This is a review of approaches to the design of peptides and small molecules that selectively block the oncogenic RAS-p21 protein in ras-induced cancers. Single amino acid substitutions in this protein, at critical positions such as at Gly 12 and Gln 61, cause the protein to become oncogenic. These mutant proteins cause over 90 percent of pancreatic cancers, 40–50 percent of colon cancers and about one third of non-small cell cancers of the lung (NSCCL). RAS-p21 is a G-protein that becomes activated when it exchanges GDP for GTP. Several promising approaches have been developed that target mutant (oncogenic) RAS-p21 proteins in these different cancers. These approaches comprise: molecular simulations of mutant and wild-type proteins to identify effector domains, for which peptides can be made that selectively inhibit the oncogenic protein that include PNC-1 (ras residues 115–126), PNC-2 (ras residues 96–110) and PNC7 (ras residues 35–47); the use of contiguous RAS-p21 peptide sequences that can block ras signaling; cyclic peptides from large peptide libraries and small molecule libraries that can be identified in high throughput assays that can selectively stabilize inactive forms of RAS-p21; informatic approaches to discover peptides and small molecules that dock to specific domains of RAS-p21 that can block mitogenic signal transduction by oncogenic RAS-p21; and the use of cell-penetrating peptides (CPPs) that are attached to the variable domains of the anti-RAS-p21 inactivating monoclonal antibody, Y13 259, that selectively enters oncogenic RAS-p21-containing cancer cells, causing these cells to undergo apoptosis. Several new anti-oncogenic RAS-p21 agents, i.e., Amgen’s AMG510 and Mirati Therapeutics’ MRTX849, polycyclic aromatic compounds, have recently been FDA-approved and are already being used clinically to treat RAS-p21-induced NSCCL and colorectal carcinomas. These new drugs target the inactive form of RAS-p21 bound to GDP with G12C substitution at the critical Gly 12 residue by binding to a groove bordered by specific domains in this mutant protein into which these compounds insert, resulting in the stabilization of the inactive GDP-bound form of RAS-p21. Other peptides and small molecules have been discovered that block the G12D-RAS-p21 oncogenic protein. These agents can treat specific mutant protein-induced cancers and are excellent examples of personalized medicine. However, many oncogenic RAS-p21-induced tumors are caused by other mutations at positions 12, 13 and 61, requiring other, more general anti-oncogenic agents that are being provided using alternate methods. Ras oncogenes that encode the RAS-p21 protein  are known to be important causative factors in a large number of human cancers. Over 90 percent of pancreatic cancers, 40–50 percent of colon cancers, and about one-third of non-small-cell carcinomas of the lung express the ras oncogene RAS-p21, which transforms cells with a high efficiency, and the other is RAS-p21 which is non-transforming . Since nIn line with this conclusion, conformational studies  have indIn the next three sections, we present four ways in which specific peptides and small molecules block oncogenic RAS-p21, making them highly useful in treating RAS-p21-induced tumors. These include: 1. the identification of peptides corresponding to domains of RAS-p21 that selectively inhibit oncogenic RAS-p21 using molecular simulations and peptide library approaches in which overlapping peptide sequences from RAS-p21 are assayed for their abilities to block oncogenic RAS-p21 selectively; 2. a cyclic peptide library approach that generates peptides based on their abilities to bind to grooves in G12D-RAS-p21 that stabilize an inactive form of this protein bound to GTP, and a small molecule library screening approach for poly-aromatic compounds that can insert into a groove present specifically in the GDP-bound form of G12C-RAS-p21 that permanently locks this oncogenic form of RAS-p21 in its inactive GDP-bound state; 3. an informatic approach in which candidate peptides are computed for their abilities to dock to specific domains of wild-type and oncogenic forms of RAS-p21 and the concurrent use of a small molecule library to generate candidate analogues of GTP; and 4. a novel immunologic technique in which a peptide that targets cancer cell membranes is fused to the anti-RAS-p21 antibody Y13 259 that inactivates oncogenic RAS-p21.To determine the possible structural effects of oncogenic amino acid substitutions in RAS-p21 on its three-dimensional structure, conformational energy calculations, using two types of sampling methods, i.e., molecular dynamics  of RAF, and with GAP, SOS and PI3K proteins; the switch 2 region involving residues 55–71; and residues 96–110 and 115–126, both found to be involved the binding of RAS-p21 to SOS [Xenopus laevis oocytes. Three peptides were found to block microinjected G12V-RAS-p21 but were found to cause low or absent inhibition of insulin-induced oocyte maturation, i.e., 35–47 , 96–110  and 115–126 . All three of these peptides were further tested against progesterone-induced oocyte maturation which, in contrast