Recent technological advances, together with an increasing body of evidence from randomized trials, have placed coronary computer tomography angiography (CCTA) in the center of the diagnostic workup of patients with coronary artery disease. The method was proven reliable in the diagnosis of relevant coronary artery stenosis. Furthermore, it can identify different stages of the atherosclerotic process, including early atherosclerotic changes of the coronary vessel wall, a quality not met by other non-invasive tests. In addition, newer computational software can measure the hemodynamic relevance  of a certain stenosis. In addition, if required, information related to cardiac and valvular function can be provided with specific protocols. Importantly, recent trials have highlighted the prognostic relevance of CCTA in patients with coronary artery disease, which helped establishing CCTA as the first-line method for the diagnostic work-up of such patients in current guidelines. All this can be gathered in one relatively fast examination with minimal discomfort for the patient and, with newer machines, with very low radiation exposure. Herein, we provide an overview of the current technical aspects, indications, pitfalls, and new horizons with CCTA, providing examples from our own clinical practice. Coronary artery disease (CAD) remains an important cause of morbidity and mortality worldwide . CurrentAmong all non-invasive diagnostic methods employed in the work-up of patients with CCS, coronary computer tomography angiography (CCTA) stands out as an excellent integrative tool that provides information regarding calcium burden, presence and degree of coronary stenosis, type of atherosclerotic plaques, and functional relevance of such plaques, all in one examination ,6. In adMoreover, in the era of the ORBITA and ISCHEMIA trials, the optimal therapeutic approach for patients with significant CAD became a subject of debate, and in many situations shared decision approaches are recommended ,9. In thCT) can be employed for the evaluation of the functional significance of coronary artery stenoses [The technological advances seen in the last decades, together with a growing body of research confirming the relevance of this diagnostic method in the clinical routine, have made CCTA a first-line diagnostic tool for the diagnostic work-up of CAD patients . These dstenoses . The diagnostic performance of CCTA was also confirmed in patients with acute symptoms and suspected acute coronary syndromes (ACS) . PatientClinicalTrials.gov Identifier: NCT03920176) will provide answers to this question.The evaluation of asymptomatic patients with CCTA still remains an unelucidated question. Although the assessment of calcium scoring (CAC) was shown to significantly improve the risk stratification of such patients and a CCTA might be useful for patients with relevant cardiovascular risk factors, no prognostic data are available in this population and thus no clear recommendation can be made ,21. PossMoving coronary arteries are challenging structures to visualize non-invasively. This is related to their relatively small diameter and their continuous movement throughout the cardiac cycle. The ideal diagnostic tool for optimally depicting the coronary arteries should be able to synchronize to the cardiac cycle (“gating”), visualize structures at sub-millimeter resolution , and acquire the entire dataset at time frames of a couple dozens of milliseconds  . With thModern scanners are versatile in regard to the type of protocol employed for acquiring the data . This sh2 due to the radiation scatter effect [Radiation and contrast agent exposure constitute the main risks of a CCTA. With regard to radiation exposure, the radiation received by the patient is mainly dependent on the protocol employed and patient’s habitus. A retrospective ECG-gated protocol using a single source 64-slice CT yields radiation doses between 9 and 22 mSv ,41. A sir effect . 2 are at higher risk for developing contrast-induced nephropathy [The application of contrast agent can have adverse effects on the renal and thyroid function, and the function of these two organs should be evaluated prior to performing the CCTA. Patients with diabetes and a reduced renal function defined as eGFR < 45 mL/min/1.73 mhropathy . Newer shropathy ,50. Two hropathy ,52. An ihropathy . 2 with an intensity >130 HU in three adjacent pixels [A hallmark of atherosclerosis is the presence of calcium deposits. Thus, the identification of calcium in the coronary arteries correlates with the atherosclerotic burden of coronary arteries ,55,56. Tt pixels . The most pixels ,59. The t pixels ,61. CAC t pixels . Converst pixels . The mait pixels ,64. In tt pixels . Currentt pixels . Care muCCTA has been extensively compared to invasive coronary angiography for the diagnosis of luminal coronary stenosis ,67. The In addition, if modifiers are present, they should be added at the end of the classification. G is graft, S is stents, and V is plaque vulnerability.2) could reduce the accuracy of a CCTA scan, mainly due to reduced signal to noise ratio secondary to increased X-ray scatter. Several approaches such as increased tube voltage, very good heart rate preparation, and administration of contrast with a higher rate (up to 7 mL/s) can improve the quality of the CCTA [The relatively low positive predictive value can be traced to several pitfalls in interpreting CCTA acquisitions . Severe the CCTA . Of notethe CCTA . Dependithe CCTA . This bethe CCTA .The diagnostic value of CCTA was compared to other non-invasive functional tests in several studies. Thus, the EVINCI study evaluated 475 patients with stable chest pain with CCTA, stress perfusion imaging, and wall motion imaging by either stress echocardiography or stress cardiovascular magnetic resonance imaging and used the results of invasive coronary angiography as reference standard. CCTA provided the best accuracy in identifying hemodynamically relevant CAD (area under the curve (AUC) 0.91) compared to all other methods . These dA major advantage of CCTA over conventional ICA is its ability to non-invasively provide detailed characterization atherosclerotic plaque composition and volume . This isLow attenuation plaques are considered to mirror the vulnerable plaques characterized by necrotic lipid rich core. As CCTA can distinguish between lipid and fibrotic tissue in terms of HU, it is conceivable that analyzing plaques in terms of HU can identify those with a predominant lipid composition. In this regard, several studies used intravascular ultrasound (IVUS) as reference standard for the characterization of low attenuation plaques ,96. AlthPositive remodeling is a process that occurs in the early stages of atherosclerosis and is considered as a compensatory mechanism of the vessel to maintain a sufficient non-stenotic area in the context of atherosclerotic plaque progression . The preSpotty calcifications are more commonly seen in plaques with thin fibrous cap than in stable plaques . MicrocaThe napkin-ring sign is a form of low attenuation plaque that exhibits a heterogenous pattern of attenuation. Thus, the core shows low attenuation pattern indicative of lipid rich necrosis and the cap displays high attenuation indicative of the fibrotic cap . This tyCCTA is an excellent tool for identifying various stages of the atherosclerotic process and especially for identifying vulnerable plaques, which are considered as precursors of ACS. On the basis of the amount of calcium present, we can classify plaques as calcified, partially calcified, and non-calcified . SeveralThe role of high-risk features in predicting future cardiovascular events was confirmed in several trials. The PROMISE trial highlighted the relevance of high-risk plaque features for predicting cardiac events . In the The role of lipid-lowering therapies in the primary and secondary prevention of patients with CAD is widely recognized ,115. HMGBesides lipids, inflammation plays a crucial role in the development of the atherosclerotic lesion, especially in the early stages. Peri-coronary adipose tissue (PCAT) appears to play an active role in the atherosclerotic process . Thus, GCT values across the entire coronary tree can be derived using a commercially available software [CT to accurately identify hemodynamically relevant stenoses was tested in several studies that used invasive FFR as the reference standard. In this regard, the DISCOVER-FLOW study was the first study to find a good accuracy (AUC of 0.9) between FFRCT and invasive FFR [CT compared to standard CCTA for the identification of hemodynamically relevant lesions in 254 patients [CT measurements were used for clinical decisions, without a change in terms of prognosis between the two study arms [CT against invasively measured FFR as well as PET and SPECT and found superior AUC in the diagnosis of relevant coronary stenoses compared to standard CCTA, PET, and SPECT [CT . As clinical experience increases with this method, several approaches related to interpreting data from FFRCT should be taken into consideration. Thus, it appears that due to model constraints, very distal segments could exhibit values <0.8 without the presence of a hemodynamically relevant stenosis. In addition, values between 0.75 and 