDI (i.e., Asp-174-->Ile) could impart nearly full (70%) Rho GDI activity on the LD4 molecule.

Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the beta subunit of the high-affinity IgE receptor: evidence for a family of proteins with four membrane-spanning regions.

We report the cloning of the cDNA for a human gene whose mRNA is expressed specifically in hematopoietic cells. A long open reading frame in the 1.7-kb mRNA encodes a 214-aa protein of 25 kDa with four hydrophobic regions consistent with a protein that traverses the membrane four times. To reflect the structure and expression of this gene in diverse hematopoietic lineages of lymphoid and myeloid origin, we named the gene HTm4. The protein is about 20% homologous to two other "four-transmembrane" proteins; the B-cell-specific antigen CD20 and the beta subunit of the high-affinity receptor for IgE, Fc epsilon RI beta. The highest homologies among the three proteins are found in the transmembrane domains, but conserved residues are also recognized in the inter-transmembrane domains and in the N and C termini. Using fluorescence in situ hybridization, we localized HTm4 to human chromosome 11q12-13.1, where the CD20 and Fc epsilon RI beta genes are also located. Both the murine homologue for CD20, Ly-44, and the murine Fc epsilon RI beta gene map to the same region in murine chromosome 19. We propose that the HTm4, CD20, and Fc epsilon RI beta genes evolved from the same ancestral gene to form a family of four-transmembrane proteins. It is possible that other related members exist. Similar to CD20 and Fc epsilon RI beta, it is likely that HTm4 has a role in signal transduction and, like Fc epsilon RI beta, might be a subunit associated with receptor complexes.

Cloning of human RTEF-1, a transcriptional enhancer factor-1-related gene preferentially expressed in skeletal muscle: evidence for an ancient multigene family.

Transcriptional Enhancer Factor-1 (TEF-1) is a transcription factor required for cardiac muscle gene activation. Since ablation of TEF-1 does not abolish cardiac gene expression, we sought to identify a human gene related to TEF-1 (RTEF-1) that might also participate in cardiac gene regulation. A human heart cDNA library was screened to obtain a full-length RTEF-1 cDNA. Fluorescence in situ hybridization assigned the RTEF-1 gene to chromosome 12p13.2-p13.3. In contrast, PCR screening of human/rodent cell hybrid panels identified TEF-1 on chromosome 11p15.2, between D11S1315 and D11S1334, extending a region of known synteny between human chromosomes 11 and 12 and arguing for an ancient divergence between these two closely related genes. Northern blot analysis revealed a striking similarity in the tissue distribution of RTEF-1 and TEF-1 mRNAs; skeletal muscle showed the highest abundance of both mRNAs, with lower levels detected in pancreas, placenta, and heart. Phylogenetic analysis of all known TEF-1-related proteins identified human RTEF-1 as one of four vertebrate members of this multigene family and further suggests that these genes diverged in the earliest metazoan ancestors.

Polymorphisms in the coding and noncoding regions of murine Pgk-1 alleles.

The mouse X-linked Pgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, the Pgk-1b allele causes the appearance of mouse PGK-1b enzyme activity. We describe here cloning of mouse Pgk-1a, an allele of Pgk-1 which encodes an enzyme, PGK-1a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encoding Pgk-1b and Pgk-1a and transfected these constructs into human cells to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1b and PGK-1a, respectively. A number of other DNA sequence polymorphisms exist between Pgk-1b and Pgk-1a including part of an L1 repeated element unique to Pgk-1a.

Identification of a novel protein with GDP dissociation inhibitor activity for the ras-like proteins CDC42Hs and rac I.

We have recently cloned the human cDNA for a gene, denoted D4, that encodes a protein 67% identical to the bovine rhoGDI protein, a GDP dissociation inhibitor (GDI) for the ras-related rho-subtype proteins. We now present data on the cloning and structural analysis of the murine D4 cDNA and confirm its preferential expression in hematopoietic tissues. The predicted murine and human D4 proteins are almost 90% identical, indicating that D4 and rhoGDI are different genes and that they are probably members of a related family of genes. Functional studies with the human D4 protein demonstrate that D4 has GDI activity against the CDC42Hs and rac I proteins, but binds to these proteins with a significantly weaker affinity than does the rho-subtype GDI. These data suggest that D4, which will in subsequent communications be denoted as GDI.D4, might be a GDI for other known or as yet unidentified ras-like GTP-binding proteins. Alternatively, D4 could have other biochemical functions. During murine embryogenesis, D4 transcripts are detected in yolk-sac cells, where the earliest hematopoietic precursors are found. When these precursors undergo proliferation and differentiation in vitro, a dramatic increase in D4 expression is seen. D4 probably has a significant function during the growth and development of hematopoietic precursors.

RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas.

GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIgamma, is approximately 50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIgamma functions as a GDI for CDC42 but with approximately 20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIgamma, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIalpha and GDID4 be renamed RhoGDIbeta. The new GDI should widen the scope of investigation of this important class of regulatory protein.

Targeted disruption of guanosine diphosphate-dissociation inhibitor for Rho-related proteins, GDID4: normal hematopoietic differentiation but subtle defect in superoxide production by macrophages derived from in vitro embryonal stem cell differentiation.

The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.