to insulin, is known to occur by ras-independent pathways [Based on these results, peptides corresponding to these domains were synthesized and tested for their abilities to inhibit oncogenic RAS-p21 in Since PNC-7 is from the switch 1 domain of RAS-p21 and known to interact with RAF as a downstream target, to localize its site of action, full length c-RAF was injected into oocytes and found to induce maturation that was blocked by PNC-7. In contrast, an oncogenic RAF form, RAF BXB, that has its amino terminal regulatory domain deleted, induced oocyte maturation that was not inhibited by PNC-7 . These rEvidence has accumulated that supports this conclusion. In oocytes injected with oncogenic RAS-p21, there was a dramatic increase in the level of phosphorylated (activated) MAPK (ERK) and jun kinase (JNK) ,21, presinsulin-treated oocytes [Several of these putative targets of activated wild-type RAS-p21–RAF complexes not utilized by oncogenic RAS-p21 proteins were subsequently identified, using differential gene expression libraries for oocytes that were induced to mature either with oncogenic -ras p21 or insulin. In the wild-type RAS-p21-matured oocytes but not in the oocytes induced to mature with Val 12-RAS-p21, two dual specificity kinases, TOPK (T-Cell Origin Protein Kinase) and Dyrk1A , were expressed . The for oocytes . These fMolecular dynamics calculations on the structures of wild-type- and G12V-RAS-p21 bound to the ras-binding domain (RBD) of RAF (residues 25–131) show that there are major shifts in domain positions in the RBD, especially in a surface loop involving residues 97–110 of the RBD , as showNeither peptide was found to inhibit c-RAF-induced oocyte maturation, excluding RAF as a site of action for these peptides. In separate studies, oncogenic RAS-p21 was found to bind to jun-N-terminal kinase (JNK) that activates the transcription factor, jun see  with an Drosophila antennapedia protein that enables the peptides to be transported across cell membranes—referred to as cell-penetrating peptides or CPP [Antennapaedia protein, that cause peptides or whole proteins or other types of molecules bound to them, usually covalently but sometimes non-covalently, to cross cell and nuclear membranes. After entry into the cell, the CPPs are often cleaved, allowing for their cargo to exert their effects intracellularly. Generally, CPPs contain a high percentage of positively charged amino acid residues, such as Lys, Arg and sometimes His. Other types of CPPs contain a predominance of non-polar, hydrophobic amino acids such as Leu, Ile,Val, Phe and Met or alternating segments of polar and non-polar residues. The mechanisms by which trans-membrane penetration occurs are largely unknown but are thought to involve endocytosis, using a variety of possible mechanisms such as pore formation from interactions of the positively charged amino acids with negatively charged membrane moieties such as phospholipids and heparan sulfates and formation of inverted micelles, among a number of other proposed mechanisms.Both PNC-2 and PNC-7 were synthesized and attached on their carboxyl termini to a leader transmembrane-penetrating sequence (KKWKMRRNQFVKVQRG) from s or CPP . As discs or CPP  and refeThese two modified peptides were incubated with a normal pancreatic acinar cell line (BMRPA1) and its k-ras (G12V) transformed counterpart cell line TUC-3. The transplantation of TUC-3 but not BMRPA1 cells in nude mice resulted in metastatic pancreatic cancer with peritoneal carcinomatosis . IncubatThese results were attributed to the zygosity of the different cell lines. Both TUC-3and HT1080 cells are heterozygous for oncogenic RAS-p21. It was postulated that, if a blockade of the oncogenic pathway occurs, a normal RAS-p21 protein can utilize alternate pathways, resulting in reversion to the untransformed phenotype. Additionally, since MIA-PaCa-2 cells are homozygous for oncogenic RAS-p21 and do not have normally functioning RAS-p21, a blockade of the oncogenic pathway blocks cell growth, resulting in cell death. In all cancer cell lines treated with either peptide, JNK-P levels were continuously reduced as a function of time, as found in the oocyte studies ,25. ThesA comparison of the average structures for G12V-RAS-p21-GppNH-p and wild-type RAS-p21-GppNHp bound to the ras-binding domain (RBD) of RAF revealed a major 90° flip in a surface loop involving in the RBD at its 97–110 sequence, as shown in In separate studies, the enzyme GST-π, which converts xenobiotic compounds into thio ether conjugates of glutathione, has been found to bind to JNK-jun complexes , and oncSince a major signal transduction pathway activated by RAS-p21 involves the binding of RAS-p21 to RAF at the cell membrane, resulting in the downstream activation of MEK and ERK, agents have been devised to block this interaction. As an alternative to using conformational analysis to design ras peptides that could block this interaction as described above, the use of overlapping 12-mer (dodecapeptides) from the RAS-p21 protein was introduced. These peptides were then assayed for their abilities to serve as decoys to RAF, causing the