0.8 lie in a gray area and should be clarified using additional testing [The FAME studies elegantly showed that patients undergoing PCI exhibit a benefit in terms of cardiovascular endpoints only when hemodynamically relevant stenoses (FFR < 0.8) are treated ,128. Thusoftware . The maisive FFR . These rpatients . Furtherudy arms . In addind SPECT . All the testing . Other computational methods were developed to extract hemodynamic data from three-dimensional CCTA datasets. The virtual functional assessment index (vFAI) can be employed in establishing the functional relevance of a coronary stenosis. It was firstly developed from ICA datasets and essentially mimics the invasively obtained FFR values . The vFAAnother function of computational fluid dynamics is to clarify the underlying mechanical mechanisms that determine the evolution of coronary plaques ,143. ThuCT, namely, the opportunity to perform myocardial perfusion under hyperemia (CTP). The principle is similar to that employed in other non-invasive stress tests such as CMR, SPECT, or PET and is based on inducing “forced” vasodilation of the distal arterioles using hyperemia inducing agents such as adenosine, dipyridamole, or regadenoson [CT on top of standard CCTA using invasively obtained FFR as standard of reference in 147 patients. The authors found similar diagnostic accuracies of CCTA-CTP vs. CCTA-FFRCT for identifying relevant CAD. Furthermore, both CTP and FFRCT improved the diagnostic strength of CCTA alone [CCTA offers another approach in establishing the relevance of a coronary stenosis besides FFRadenoson ,150. Sevadenoson . The valadenoson . FurtherTA alone . MoreoveTA alone . p = 0.004). Although patients in the CCTA arm had more invasive procedures in the first two years, at the five-year follow-up no difference in the number of invasive procedures was noted between the two arms. The SCOT-HEART study was a landmark study for CCTA, and with regards to this study, CCTA was found to be the only non-invasive diagnostic method with the “back-up” of a randomized trial that had a positive effect on prognosis when used in the routine work-up of patients with known or suspected CAD. Another important study was the PROMISE trial [There is mounting evidence related to the prognostic value of CCTA. Perhaps the most representative analysis in this regard was the SCOT-HEART trial . In thisSE trial . In thisSE trial . Thus, pSE trial ,159. FurSE trial . In-stent restenosis (ISR) still remains a relatively rare but clinically relevant problem, even in the era of modern drug-eluting stents . The evaAlthough major advances were seen in PCI technique in the last decades, coronary artery bypass graft (CABG) surgery still remains the standard of care in advanced multi-vessel CAD . DependiThe value of CCTA in establishing the appropriate treatment strategy—interventional or operative—in patients with left main or three vessel coronary artery disease was tested in the SYNTAX III trial . A totalCoronary anomalies are a relative common finding in CCTA studies, although they affect less than 1% of the general population . In mostThe field of transcatheter interventions for valvular heart disease has seen tremendous developments in the last decades. Cardiac CT currently plays a pivotal role in planning for a transcatheter aortic valve replacement providing reliable information related to coronary anatomy, severity of the aortic valve stenosis, and accurate anatomic characterization of the aortic root . FurtherCoronary computer tomography angiography is an excellent method for the evaluation of patients with known or suspected coronary artery disease, with a large body of evidence to support this. It currently provides a vast array of information, both morphological and functional, and helps in establishing an appropriate treatment strategy in these patients.


Antimicrobial resistance is the failure of antimicrobial’s effect against the growth and multiplication of microorganisms. Imprudent and over antimicrobial use (AMU) aggravates antimicrobial resistance (AMR). Antimicrobials are massively used in animal production as compared with AMU in human health sectors. This research was done with the objective of assessing the knowledge, attitude, and practice (KAP) status of animal producers towards AMU and AMR. A Cross-sectional study design and questionnaire were conducted and both qualitative and quantitative data analyses were used. The logistic regression was used to test the effect of each predictor variable on the knowledge, attitude, and practice of the participants. Out of 571 animal producers, the majority (80.2%) of them were not knowledgeable and 85.3% of the animal producers had a negative attitude towards the AMU and AMR. Likewise, the practice of 78.5% of the animal producers were practice improperly towards AMU and AMR. All the questions that were designed to assess the KAP of the animal producers were significantly associated (P<0.05) with each respective category of KAP. The educational status of animal producers was negatively correlated (OR = 0.38) with all their knowledge, attitude, and practice of AMU and AMR, but sex has a positive correlation (OR = 2.89) with both the knowledge and practice of animal producers. In conclusion, the animal producers in the Oromia zone had unsatisfactory knowledge regarding AMU and AMR. The animal producer’s attitude and their practices were negative and improper respectively. As a result, consecutive awareness creation on both AMU and AMR is recommended and integrated AMU governance in animal production is recommended to be applied. According to the definition of the American Veterinary Medical Association , “antimiEven if different factors aggravates AMR, over-use, and misuse of antimicrobials play a major role. Indeed, AMR emergence and diffusion correspond to a selective process allowing microbial populations to adapt to their environment. Therefore, AMR is inextricably tied to all forms of antimicrobial use (AMU) and will be favored when this use is sub-optimal or widespread. In particular, avoidable practices that are recognized as key contributors to AMR are AMU in animal production for growth promotion, prophylaxis, and metaphylaxis; AMU without professional oversight; and or AMU after poor diagnostic techniques . As explAntimicrobial-resistant pathogens can circulate in populations of humans and animals, through food, water, and the environment . TransmiAs recommended by the American Veterinary Medical Association , “judiciTherefore, the objective of this study was to assess the status of the livestock animal producers’ knowledge, attitude, and practice towards AMU and AMR in the Oromia zone.The study was conducted in the Oromia zone, which has an area of 3,470 km2 with a population of 457, 278 . AccordiStructured questionnaire interviews were conducted to assess the knowledge, attitude, and practice (KAP) of AMU and AMR of animal producers in the Oromia zone. In each selected district of this zone, the largest open livestock market was chosen and the animal producers in these markets were invited for interview. All animal producers regardless of their educational status and sex were included as information sources for the study.The questionnaire had four sections and contained close-ended questions . The firet al. [A cross-sectional study was conducted on the animal producers’ KAP towards AMU and AMR in selected districts of the Oromia zone. The sample size for assessing animal producers’ KAP towards AMU and AMR was calculated based on the suggestions of Bartlett et al. . BartletWhere n = is the required sample sizep = is the percentage occurrence of a state or conditionz = is the value corresponding to level of confidence requirede = is the percentage maximum error requiredSince there was no study done about the KAP of animal producers towards AMR and AMU in the study area, 50% for p-value, 95% (1.96) for z-value, and 5% for e-value were taken. As a result, the sample size was calculated as follows.The researchers collected a higher number of samples than the calculated minimum number of samples (384). The total sample size from Bati, Dewa Chefa, Jule Timuga, and Kemissie were 182, 135, 120, and 134 respectively. As a result, the total sample size for this study was 571.The ethics concerned with this research activity were evaluated by the institutional review board (IBR) of Wollo University. For conducting this research the researchers were requested a support letter from Wollo University, school of veterinary medicine and received a letter with the reference number WU/SVM/212/10. Participants of the study were given all the information regarding the study and signed the informed consent form before they were recruited into the study.In this study, both descriptive and inferential analyses were used. After the required number of samples were collected, it was administered in Microsoft Excel 2013. The dataset was composed of 24 categorical variables . Based on Likert’s scale, dependent binary variables were produced for each category of KAP from each respective variable. The participants who correctly answer all knowledge-oriented questions were categorized as knowledgeable and those who fails to answer one or more were grouped as not knowledgeable. Likewise, all respondents whose