displacement of oncogenic RAS-p21 from binding to RAF . These pVKKKIKAEIKIKMSKDGKKKKKKSRTRCTVM, called Mut3DPTSh1-Ras, where the italicized sequence is the CPP and the bold sequence is K-ras residues 169–188. This peptide was shown to compete for the binding of ras to RAF in MDA-MB-231 breast cancer cells [The RAS-p21 169–188 peptide was synthesized with a cell-penetrating peptide or CPP (leader sequence) on its amino terminus, termed Mut3DPTSh1, the whole sequence being: er cells . Unfortuer cells . This coer cells . ResultsMut3DPTSh1-Ras was then assayed for its ability to induce cancer cell death in a number of different cancer cell lines and was found to induce significant apoptosis in MDA-MB-231 breast cancer, SW626 ovarian cancer, SW480 colon cancer and HBCx17 breast cancer cell lines, but only minimally affected the cell viability of HCT-116 and HT-29 colon cancer cell lines, even though they carry K-ras mutations . PeculiaMut3DPTSh1-Ras was also evaluated on human chronic lymphocytic leukemia (CLL) cells from CLL patients as well as peripheral blood mononuclear cells from healthy donors . There wThis peptide was further tested in nude mice against hematopoietic tumor cell lines of B-cell origin, such as Burkitt’s lymphoma (Daudi cell line) and hairy cell leukemia (Jok 1 and 5.3 cell lines), the former of which the study claims is chronic lymphocytic leukemia-like . None ofThe investigators in this study attempted to contrast their Mut3DPTSh1-Ras peptide results with those concerning the peptides employed to block oncogenic RAS-p21 selectively based on the results of molecular modeling as discussed in As noted in the Introduction section, X-ray crystallographic structures of wild-type and oncogenic RAS-p21 bound to GDP and GTP suggested that there is an absence of surface-exposed clefts to which drug molecules can bind, suggesting that RAS-p21 may be “undruggable ,13”. ThiX-ray crystallographic studies on the G12D-RAS-p21 mutant protein bound to the non-hydrolyzable GTP analogue, GppNHp, indicated that a pocket  was present between the structure of residues 55–65 of the switch 2 domain and the α-2 helix involving residues 67–73 and was not present in this RAS-p21 protein bound to GDP . This fi31P-NMR studies on mutant T35S-RAS-p21 bound to GppNHp indicated that there were in fact two bound conformational forms called state 1 and state 2. In state 1, the switch 1 domain is not well-defined crystallographically and does not bind to the RBD of RAF (see above) [Prior X-ray crystallographic and e above) . In state above) . Thus, t12 encoded compounds was employed [50 = 12.5 uM) and blocked the SOS-mediated nucleotide exchange of the GDP-loaded protein. On the other hand, KD2 did not block wild-type RAS-p21 charged with GppNHp from binding to the RAF RBD, suggesting that this cyclic peptide will block only oncogenic G12D-RAS-p21 and would therefore not affect normal cells.To this end, a high diversity cDNA expression library of over 10employed , and a sThe X-ray crystal structure of KD2 bound to G12D-RAS-p21-GppNHp has been determined  and supe50 compared with that for KD2, although these cyclic peptides were found to block wild-type RAS-p21 at higher concentrations (100 Um range). In addition, bicyclic forms of the cyclic peptide, such as the disulfide bicycle in which two SH groups at positions 3 and 9 form a disulfide bridge, have been synthesized and found to block the binding of G12D-RAS-p21-GppNHp to the RAF RBD with a ten-fold reduction (for the disulfide bicycle) in the IC50 [Other cyclic peptides containing substitutions for Thr 11 in KD2 were found to have an enhanced effect on the binding of the peptides to G12D-RAS-p21-GppNHp. In particular, the substitution of an unnatural amino acid with a 4-methyl piperidine ring  for Thr 11 was found to have about a fifteen-fold decrease in its ICthe IC50 .KD2 and other cyclic peptides that specifically bind to the GTP-bound state of oncogenic G12D-RAS-p21 have thus far not been found to inhibit cancer cell growth; this is most likely due to the fact that they do not readily penetrate the cell membranes of these cells . At presAnother X-ray structure of RAS-p21 with G12C (CysH replaces Gly 12) bound to GDP has also been found to have a “druggable” pocket or groove in a similar domain to the one described above in 50 = 6 and 9 nM, respectively). It blocks the viability of most cancer cell lines with the G12C mutation except for a lung alveolar cell carcinoma (SW1573) line. It has also been found to cause a reduction in tumor burden in mice with syngeneic G12C-RAS-p21-induced tumors and has been found to eradicate these tumors in a dose-dependent manner. The structure of AMG510 bound to G12C-RAS-p21-GDP is shown in Of the compounds synthesized and tested, the one with highest affinity, in the picomolar range, for G12C-RAS-p21-GDP was produced at Amgen called AMG510 ,35,36, nThis drug appears to synergize with other anti-cancer agents such as carboplatin, MAPK inhibitors and with immunotherapeutic agents, particularly anti-PD-1 . In thisOf prime importance, as noted above, AMG-510 has received FDA approval for treating G12C-RAS-p21-induced human tumors. At present, the treatment of four patients with NSCCL resulted in 34 and 67% remission in two of the patients and stable disease in the other two patients. A follow-up scan of the second responder patient after 18 weeks of treatment revealed a complete resolution of target remissions. These findings suggest that AMG-510 is a major advance in treating ras-induced human tumors.However, further studies indicate that human colorectal carcinomas (CRC) with G12C-RAS-p21 mutations do not respond as favorably . FurtherOther compounds that act in a similar manner to AMG510 have been developed by Mirati Therapeutics including MRTX849, now called Adagrasib, shown in An informatic approach to the design of peptides and small molecules that block RAS-p21-induced cell transformation has been carried out using peptide and small molecule libraries . The X-rIn the docking procedures for each of the 19 peptides, an “affinity maturation” procedure was further performed in which the docking procedure was re-performed on each peptide in which each residue of the peptide was replaced with all other naturally occurring amino acids. Binding energies for final peptide structures bound to wild-type and mutant forms of RAS-p21 were computed with a formula that uses the number of charge–charge, charge–apolar, polar–polar, polar–apolar interactions between peptide and RAS-p21 and (presumably isolated) apolar residues and charged residues on the peptide and RAS-p21 . The intConcurrently, the ZINC database of small molecules was screened for compounds that had similar structures to GppNHp. This search was further screened by the FAF-Drugs4 server that utilizes a number of pharmacological criteria such as absorption, distribution, metabolites, half-lives, toxicities and other attributes and other “rule of thumb” criteria. Of 7305 structures screened, 474 were used for docking studies against the RAS-p21 protein structures .CRRWQWRMKK) and Retro (LGGIVSAVKKIVDFLG), were computed to have the lowest binding energies and lowest dissociation constants (Kds) to G12D- and G13D-K-RAS-p21 (in the nM range) both at the P1 loop (mutation site) and the switch 1 and 2 domains, the RAF binding sites. Other peptides were computed to have similar but slightly high binding energies and Kds. The lowest energy docked GTP analogues structure from the ZINC database (Zinc 12502230) was computed to have the highest affinity for both structures [Of the 19 final peptides, two, called LfcinB (FKructures .RRRWQWRRKK for LfcinB and LGRIVSAVKKIVRFLG for Retro (substitutions shown in red), to allow for peptide transport across cell membranes [Since the both LfcinB and Retro peptides were computed to bind in different regions of the two mutant RAS-p21 proteins, a new peptide containing both peptides was constructed in which the two peptides were connected with a presumably flexible GGGGS connecting or linker peptide. This constructed peptide, Retro-linker-LfcinB, was further modified to contain more positively charged arginine residues, i.e., FKembranes . This peembranes .These two agents were found to reduce the cell viability of both cell lines by about 60 percent at high micromolar concentrations despite their computed dissociation constants being in the nanomolar range . In viewAs a test for the specificity of the linked peptides and the GTP analogue for inhibiting oncogenic RAS-p21 in cells, their effects on the expression of two downstream targets of RAS-p21, cyclin D and catenin beta-1,encoded by the CCND1 and CTNB1 genes, respectively, were determined . CCND1 eThis rather unique approach  is basedSignificantly, the BR2 peptide was found to enter all three cancer cells but did not appear to enter any of the three normal control cell lines using immunofluorescence, although flow cytometry indicated about 25–30 percent penetrance (as opposed to 100 percent penetrance for the cancer cells) . The conH-(GGGGS)3-VL-H6 and tested for its ability to kill RAS-p21-transformed tumor cells [Using the plasmid expression of BR2 linked to the single chain variable fragment or scFv of the anti-RAS-p21 Y13–259 antibody, a fusion polypeptide or BR2-scFv Y13 259 was synthesized as BR2-Vor cells . The speThis construct was tested on HCT 116 (human colon cancer) cells which contain oncogenic substitutions for Gly 13 . The resConsidering that RAS-p21 has been considered to be “undruggable,” there have been noticeable advances in therapeutic modalities for ras-induced tumors. Careful studies of the X-ray crystal structures of different oncogenic forms of RAS-p21 bound to GDP or GTP have revealed grooves that allow for the insertion of specific (cyclic) peptides or polyaromatic small molecules such as KD2, AMG510 and MRTX 849 (and 1139) that selectively block signal transduction by specific mutations in RAS-p21. AMG510 (Sotorasib) and MRTX 849 (Anagrasib) have already been FDA-approved for the treatment of ras-induced tumors, provided that they have the specific G12C mutation and promise to be effective agents in oncological treatment. If KD2 and similar cyclic peptides can be modified to cross cell membranes, these may prove likewise