attitude was towards the mitigation of AMR development in all attitude-based questions were grouped into a positive attitude and the respondents who had attitudes that promote AMR development were categorized into a negative attitude. In the same way, all the participants who were practiced towards the reduction of AMR were grouped into good practice and those who practice in the opposite were categorized into bad/improper practice. Each participant was assigned to the newly formed KAP categories by using a pivot table. The purpose of producing these three dependent variables was for assessing the effect of each predictor variable on KAP using logistic regression. Since 3 binary variables (KAP variables) were formed in addition to the 24 categorical variables, the dataset totally contains 27 variables The binary logistic regression was used to see which explanatory variables are predictive for the result, knowledgeable or not knowledgeable; good practice or improper practice; and positive attitude or negative attitude. Our investigation of the animal producers’ knowledge, attitude, and practice on AMU and AMR follows three steps. The first step was assessing the relationship between potential predictor variables with the animal producers’ KAP one by one. Secondly, we adjust the relationship for the potential confounding effects. Finally, we consider the possibility of interaction effects among the variables.After descriptive investigations using crosstabs, the association between KAP categories  and each predictive variable was conducted. Likelihood ratio test (LR test), and probability values from SPSS software version 26 were used to see the association between dependent variables  and predictive variables. The effect level of demographic variables on knowledge, attitude, and practice was shown by the odds ratio. Wald’s chi-square test and probability value were used to see the association between demographic variables with KAP.Half (50.3%) of the participants were semi-urban residents, and 47.7% of the respondents were in the age group of 31–40 years. There was a higher number (56.9%) of male participants than females (43.1%). In the study area, the majority (34.5%) of animal producers had reared goats and a small number (6.5%) of the participants were reared sheep while 22.4% of the respondents were reared all food animals. Food animals in the study area are ruminants , camels, and poultry only. Due to cultural and religious issues, other species of animals are not allowed to eat in the study area. The highest number (32.9%) of the participants were not educated (illiterate) and others were attended from primary to tertiary education levels . The detThe majority (80.2%) of the participants were not knowledgeable about antimicrobial use in animal productions and its negative consequence (AMR) . A largeThe majority (55.9%) of the animal producers didn’t know about rational AMU and the prevention of AMR formation. Thirty-four percent of the animal producers were not aware of AMU in animal production can able to aggravate AMR. Forty-one percent of the participants know that their imprudent use of antimicrobials in animal production can have negative externality to society in the form of AMR. The details of the animal producers’ knowledge and the statistical associations are presented in As showed in As indicated in The animal producers’ AMU and AMR practices were tested using six practice-related questions . The majThe residence, district, age, sex, educational level of the animal owners, and the animal type they reared were the demographic categorical variables. The categories under each categorical variable were taken as the dummy variables and one of them was considered as a reference dummy variable. All the dummy variables of the variable “educational level” were significantly associated (p<0.05) with all the knowledge, attitude, and practice of the participants. The educational level of animal producers was negatively correlated (OR < 1) with their KAP towards AMU and AMR . That meOut of the total participants (571) of this study, the majority (80.21%) of the animal producers were not knowledgeable about the AMU and AMR in animal production. Comparable with this finding, 70% of the livestock keepers in Ethiopia , 90% of In the present study, 55.9% of the respondents didn’t have prior knowledge about antibiotic resistance. In the opposite of this finding, a high proportion (77%) of the livestock producers in Vietnam , and 35.A large proportion (39.8%) of the participants in the present study had full information about the withdrawal period of antimicrobials from animal products. Larger than our finding, 97% of animal producers in Vietnam  were knoThe animal producers who were not knowledgeable about AMU and AMR had improper AMU and AMR practices in animal production. In the opposite of our finding, the study conducted in Vietnam  revealedIn our finding large number (85.29%) of farmers had a negative attitude (have an attitude of increasing AMR) regarding the use of antimicrobials and AMR in animal production. Lesser (51%) than the present study, the layer and pig farmers in Thailand  had a neIn the present study, 28.9% of the animal producers disagreed that their irrational AMU in animal production could affect the health of others in the form of AMR. Unlike our finding, 63.7% of the dairy farmers in Swiss  and 95% In the present study, a large proportion (44.1%) of the animal producers disagreed with the contribution of alternative AMU in animal production  in the reduction of AMR formation. On the contrary, 96% of the livestock producers in Vietnam  and 82% In our study, 23.1% of the farmers didn’t believe that using antimicrobials in animal production is abusing antimicrobials. On the contrary, a large proportion (69.4%) of the livestock farmers in Vietnam  believedThe AMU and AMR practices of the animal producers in this study were improper  (78.46%). Comparable with this finding, 72.3% of the livestock farmers had improper practices in the study conducted in Ethiopia . Based oIn the present study, the majority (53.9%) of the animal producers had a trend of using prescriptions before purchasing antimicrobials. Comparable to our finding, 48% of turkey and rabbit farmers in Italy , 48% of In the current study, a large proportion (41.5%) of the animal producers have used antimicrobials for treatment purposes. Likewise, a high percentage (69%) of the livestock farmers in Vietnam , 38% of In the present study, the majority (56.7%) of the farmers had the trend of using AMU guidelines/regulations. Higher than the current finding, 70% of the livestock farmers in Ethiopia , 93% of The educational status of the animal producers in this study was significantly associated (p<0.05) with their knowledge, attitude, and practice towards AMU and AMR. The present finding is comparable with the studies conducted in Bingol, Turkey , ShandonThe dummy variables of educational level  are negatively correlated with the knowledge of animal producers . In the opposite to this finding, the study done in Vietnam  showed tIn the present study, the sex of animal producers is significantly associated (p<0.05) with their knowledge and practice towards AMU and AMR. The sex of the animal producers in the current study was positively correlated with both farmer’s knowledge  and practice . The reason why male animal producers were more knowledgeable towards AMU and AMR than females might be due to the exposure of males to meetings, training, and media in the study area. Participation of females in meetings, training, and others is not culturally common in the study area. Likewise, the study conducted in Shandong province, China  showed tFrom this study, the mitigation of AMR formation and effective governance of AMU along the animal origin food chain requires a thorough understanding of the animal producers’ knowledge, attitude, and practice towards AMU and AMR in animal production, using a One Health approach. The animal producers in the Oromia zone had unsatisfactory knowledge of AMU and AMR. Alike their knowledge their attitude and practice regarding AMU and AMR were majorly negative and improper respectively. Overall, the practical recommendations based on the findings from the study include:Continuous awareness creation should be done to animal producers and the general community.Implementable and inclusive AMU policy formulation and enforcement are recommended.Integrated AMU governance among all individuals, sectors, and actors regardless of their geographic locations and economic status should be applied.All GOs and NGOs should have a special focus to control imprudent AMU and AMR in animal production.The study might be liable to social desirability. In addition, the nature of the study design  can influence the cause and effect relationship of the predictor variables and the dependent binary variables  of the animal producers.S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Successful pregnancy establishment in mammals depends on proper embryo-maternal communication. Prokineticin 1 (PROK1) is a secretory protein that exerts pleiotropic functions in various tissues. Despite the studies that have primarily been performed with human cell lines and mice, the function of PROK1 in trophoblasts has still