to be effective agents against G12D-RAS-p21-induced cancers. In this regard, MRTX1139 is another promising drug that blocks G12D-K-RAS-p21 by freezing it in the inactive GDP state. Thus, both KD2 and MRTX1133 target G12D mutant RAS-p21, the former in an inactive GTP-bound state, the latter in the inactive GDP-bound state, suggesting that these agents can act synergistically.It should be noted that the two FDA-approved drugs, AMG510 and MRTX849, target exclusively G12C-K-RAS-p21. This oncogenic form occurs most frequently in adenocarcinomas of the lung. About 40 percent of ras-induced tumors of this type harbor this mutation . Its occOn the other hand, a significant number of solid tissue tumors that harbor ras mutations express G12V-K-RAS-p21. As a percentage of all ras-induced tumors, G12V-K-RAS-p21 occurs in about 22 percent of lung adenocarcinomas, 30 percent of colon cancers and 40 percent of pancreatic cancers . Thus, tAs discussed above, other peptide agents and approaches to design additional peptide agents, although less fully developed, show promise in being able to block ras-induced tumor growth irrespective of the mutations at position 12 and other positions in the RAS-p21 sequence. Among these peptides, PNC-1, 2 and 7 peptides linked to the cell-membrane-penetrating leader sequence block oncogenic RAS-p21-induced tumors selectively by interfering with signal transduction pathways that are unique to mutant RAS-p21. It is vital to investigate these pathways further as there appears to be “escape” pathways that allow for the wild-type RAS-p21 protein to function normally in normal cells. These agents induced either the phenotypic reversion of cancer cells heterozygous for mutant RAS-p21 or tumor cell necrosis for cancer cells that are homozygous for mutant RAS-p21. None of these peptides have a demonstrable effect on the viability or growth of untransformed cells. These peptides remain to be tested in vivo.The immunological approach involving the attachment of the anti-ras Y13 259 scFv heavy and light chain segments to the tumor cell membrane recognition peptide, BR2, seems to be another productive approach in the design of specific anti-RAS-p21 agents. Using this agent, specificity for cancer cells is provided by the BR2 peptide that allows for the anti-RAS-p21 antibody to enter ras-transformed cancer cells. Again, in vivo studies with this construct remain to be performed.Finally, much of the successful development of anti-ras peptides and small molecules has resulted from library searches for these molecules. These were involved in the development of AMG510 and MRX849 and KD2, the latter resulting from a combination of library searches, use of expression vectors to produce peptides and use of cyclization methods to produce the cyclic peptides. The screening procedures for all of these peptides and small molecules are based upon the ability of these agents to bind to specific forms of oncogenic RAS-p21. In view of the need for more general anti-ras agents, the use of informatic approaches such as the ones described in


To assess the effects of using an animated local language educational video to counsel postpartum adolescents on the benefits and use of long-acting reversible contraception (LARC).A two-stage, single group, experimental study was conducted. A total of 124 female adolescents aged < 20 years who had given birth within the last six weeks participated in the study. An educational video and a questionnaire were developed and validated. Participants were asked to fill out a questionnaire on basic demographic data, contraception preferences, and 10 true/false statements to test general contraceptive knowledge, after which they were shown an educational video in a private room. Following this, participants completed the second part of the questionnaire that assessed their knowledge using the same true/false statements and contraception preferences administered earlier.The mean age (standard deviation) of participants was 18.1 (1.5) years. Participants’ mean age (SD) at the time of their first sexual intercourse was 16.2 (1.6) years. Among the 124 participants, 31 (25%) indicated that they would use LARC before viewing the educational video. After viewing the educational video, this number increased to 48 (38.7%). The participants’ knowledge score was independently associated with their preference to select LARC .This study demonstrated that counseling tools such as animated local language educational video might effectively improve contraceptive knowledge and the preference for LARC in postpartum adolescents. An educational video regarding LARC could be used as a counseling tool for postpartum adolescents. Unintended pregnancy in teenagers is a major problem worldwide. Each year, at least 10 million unintended pregnancies occur among adolescent girls aged 15–19 in developing regions . The WorThis was a two-stage, single-group, experimental study conducted at the Family Planning and Reproductive Health Clinic of the King Chulalongkorn Memorial Hospital. During the study period, LARC, including contraceptive implant(s) and copper intrauterine devices (IUDs), became freely available through the national health care program for all adolescents. We recruited women aged < 20 years who had given birth within the last six weeks and were fluent in Thai. Women who had an absolute contraindication for one of the following contraceptives were excluded: combined/progestin oral contraceptive pills, depot medroxyprogesterone acetate (DMPA), contraceptive implant (s), and copper IUDs. Based on these criteria, a total of 124 female adolescents participated in the study.This study was conducted in accordance with the principles of the Declaration of Helsinki. This study was approved by the Ethics and Institutional Research Committees of the Faculty of Medicine (IRB No.135/57) and registered in the Thai Clinical Trials Registry (TCTR) #: TCTR20150715001. Written informed consent was obtained in Thai from participants before starting any procedures.An animated 10-min Thai language educational video was developed according to the WHO's family planning handbook . The conThe sample size was calculated based on a pilot study of 30 postpartum adolescents conducted at the same institution. To achieve 80% power at a 5% significance level, a sample size of 124 participants was required.p-values, and 95% confidence intervals (CIs) were calculated to estimate factors that were independently associated with preference to select LARC. Statistical significance was set at p < 0.05.Data analysis was performed using the IBM SPSS for Windows . Means, standard deviations (SDs), and percentages were used to describe the demographic characteristics. McNemar’s test was used to compare the selection of LARC before and after viewing the educational video. Multivariable models were analyzed, which included variables that were potential confounders based on both previous literature and univariate analysis (p ≤ 0.2) , 13. OddA total of 124 female adolescents were enrolled in the study. The study flow is shown in Fig. Approximately 75% (93/124) of the participants initially selected non-LARC, but after viewing the educational video, 21.5% (20/93) changed their minds and reported they would prefer to use LARC.. However, three participants  who initially said they would prefer to use LARC before viewing the educational video later changed their preference to a non-LARC method because they were afraid of the pain associated with the procedure. The mean (SD) contraceptive knowledge scores increased from 6.8 (1.2) points to 7.9 (1.4) points after viewing the educational video. Participants who selected LARC after viewing the educational video said they had changed their opinion due to benefits such as effectiveness  and the need for long-term contraception . Half of the participants  who did not select LARC stated that they were afraid of pain associated with the procedure. Approximately 35% (27/76) of the participants stated that they had some concerns about the side effects of LARC.p = 0.02) as described in Table p = 0.01).Univariable analysis showed that knowledge score after counseling/viewing the educational video was significantly associated with the preference to use LARC (Yes) (OR 1.42, 95% CI 1.07—1.88, Our study shows that the use of an animated 10-min Thai language educational video as an adjunct to the traditional in-office counseling method can significantly increase the knowledge about LARC and contribute to the subsequent preference for LARC methods. This finding is consistent with the results of a randomized controlled clinical trial conducted by Burapasikarin et al., which showed that an educational video on LARC can increase its utilization during the postpartum period , AnotherThe results of the multivariable analysis showed that participants’ general contraceptive knowledge regarding LARC might have affected their preference for LARC. Providing more information on LARC in layman’s terms may help increase the preference for selecting this contraceptive method. This finding corroborates previous reports that improved knowledge of LARC is associated with its increased use –17.Our analysis shows that sources of income and health insurance were not associated with LARC selection. One possible explanation is that LARC is free of charge in our setting. This result may not be generalizable as the cost can be a barrier in other locations and could decrease the preference for LARC . The resThis study successfully demonstrated the effectiveness of using an animated 10-min Thai language educational video as a counseling tool to significantly increase the preference for LARC among postpartum mothers. Additional studies with longer follow-up periods are necessary to evaluate how this educational video subsequently affects patient decision-making to choose LARC over other methods of contraception.