not been fully elucidated. Hence, the aim of this study was to establish the role of PROK1 in trophoblasts during implantation and placentation. Prokineticin 1 mRNA was elevated in porcine trophoblasts during implantation and the early placentation period. Furthermore, we reveal that PROK1–PROKR1 signaling induces the expression of genes involved in the regulation of angiogenesis, immunological response, trophoblast cell adhesion, invasion, and proliferation, as well as stimulating phosphorylation of MAPK and PTK2. Ingenuity Pathway Analysis identified the aforementioned and also other functions associated with PROK1-regulated genes/proteins, such as cell-to-cell contact, epithelial tissue differentiation, Ca2+ release, lipid synthesis, and chemotaxis. We also showed evidence that PROK1 acting via PROKR1 increased trophoblast cell proliferation and adhesion. The PROK1-stimulated cell proliferation was mediated by PI3K/AKT/mTOR, MAPK, and cAMP, whereas adhesion was mediated by MAPK and/or PI3K/AKT signaling pathways. Concluding, our study suggests that PROK1 plays a pleiotropic role in trophoblast function during implantation and early placentation. At this time, porcine conceptuses secrete increased levels of estrogens  and undergo elongation from spherical forms to tubular forms and finally to filamentous forms2. This transformation allows for an increased contact surface between the conceptus trophoblast cells (trophectoderm) and uterine luminal epithelium (LE) which facilitates conceptus attachment to the endometrium. This, in turn, is the first and critical step for implantation in all mammalian species. In pigs, conceptus attachment occurs between days 14 and 19 of pregnancy1. Implantation involves pregnancy-specific interactions between integrins and extracellular matrix proteins (ECM) which contribute to stable adhesion to the endometrium4. Integrin binding in the LE and trophoblast tissue is mediated by secreted phosphoprotein 1 (SPP1/osteopontin)5.In all placental mammals, including pigs, the most important processes during early pregnancy are the development of endometrial receptivity, maternal recognition of pregnancy, implantation, and placentation. The maternal recognition of pregnancy, which in pigs is initiated on days 11–12 of pregnancy, is a process during which the conceptuses (embryos with associated membranes) signal their presence to the maternal system and prolong the lifespan of the corpus luteum (CL)3. During this time, the expression and secretion of inflammatory mediators, such as interferon γ (IFNG) and δ (IFND), prostaglandins F2α (PGF2α) and E2 (PGE2), tumor necrosis factor α (TNF), interleukin 1B (IL1B), and cytokines from the interleukin 6 family, namely, interleukin 6 (IL6), interleukin 11 (IL11), and LIF interleukin 6 family cytokine (LIF), by the porcine endometrium and conceptus are increased6.Implantation and the establishment of pregnancy in pigs, as well as in other mammalian species, display hallmark signs of inflammation1. One of the most vital processes for placenta development is the formation of the local blood circulation system at the embryo–maternal interface, i.e., angiogenesis. Recent findings indicate that prokineticin 1 (PROK1) may serve as a novel angiogenic factor involved in the regulation of implantation and placentation9. This molecule  is a 11-kDa protein and its expression in humans has been reported, among others in the endometrium, placenta, trophoblast, ovary and fallopian tube10. Prokineticin 1 acts through two related receptors, namely, PROKR1 and PROKR2. Interestingly, in human trophoblast cells, PROKR1 mRNA expression has been found to be 80 times higher when compared to the expression of PROKR2 mRNA11. Besides angiogenesis, PROK1 may regulate a number of biological processes, including cell–cell contact, cell migration, proliferation, and inflammatory response12. Our recent study revealed that PROK1 could be an embryonic signal mediator which, acting via PROKR1, stimulates angiogenesis in the porcine endometrium during implantation and the early placentation period7. Moreover, PROK1 regulates the endometrial expression of pregnancy-related genes and proteins, as well as the prostaglandin and cytokine secretion in pigs8. In humans, the peak of PROK1 and PROKR1 expression occurs in placenta at 8–11 weeks of gestation and then gradually decreases by the end of the first-trimester of pregnancy11. Interestingly, impaired PROK1 signaling has been linked with placental pathologies such as preeclampsia and intrauterine growth restriction12.The implantation is followed by placentation. Pigs have a true epitheliochorial placenta in which the luminal epithelium remains intact throughout the entire pregnancy . The main function of the placenta is the fetal–maternal exchange of gases, nutrients, hormones, cytokines and other regulatory molecules that support the growth and development of the fetus14 or mice15. Thus, the function of PROK1 in conceptuses/trophoblasts during early pregnancy has not been fully elucidated yet. Based on our recent data and previous studies, we hypothesize that during early pregnancy, PROK1, acting through its receptor, contributes to trophoblast attachment/implantation and early placentation. Therefore, the objectives of the present study were the following: (1) to determine the gene expression profiles of PROK1 and PROKR1 in porcine conceptuses/trophoblasts during early pregnancy; (2) to study the expression profiles of pregnancy-related genes such as SPP1, biglycan (BGN), fibroblast growth factor 9 (FGF9), and IL11 in porcine trophoblasts; (3) to identify pregnancy-related genes activated by PROK1–PROKR1 interaction in trophoblasts; and (4) to elucidate the effect of PROK1 on porcine trophoblast cell proliferation and adhesion to ECM proteins and to identify the signaling pathways involved in these processes.Studies on PROK1 function during implantation and placentation have mainly been performed on human cell lines  and rarely with ex vivo placenta tissuesPROK1, PROKR1, BGN, IL11, and SPP1 mRNA we used conceptus/trophoblast tissue collected from pregnant gilts between 10 and 25 days of gestation. The expression of the PROK1 gene in the conceptus (days 10–17) and trophoblast (days 18–25) increased gradually in the studied stages of pregnancy. Trophoblast expression of PROK1 mRNA was significantly upregulated on days 17–19 (implantation stage) compared to days 10–12 (preimplantation stage) of pregnancy. The abundance of PROK1 mRNA reached the highest levels on days 20–25 of pregnancy (the onset of placenta development) compared to earlier stages . Trophoblast expression of these genes was significantly upregulated on days 14–16, and 17–19, compared to days 10–12 of pregnancy and reached the highest levels on days 20–25 of pregnancy  for 24 h in the presence/absence of a PROKR1 antagonist .FGF2) and vascular endothelial growth factor A (VEGFA) genes  gene  gene , cadherin 13 (CDH13), fms-related receptor tyrosine kinase 1 , kinase insert domain receptor , nuclear factor of activated T cells 2 (NFATC2) and regulator of calcineurin 1 (RCAN1) genes , and TNF genes  gene  and Wnt family member 7A (WNT7A) genes , FGF9, matrix metallopeptidase 9 (MMP9), mucin 4 (MUC4), SPP1, transforming growth factor beta 3 (TGFB3), and Wnt family member 4 (WNT4) genes  but not by the day of pregnancy on which the cells were collected. PROK1 induced the phosphorylation of MAPK1/3 and PTK2 proteins in trophoblast cells collected on days 15 and 20 of pregnancy  and protein tyrosine kinase 2  phosphorylation we used an in vitro model of porcine trophoblast cells. Primary trophoblast cells collected on days 15 and 20 of pregnancy were incubated with the PROK1 (40 nM) for 10 min in the presence/absence of a PROKR1 antagonist . Phosphorylation of MAPK1/3 and PTK2 in porcine trophoblast cells was affected by treatment with PROK1  for 24 h in the presence/absence of a PROKR1 antagonist  and inhibitors of MAPK , phosphoinositide 3-kinase —for adhesion and proliferation assay; and inhibitors of protein kinase B , mammalian target of rapamycin , and cAMP synthesis SQ 22536; 25 μM)—only for proliferation assay. PROK1 after 24 h of incubation increased the adhesion to fibronectin by the trophoblast cells collected on days 15 and 20 of pregnancy  such as: cell attachment, migration and invasion, cell–cell contact, cell viability, organismal death, apoptosis, the differentiation of epithelial tissue, MAPKKK cascade, release of Ca2+, angiogenesis, vasculogenesis, inflammatory response, the recruitment of blood cells, chemotaxis, and the synthesis of lipids 16.Although the function of genes or protein selected for expression studies has been already known it is difficult to make a clear conclusion about the involvement of PROK1 in processes in which PROK1-regulated genes are involved. It is related to fact that some genes may be involved in stimulation whereas other genes—in inhibition of particular process. Thus, to minimize the bias the whole gene set was analyzed using Ingenuity Pathway Analysis (IPA) software to investigate the processes potentially related to PROK1 action. Functional annotation analyses performed using IPA revealed biological function categories with 4 or more molecules/genes significantly regulated by PROK1  when