This article discusses perspective and frame of reference in the metaphorical description of mathematical concepts in terms of motions, gestures, and embodied actions. When a mathematical concept is described metaphorically in terms of gestures, embodied actions, or fictive motions, the motor system comes into play to ground and understand that concept. Every motion, gesture, or embodied action involves a perspective and a frame of reference. The flexibility in taking perspective and frame of reference allows people to embody a mathematical concept or idea in various ways. Based on the findings of past studies, it is suggested that the graphical representation of a mathematical concept may activate those areas of the motor system that are involved in the production of that graphical representation. This is supported by studies showing that when observers look at a painting or handwritten letters, they simulate the painter’s or writer’s hand movements during painting or writing. Likewise, the motor system can contribute to the grounding of abstract mathematical concepts, such as functions, numbers, and arithmetic operations. Beat gestures do not depict semantic content of concepts but accompany speech as a rhythmic alignment. Iconic and metaphoric gestures are often put in one category and are called representational or depictive gestures  never goes beyond 1, and f(x) oscillates between-1 and 1 are two examples in which a mathematical concept is metaphorically understood in terms of a fictive motion. Limit and continuity are two fundamental concepts in calculus that are conceptualized as dynamic concepts and fictive motions, although they have been formally defined in entirely static terms  into another representation (target representation) and understanding the target representation in terms of the base representation. This is a metaphorical process as the former representation is structured and understood in terms of the latter representation . Mathemaic terms . Crucialic terms . Describic terms . This isf(x) oscillates between-1 and 1. This could happen not only in the process of fictive motion metaphors but also for visual processing of images that contain implied motion. For example, there is some evidence suggesting that visual processing of images that contain traces of brushstroke could activate sensorimotor cortical circuits  of the observer, the embodying agent who performs the gestures or embodied actions, and frame of reference are three of these factors. This is particularly the case in understanding mathematical concepts through motion-based metaphors. When a mathematical concept or idea is metaphorically described in terms of a motion event, depending on the observer’s perspective and frame of reference of the motion, the form of gestures or embodied actions varies. In other words, from a given perspective and within a given frame of reference, that concept or idea is described by a certain form of gesture or embodied action. An example could make the point clearer. The visual representation of a function may be shown as a curve in a Cartesian coordinate system. This curve can be understood as the trace of a fictive motion. This fictive motion has a starting point and an ending point. From one perspective, the observer can see the starting point and the ending point on her left and right, respectively. From an opposite perspective, the observer would see the starting point and the ending point on her right and left, respectively. However, from another perspective, the observer would see the starting point and the ending point along a hypothetical line that goes straightly into her eye. In fact, there is an infinite number of perspectives from which the fictive motion can be seen. Therefore, an algebraic representation of a function can be embodied in terms of a fictive motion in an infinite number of ways. The key point is that various perspectives could lead to a shared understanding of a concept. Understanding a concept from a certain perspective does not mean that the concept cannot be understood from other perspectives. In fact, an individual can understand a concept from a certain perspective and at the same time simulate that concept from other perspectives.When a teacher describes a concept through gesture, she creates the visual representation from her perspective within a certain frame of reference. The student sees the visual representation as an external observer. The formation of a mental representation of that concept takes place through a mapping process. In this process, the student maps the received representation into a new representation from her perspective within her frame of reference. In fact, this process is a mapping of perspective and a mapping of frame of reference. In an interesting study, In collaborative gestures and embodied actions, mapping of perspective and mapping of frame of reference becomes more complex, as two or several individuals with different perspectives and different frames of reference interactively create a model for understanding a single concept or idea. Although that concept or idea may be embodied differently from different perspectives and within different frames of reference, these different embodied representations are isomorphic with each other, as they represent a single concept or idea. Here, what is important is the embodied representation that is shared by all collaborators (with different perspectives and different frames of reference). As two or more people collaborate through