compared with late gestation (days 14.5 and 17.5)15. In contrast to studies in mice and humans, PROKR1 mRNA expression in porcine conceptuses remained constant during the studied stages of pregnancy. The upregulation of PROK1 mRNA abundance in porcine trophoblasts during implantation and the early placentation period corresponds with an increased expression of PROK1 and its receptor in the endometrium7, which implies that PROK1 may be an important factor during conceptus development and pregnancy establishment.We demonstrated, that the expression of 19; however, there is no information regarding such mechanism in trophoblast cells. Herein, we demonstrated that PROK1, acting through PROKR1 in the porcine trophoblast, stimulated the expression of genes involved in angiogenesis, immunological response, trophoblast cell adhesion, invasion, and proliferation, in addition to activating signaling pathways related to cell adhesion and proliferation. Moreover, the results from the Ingenuity Pathway Analysis linked the PROK1-affected expression of genes/proteins to processes and functions such as cell attachment, migration and invasion, cell–cell contact, cell viability, organismal death, apoptosis, the differentiation of epithelial tissue, MAPKKK cascade, release of Ca2+, angiogenesis, vasculogenesis, inflammatory response, the recruitment of blood cells, chemotaxis, and the synthesis of lipids. These results indicate a pleiotropic role for PROK1 in trophoblast functions. It has been well documented that gene and/or protein expressions of PROK1-regulated factors such as FGF220, IFNG21, IGF122, IL1B24, MMP925, TBFB324, WNT426, VEGFA, and FLT127 are elevated in porcine trophoblasts during implantation and early pregnancy. Moreover, other PROK1-induced genes or proteins determined in the present study – namely, ANGPT1, and ANGPT228, BGN25, CDH126, MAPK1/MAPK3, and PTK229, IL6, LIF, LIFR30, SPP131, TNF32, KDR32, WNT5A, and WNT7A26 have been shown to be expressed and play important roles in porcine trophoblasts or placenta during early pregnancy. In present study, we demonstrated that the expression of BGN, IL11 and SPP1 mRNA increased gradually during conceptus/trophoblast development and reached the highest levels at the onset of placentation. Moreover, the present findings indicated that the expression of FGF9 increased since the beginning of implantation, and remained higher at the onset of placentation when compared to the preimplantation stage.Proper communication between the conceptus and uterus is essential for successful pregnancy establishment and development. It has been shown that PROK1 regulates the expression of numerous pregnancy-related genes in the human and porcine endometrium33; however, recent studies have shown an emerging role of PROK1 during pregnancy12. Our previous study has demonstrated that PROK1 is involved in endometrial angiogenesis by the stimulating endothelial cell proliferation and network formation and by increasing the secretion of VEGFA and PGF2α7. In the present study, we showed that PROK1, acting through PROKR1 in trophoblast cells, regulated the gene expression of several angiogenic factors and their receptors, including ANGPT1, ANGPT2, CCN2, CDH13, FGF2, FLT1, KDR, NFATC2, RCAN1, and VEGFA. These findings are in agreement with our previous results indicating that PROK1 increases ANGPT1, ANGPT2, CCN2, CDH13, and NFATC2 mRNA expression in the porcine endometrium8. FGF2 and VEGFA and its receptors are well known to play an important role in peri-implantation vascular events and conceptus development in pigs34. During angiogenesis angiopoietins, CCN2, CDH13, FGF2, NFATC2, and RCAN1 acting together with VEGFA and their relevant receptors, are involved in endothelial cell survival, migration, adhesion, and vessel remodeling37. The aforementioned studies and the findings of the present study confirm our assumption that PROK1–PROKR1 signaling can participate in angiogenesis during placenta development. Moreover, the PROK1-induced expression of ANGPT1, ANGPT2, CCN2, FGF2, FLT1, KDR and VEGFA may be involved in the regulation of trophoblast cell proliferation, migration, and invasion42.In mammalian species, the placenta is one of the most vascularized organs in the whole organism. In the formation of the capillary network in the placenta, one of the most important processes is angiogenesis, i.e., the process of new blood vessel formation from pre-existing blood vessels. Angiogenesis is tightly controlled by numerous angiogenic regulators, the most common of which are VEGFA, FGF2, ANGPT1, and ANGPT24. Moreover, during early pregnancy in all placental mammals, including pigs, there is an extensive recruitment of lymphocytes, particularly natural killer cells, dendritic cells, and macrophages at the maternal–fetal interface43. Therefore, implantation is associated with signs of inflammation and increased expression of inflammatory mediators such as cytokines, growth factors, and lipids3. In the present study, we showed that PROK1–PROKR1 signaling increased the gene expression of cytokines IFNG, IL1B, IL6, IL11, LIF, LIFR, and TNF in trophoblast. Our findings are consistent with previous reports, demonstrating that PROK1 increases IFNG, IL6, LIF, LIFR, and TNF mRNA expression in the porcine endometrium8. Prokineticin 1-stimulated cytokines such as IL1B, TNF and cytokines belonging to the IL 6 family , together with their receptors, are important for pregnancy establishment and placentation45. These cytokines are involved in trophectoderm remodeling, proliferation, attachment to the endometrium, and the regulation of trophoblast cell migration and invasion44. Moreover, during implantation, IL1B and IFNG play important roles in the regulation of immunotolerance at the maternal–fetal interface46. In pigs, IFNG stimulates endometrial expression of IFN-regulated genes that are involved in the release of histotroph, cell proliferation, cell development, and attachment of the conceptus46. The present results confirmed our hypothesis that PROK1–PROKR1 signaling contributes to the regulation of immune response and the creation of proinflammatory environment in uterus by increasing the expression of cytokines in the porcine trophoblast during attachment and early placentation.Successful pregnancy establishment and development requires a precise immunological balance between the conceptus carrying paternal antigens and the maternal immune system. The modulation of the immune system prevents conceptus rejection by the maternal organism and is vital for conceptus growth, survival, and implantation13 and adhesion19. Using specific inhibitors, we demonstrated that PROK1–PROKR1 signaling elevated trophoblast cell proliferation by activating MAPK, PI3K/AKT/mTOR kinases, and cAMP pathways on days 15 and 20 of pregnancy. Trophoblast cell adhesion was induced by activation of MAPK (on days 15 and 20 of pregnancy) and PI3K/AKT signaling pathways (on day 20 of pregnancy). We also indicated that in the porcine trophoblast cells PROK1 induced MAPK1/3 and PTK2 phosphorylation and stimulated the expression of genes involved in the regulation of trophoblast cell adhesion, invasion, and proliferation, such as BGN, CDH1, FGF9, IGF1, MMP9, MUC4, SPP1, TGFB3, WNT4, WNT5A, and WNT7A. Our results are consistent with previous studies indicating that PROK1 increases MAPK1/3 phosphorylation in the BeWo choriocarcinoma cell line13. Moreover, the present results are in agreement with our previous findings revealing that PROK1 stimulates MAPK and PTK2 phosphorylation and FGF9, MUC4, SPP1, and TGFB3 gene expression in the porcine endometrium8.Proper conceptus development and placentation also depend on cell differentiation, proliferation, adhesion, migration, and invasion. The present study reveals that PROK1 increases the proliferation of porcine trophoblast cells on days 15 and 20 of pregnancy and stimulates their adhesion to the ECM. Moreover, using a specific PROKR1 antagonist, we demonstrated that the stimulatory effects of PROK1 on conceptus cell proliferation and adhesion depended on PROK1–PROKR1 signaling. Our results are in agreement with previous studies using BeWo and JEG-3 choriocarcinoma lines suggesting that PROK1 promotes human trophoblast cell proliferationBGN, CDH1, FGF9, IGF1, MMP9, MUC4, SPP1, TGFB3, WNT4, WNT5A, and WNT7A, which are implicated to participate in regulating processes such as cell differentiation, proliferation, adhesion, migration, or invasion during conceptus development and implantation48. In addition, in humans, TGFB3, CDH1, and WNT5A are involved in the inhibition of trophoblast invasiveness51. Moreover, in the porcine endometrium, MUC4 gene expression is upregulated during early pregnancy and is supposed to be responsible for modulating the proteolytic capacity of the conceptus and protecting the LE from trophoblast invasion52. It can be suggested that the regulation of TGFB3, CDH1, MUC4, and WNT5A expression via PROK1 in trophoblasts may be related to the non-invasive type of placentation in pigs. Another important gene induced by PROK1 is MMP9, which has been demonstrated to regulate invasion and the migration of human trophoblast cells53. Our previous studies show evidence that PGF2α stimulates MMP9 mRNA expression in the porcine endometrium, suggesting its involvement in ECM remodeling54. In porcine conceptuses, MMP9 gene expression increases gradually and reaches the highest level on days 20–25 of pregnancy. This in turn suggests the participation of MMP9 in trophoblast ECM remodeling during placentation. Secreted phosphoprotein 1 binds to cell surface integrins that promote cell–cell and cell–ECM adhesion which further leads to cytoskeletal reorganization and the stabilization of adhesion1. The present study indicated that expression of SPP1 mRNA in porcine conceptuses increased gradually and reached the highest level on days 20–25 of pregnancy. Interestingly, SPP1 has also been reported to regulate the fetal/placental development in pigs and has been suggested to play an important role in conceptus attachment and placentation31. The aforementioned findings, together with our present results, confirm our assumption that PROK1, acting by PROKR1, can stimulate the proliferation and adhesion of porcine trophoblast cells and regulate trophoblast cell invasiveness. In addition, it may play an important role in conceptus development.PROK1 induced the trophoblast expression of the genes PROK1 mRNA increases gradually and reaches its highest level on days 20–25 of pregnancy (beginning at placentation) in pigs. Our study indicates that PROK1, acting by PROKR1, regulates the expression of the pregnancy-related genes ANGPT1, ANGPT2, CCN2, CDH13, FGF2, FLT1, KDR, NFATC2, RCAN1, VEGFA, IFNG, IL1B, IL6, IL11, LIF, LIFR, TNF, BGN, CDH1, FGF9, IGF1, MMP9, MUC4, SPP1, TGFB3, WNT4, WMT5A, and WNT7A which are involved in processes such as angiogenesis, immunological response, trophoblast cell adhesion, invasion, migration and proliferation, cell–cell contact, apoptosis, the differentiation of epithelial tissue, MAPKKK cascade, the release of Ca2+, angiogenesis, vasculogenesis, inflammatory response, the recruitment of blood cells, chemotaxis, and the synthesis of lipid , days 14–16 , days 17–19  and days 20–25 . Dissections of trophoblast tissues from conceptuses were performed since day 18 post mating29. Therefore, expression of studied genes was analyzed in 10- to 17-day conceptuses and 18- to 25-day trophoblast tissues. Collected conceptuses/trophoblasts were snap-frozen in liquid nitrogen and stored at − 80 °C. The expression profiles of PROK1, PROKR1, BGN, FGF9, IL11, and SPP1 genes in collected conceptuses/trophoblasts samples were determined using real-time RT-PCR as described in the subchapter Real-time RT-PCR.Peripubertal crossbred gilts of similar age (6–6.5 months) and genetic background, after two natural estrous cycles were bred at the onset of estrus (day 0) then 12 h and 24 h later. Pregnant gilts were slaughtered at the local abattoir between days 10 and 25 of pregnancy. Samples were collected as described previously29 from conceptuses recovered from uteri of pregnant gilts on day 15 (n = 10) and 20 of pregnancy (n = 7). To assess the purity of cell cultures, isolated cells were stained for the presence of the trophoblast marker—cytokeratin as described previously25. Purity of cell cultures was assessed at 98%. After isolation, cells were plated onto plates and cultured in medium M199  containing 10% of (v/v) newborn calf serum  and antibiotics at 37 °C in a humidified atmosphere containing 95% air and 5% CO2.Porcine trophoblast cells were isolated as described previously6 cells/well in 6-well plates. After reaching 80% confluence, the cells were incubated with medium M199 supplemented with 1% (v/v) NCS and antibiotics containing the control vehicle, 0.01% ethanol) or PROK1  for 24 h in the presence/absence of a PROKR1 antagonist . The PC7 antagonist was kindly gifted by Dr. Gianfranco Balboni. The doses of PROK1 and PC7 were chosen based on previous studies8. After incubation, the cells were lysed in Fenozol buffer  and stored at − 80 °C until gene expression analyses. Details related to gene expression analyses are described in the subchapter Real-time RT-PCR.The effect of PROK1 on the expression of selected pregnancy-related genes was studied using cultured porcine trophoblast cells collected from day 15 and 20 of pregnancy. The primary trophoblast cells were seeded at 0.5 × 10Isolation and culture of porcine primary trophoblast cells in Experiment 2.To determine whether PROK1 may affect selected signaling pathways, adhesion and proliferation of the porcine trophoblast cells, an in vitro model was used. Porcine primary trophoblast cells on days 15 and 20 of pregnancy were isolated as described in the subchapter 6 cells/well in 6-well plates. After reaching 80% confluence, the cells were treated with medium M199 containing control  or PROK1 (40 nM) for 10 min in the presence/absence of a PROKR1 antagonist . Following treatment, the cells were lysed in RIPA buffer  and stored at -30 °C until phosphorylation analyses by Western blot as described below.The effects of PROK1 on PTK2, and MAPK phosphorylation were studied using porcine trophoblast cells seeded at 0.5 × 106 cells/well) with medium M199 supplemented with 1% (v/v) NCS and antibiotics containing vehicle (0.01% ethanol) or PROK1 (40 nM) in the in the presence/absence of PC7 (1 µM), MAPK inhibitor  or PI3K/AKT kinase inhibitor  for 24 h at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. After treatment, the cells were detached from the 6-well plate, and 100 μL (0.4 × 105 cells/well) of each cell suspension was transferred onto Millicoat Cell Adhesion Strips coated with human fibronectin  and incubated for 2 h at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. Following incubation, cell adhesion was measured using a colorimetric method accordingly to the manufacturer’s protocol. Experiments were repeated seven times (for each day 15 and 20 of pregnancy) in duplicates. The fold difference was determined by dividing the absorbance displayed by PROK1-treated cells by the absorbance displayed by control-treated cells.Confluent cells were incubated in 6-well plates  were incubated for 24 h with vehicle (0.01% ethanol) or PROK1 (40 nM) in the presence/absence of prokineticin receptor 1 antagonist , MAPK inhibitor , PI3K/AKT kinase inhibitor , AKT kinase inhibitor , cAMP synthesis inhibitor  and mTOR kinase inhibitor . After treatment, proliferation was assessed using the Cell Titer 96 Aqueous One Solution Proliferation Reagent . Experiments were performed in triplicates and repeated nine times or seven times . The fold difference was calculated by dividing the absorbance obtained for PROK1-treated cells by the absorbance obtained for control-treated cells.A proliferation assay was performed as described previously55. The mRNA expression of PROKR1, VEGFA, FLT1, KDR, LIF, LIFR, WNT4, WNT5A and WNT7A were analyzed using Power SYBR Green  with oligonucleotide primers  according to the manufacturer’s protocol (Supplementary Table http://ewindup.info/miner/)56. The stability of the reference genes  was examined by using the statistical algorithm Norm Finder 2.057. Gene expression values in conceptus/trophoblast tissues collected from pregnant gilts (days 10–25) was normalized against PPIA expression. Gene expression values in trophoblast cells isolated from gilts on days 15 and 20 of pregnancy after in vitro experiment was normalized against the geometric mean of ACTB and PPIA.Isolation of RNA, reverse transcription, and real-time PCR for samples from Experiment 1 and 2 were performed as described previously58. Briefly, 50 μg of total protein extracts were dissolved in SDS gel loading buffer , 4% SDS, 20% glycerol, and 2% β-mercaptoethanol), heated at 95 °C for 4 min, and separated on 8% Tris–Glycine SDS-PAGE. Separated proteins were electroblotted onto a 0.4 µm polyvinylidene fluoride membranes. The membranes were incubated overnight with primary antibodies  was evaluated by Western blot analyses as described earlierTo identify the signaling pathways, molecular networks, and biological functions of PROK1-regulated genes in the porcine trophoblast, Ingenuity Pathway Analysis  was used. Processes and functions potentially affected by PROK1 were identified using "Diseases and biofunctions" analysis and visualized in Path Designer tool. Results were summarized in graphic format.P < 0.05 was considered statistically significant.Statistical analyses were performed by GraphPad Prism software version 9.0 . All data were tested for normality and homoscedasticity. Before using parametric tests, log-transformation of data was performed when needed. The results of Experiment 1 were analyzed by one-way analysis of variance (ANOVA), followed by the Bonferroni post-hoc test. The effects of PROK1 on the expression of the selected pregnancy-related genes (Experiment 2), the phosphorylation of MAPK 1/3 and PTK2 (Experiment 3) were evaluated using two-way ANOVA with the Bonferroni post-hoc test. The statistical significance of the effect of PROK1 on trophoblast cell proliferation and adhesion was evaluated using one-way ANOVA, followed by Bonferroni post-hoc test. The materials collected were reviewed and accepted following the guidelines of the Local Ethics Committee for Experiments on Animals of University of Warmia and Mazury in Olsztyn, Poland, and conducted in accordance with the national guidelines for agricultural animal care, and the ARRIVE guidelines.Supplementary Information 1.Supplementary Table 3.