gestures to create a visual representation of a concept or an idea, they have to create a shared frame of reference. Collaborators cannot share precisely the same perspective at the same time. However, this will not create any problem in acquiring a shared understanding of a concept or an idea. The following section discusses perspective and frame of reference during the activation of affordances of objects.The concept of affordance was initially introduced by If looking at or thinking about an object  activates the motor system, it can be said that a motion event is simulated in the mind of the comprehender. This motion simulation can be done from various perspectives and within a variety of frames of reference. When an observer looks at an image showing a running man, the action of running itself or the process of observing a real running man can be simulated in the mind of the observer. In other words, the observer of the image can imagine himself as the runner or as someone who looks at a running man. This simulation can be done from a variety of perspectives. Even observing the image of a road may activate the motor system, as a road affords movement. People can walk, run, and perform many body movements on a road. In the same way that looking at a glass or the image of a glass may activate those neural substrates that are involved in the action of grasping, observing a road or the image of a road may activate those neural substrates that are involved in walking or running on a road. In more complex cases, thinking about highly abstract mathematical concepts that are metaphorically described in terms of graphical representations can activate the motor system. In such cases, it may be said that those hand movements that have produced the graphical representation of that abstract concept are simulated. The following section discusses the findings of several empirical studies that support this proposal.f(x) is described in terms of a graphical representation and described metaphorically as f(x) oscillates between-1 and 1, the oscillatory movement could be simulated in the mind of the comprehender. From the perspective of the strong version of embodiment , the comprehender may divide the graphical representation into a set of small parts and simulate each part in different stages. The division could be based on where the key features of graphical representation are located. For example, the local maximum point of the graphical representation of a function can be put in a small separate section. The simulation of those hand movements that produce this small part can help the comprehender acquire a better understanding of the local maximum point. Therefore, if a key feature of a concept is placed in one separate part of its graphical representation, the simulation of those hand movements that produce that part could help the comprehender acquire a deeper understanding of that specific feature. Manipulating some points of a graphical representation by gesture is a technique that can be used to foster the process of learning mathematical concepts. Moving a parabola along the X or Y axis in the Cartesian coordinate system, changing the parameters of the equation, and choosing another origin are some exercises that can be supported by gestures to help students acquire a deeper understanding of the equation of a parabola and its graphical representation in the Cartesian coordinate system. The aim of this article was to show how motions, gestures, and embodied actions contribute to the grounding of abstract mathematical concepts and enhance the process of mathematics teaching and learning. When a mathematical concept or idea is metaphorically described in terms of gestures, embodied actions, or a fictive motion, the motor system comes into play to ground and understand that concept or idea. This mapping process, in which an abstract concept or idea is metaphorically represented in terms of body actions and movements, can facilitate the process of understanding. It was suggested that the graphical representation of a mathematical concept may activate those areas of the motor system that are involved in producing the graphical representation. This is supported by the findings of studies that have revealed that looking at a painted image or handwritten letters can be the cause of simulating those hand movements that are involved in painting that image or writing the letters. This sort of findings emphasize the important role that is played by motions, gestures, embodied actions, and the motor system in the processing of key mathematical concepts such as functions, numbers, and arithmetic operations. Although numerous studies have been conducted in this area of research, it seems that we have a long way ahead to acquire a deeper understanding of various aspects of embodied mathematical cognition. Specifically, direct neuroscientific evidence in support of our theoretical claim is in order.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.OK-Z wrote the first draft of this manuscript. DF, ZE, and FM-R: commented on it and revised it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.