The sustained release of a water-soluble drug is always a key and important issue in pharmaceutics. In this study, using cellulose acetate (CA) as a biomacromolecular matrix, core-sheath nanofibers were developed for providing a sustained release of a model drug—metformin hydrochloride (MET). The core–sheath nanofibers were fabricated using modified tri-axial electrospinning, in which a detachable homemade spinneret was explored. A process—nanostructure–performance relationship was demonstrated through a series of characterizations. The prepared nanofibers F2 could release 95% of the loaded MET through a time period of 23.4 h and had no initial burst effect. The successful sustained release performances of MET can be attributed to the following factors: (1) the reasonable application of insoluble CA as the filament-forming carrier, which determined that the drug was released through a diffusion manner; (2) the core–sheath nanostructure provided the possibility of both encapsulating the drug completely and realizing the heterogeneous distributions of MET in the nanofibers with a higher drug load core than the sheath; (3) the thickness of the sheath sections were able to be exploited for further manipulating a better drug extended release performance. The mechanisms for manipulating the drug sustained release behaviors are proposed. The present proof-of-concept protocols can pave a new way to develop many novel biomolecule-based nanostructures for extending the release of water-soluble drugs. Sustained release, one of the most popular and common release profiles, refers to the slow release of the loaded drugs from its dosage forms in the specified release medium after oral administration ,2,3,4,5.During the past several decades, various raw materials and techniques have been expanded into the field of drug delivery, an interdisciplinary application field ,15,16,17In this nano era, more and more nanotechnologies are being introduced into pharmaceutics as new nanopharmaceutical methods ,38,39,40The sustained release of drugs from their electrospun nanofibers has developed into a strong branch today. Different types of electrospinning methods, new kinds of filament-forming polymeric matrices, and composites and hybrids comprising both polymers and inorganic nanoparticles are successively reported ,56. In lMET has the effect of lowering the blood pressure, blood fat, and weight control. It is the first choice for type II-diabetes with obesity and insulin resistance. It has a synergistic effect with insulin or sulfonylureas oral hypoglycemic agents, and can be used for the treatment of patients with insulin resistance, and can also be used for the treatment of polycystic ovary syndrome and non-alcoholic fatty liver disease ,66. It iBased on the above-mentioned background, in the present work, a new kind of core–sheath CA nanofiber is fabricated for providing a sustained release of metformin hydrochloride (MET), a popular water-soluble drug model ,64. ModiMW = 100,000, with an acetylation degree of 39.8%) were obtained from Sigma-Aldrich Co., Shanghai, China. The organic solvents, including acetone, N,N-dimethyl acetamide (DMAc), and ethanol, were purchased from the Shanghai 1st Shi-Ji Factory. Color markers for optimizing the working processes, i.e., basic fuchsin and methylene blue, were bought from Sinopharm Chem. Co., Shanghai, China. Water was doubly distilled. All chemicals were of analytical grade.Both MET and CA (w/v) MET was dissolved into the solvent mixture for preparing the middle and inner fluids, respectively. The outer fluid was the pure solvent mixture. The distance between the collector and the tip of spinneret was 20 cm during all the preparations. Two kinds of core–sheath fibers (denoted as F1 and F2) were prepared with various middle-core fluid flow rates. Meanwhile, two homogeneous nanofibers (denoted as F3 and F4) were prepared from the modified coaxial processes. The key experimental parameters are included in Three spinning solutions were prepared for implementing the tri-axial electrospinning. A mixture comprising acetone, DMAc, and ethanol in a volume ratio of 4:1:1 was exploited for preparing all three solutions. A ratio of 3% and 8% , a fiber collector, and three fluid drivers . The environmental temperature was 21 ± 4 °C, and the humidity was 47 ± 5%. All of the prepared nanofibers were stored in a vacuum drying oven to reach a constant weight before characterizations.A field emission scanning microscope  was utilized to assess the nanofibers’ morphology. The fiber samples were gold sputter-coated for 1 min under nitrogen atmosphere. The nanofibers’ structures were evaluated through a transmission microscope .−1 to 4000 cm−1, with a resolution of 1 cm−1 to disclose the compatibility of components within the nano products.An X-ray diffractometer  was utilized to measure the physical state of samples from 5° to 60° of 2θ under a voltage and current of 40 kV and 30 mA, respectively. The scan speed was 0.02 degree/s. A Spectrum 100 spectrometer  was exploited to conduct the Fourier-transform infrared (FTIR) spectroscopy from 500 cmA = 0.07589C + 0.02491 (R = 0.9998) under the maximum absorbance at λmax = 234 nm, where A and C (μg/mL) represent absorbance and drug concentration, respectively. A UV-2102PC UV-vis spectrophotometer  was exploited.A standard equation for measuring MET concentration in the solution was built using several solutions with predetermined concentrations from 1.0 to 20.0 μg/mL, which was EE, %), 50.0 mg medicated nanofibers were dissolved into 10.0 mL DMAc to free all the loaded MET. Then, 1.0 mL of the solution was dripped into 500 mL phosphate buffer saline . After scanning, the content of MET (MC) can be calculated from the standard equation. Meanwhile, the theoretical value of MET (TC) in the nanofibers can be calculated from the preparations of spinning solutions. Thus, EE of the nanofibers can be determined as follows:For determining the drug encapsulation efficiency  was measured, and the concentration can be calculated through the standard equation. Kinetics of the drug release are disclosed using the zero-order and Peppas models.In vitro drug release studies were performed using the basket method in the Chinese Pharmacopoeia (2020 Ed.). Nanofibers were placed into the baskets, which were then introduced in 600 mL of PBS  contained in 900 mL vessels of the RCZ-8A dissolution apparatus . The drug release studies were performed at 37 ± 0.5 °C and at a rotation rate of 50 rpm. At preset time intervals, an aliquot of 5 mL of the dissolution media was withdrawn and 5.0 mL fresh PBS was added to the vessels to keep a constant total volume of dissolution medium. The absorbance at λExhibited in T value in In this study, both the middle and the inner fluids have fine electrospinnability, whereas the outer solvent is unspinnable. Meanwhile, the core and middle fluids are highly compatible due to the difference between them only being their drug concentration. Their compatibility results from the small interfacial tensions between the spinning solutions at different layers and the impossibility of coagulations of solutes at the fluid–fluid boundaries. Thus, it is very convenient to adjust the middle-to-core fluid flow rate ratios, which can be exploited for manipulating the thickness of the sheath layer, i.e., the It is common sense that an electrospinning system often has four components , and that the most important part is the spinneret. A good spinneret not only guides the different kinds of spinning solutions to the electric fields for forming the designed nanofibers, but should also be convenient for setting up the whole electrospinning system and be able to save energy . In thisSome important details for implementing the modified tri-axial electrospinning are concluded in Under the selected experimental parameters, an ongoing electrospinning process for preparing nanofibers F1 is exhibited in The SEM observations of the four types of fibers and their diameters’ distributions are concluded in For comparison, nanofibers F3 and F4 were prepared using modified coaxial electrospinning. Nanofibers F3 were produced by stopping the supply of the inner fluid while the flow rate of the middle fluid was elevated to 2.0 mL/h. Similarly, nanofibers F4 were produced by switching off the middle fluid while the flow rate of the inner fluid was increased to 2.0 mL/h. Shown in The TEM images of the prepared nanofibers F1 and F2 are included in fr and cr, respectively), and also the experimental conditions , the densities of whole nanofibers , core sections (cρ), and sheath sections (sρ) can be calculated according to Equations (2)–(4), respectively:Based on the measured sizes of the nanofibers’ and their cores’ diameters  of the core-to-sheath sections of nanofibers F1 and F2 can be estimated according to Equation (5) as follows:For the sheath sections of the core–sheath nanofibersr1ρ) isr2ρ) isThen, the relative density for nanofibers F1  the more the drug is loaded, the greater the density of the drug–polymer composites; (2) the changes in fluid flow rates have little influence on the the density of the drug–polymer composites; (3) the spinning solutions’ components and compositions are the key elements in determining the nanofibers’ densities, rather than the experimental conditions.Based on the similar values of In the medicated nanomaterials, the drug’s physical state and its compatibility with the polymeric carrier are very important for functional applications. XRD patterns are often measured to detect the physical state of the components. In this study, the XRD patterns of CA, MET, and their core–sheath nanofibers F1 and F2 are presented in −1. Raw MET powders have characteristic peaks at 3360, 3274, 1673, 1588, 1381, and 1058 cm−1. However, in the core–sheath nanofibers F1 and F2, almost all of the MET peaks decreased to the point of almost disappearing. These nanofibers mainly exhibit the peaks of the polymer matrix, with some peaks slightly red shifted to a low wavenumber. These phenomena suggest that the secondary interactions happened between the CA and MET molecules. For example, the CA molecule has many —C=O groups in the —OAc groups as proton acceptors, whereas the MET molecules have —NH and —NH2 to act as proton donors. Thus, hydrogen bonding can be easily formed between them, and determine that CA and MET are highly compatible, and that the core–sheath nanofibers F1 and F2 are amorphous composites.The ATR-FTIR spectra of MET, CA, and their core–sheath nanofibers are shown in n = 3). The theoretical loading of nanofibers F1 and F2 are 31.6% and 27.8%, respectively  and release time, respectively, k is a constant, and n, as the release exponent, can be exploited to indicate the drug release mechanism.To further investigate the influences of core–sheath structures on the drug molecule release behaviors, the controlled release mechanisms are discussed. The well-known Peppas equation  Q = ktn Q1 = 0.95 + 0.79t (R1 = 0.9837), LgQ2 = 1.11 + 0.65t (R2 = 0.9933), LgQ3 = 1.57 + 0.39t (R1 = 0.9703), and LgQ4 = 1.63 + 0.36t (R1 = 0.9690), respectively (n value of 0.39 and 0.36 indicate that the drug released from the nanofibers are typically controlled by the Fickian release mechanism [n value of 0.79 and 0.65 for core–sheath heterogeneous nanofibers F1 and F2 (After treatments of the in vitro dissolution test data of the four nanofibers, the regressed Peppas equations for nanofibers F1 to F4 are Lgectively . For theechanism , i.e., t1 and F2 a suggestIn traditional pharmaceutics, the sustained release for water-soluble drugs frequently relies on hydrophobic matrices, such as insoluble polymers, lipids, and even inorganic materials ,77. CA, T” in In the present study, a new strategy is demonstrated for further manipulating drug sustained release behaviors. Shown in Oral administration is the most favorable route for the patients . ElectroA sustained release sheath and a sustained release core can be combined in the core–sheath nanofibers for synergistic actions in order to provide better drug sustained release profiles for the water-soluble drug MET. A modified tri-axial electrospinning process was developed for the smooth and robust preparation of the core–sheath nanofibers, in which both the core and sheath comprise CA and MET, but the core sections had a higher drug concentration than the sheath sections. SEM results demonstrated that all of the nanofibers had a fine linear morphology due to the electrospinnability of CA solutions. TEM results showed nanofibers F1 and F2 had the core–sheath nanostructures, owing to a higher density of core parts. XRD and FTIR tests suggested that CA and MET were compatible and MET presented in the CA matrices in an amorphous state. All the nanofibers had an EE of around 100%, suggesting no drug loss during the preparation processes. In vitro dissolution tests verified that the matrix CA, the core–sheath structures with different drug concentrations, and also the engineered sheath thicknesses were able to work together in a clearly combined mechanism to ensure a fine sustained release file from nanofibers F2, which had an initial release of 11.3 ± 2.9% in the first hour, released 95% of the loaded MET in 23.4 h, and had a very small late leveling-off release. The present proof-of-concept protocols can pave a new way to develop many novel biomolecule-based nanostructures for extending the release of water-soluble drug molecules.


VIE can be a helpful addition to CCTA when evaluating coronary arteries.Coronary computed tomography angiography (CCTA) is a widely used imaging modality for diagnosing coronary artery disease (CAD) but is limited by a high false positive rate when evaluating coronary arteries with stents and heavy calcifications. Virtual intravascular endoscopy (VIE) images generated from CCTA can be used to qualitatively assess the vascular lumen and might be helpful for overcoming this challenge. In this study, one hundred subjects with coronary stents underwent both CCTA and invasive coronary angiography (ICA). A total of 902 vessel segments were analyzed using CCTA and VIE. The vessel segments were first analyzed on CCTA alone. Then, using VIE, the segments were classified qualitatively as either negative or positive for in-stent restenosis (ISR) or CAD. These results were compared, using ICA as the reference, to determine the added diagnostic value of VIE. Of the 902 analyzed vessel segments, CCTA/VIE had sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 93.9/90.2, 96.2/98.2, 96.0/97.7, 70.0/83.1, and 99.4/99.0, respectively, in diagnosing ISR or CAD, with significantly improved specificity ( With rapid technological advancements, coronary computed tomography angiography (CCTA) has become an increasingly useful tool in the diagnosis of coronary artery disease (CAD) ,2,3. ItsMeta-analyses of CCTA performed on 64-slice multi-detector CT scanners showed a sensitivity of 86–91% and specificity of 91–93% in diagnosing in-stent restenosis (ISR), while the positive predictive value remained low at 68% ,15,16. IVirtual intravascular endoscopy (VIE), a CCTA-generated 3D visualization tool, is another approach that provides unique intraluminal views of the blood vessels, including endovascular stents or stent grafts ,26,27,28The purpose of this study is to investigate the role of VIE in delineating coronary intraluminal changes in the presence of coronary stents and plaques. By performing both CCTA and invasive coronary angiography (ICA) for each patient within 4 days, the added diagnostic value of VIE was compared with that of CCTA alone, using ICA as the gold standard.2; difficulty in cooperating with the aims of the study; hemodynamic instability; and severe tachycardia or arrhythmias. The subjects’ CCTA and ICA images were then used retrospectively for this study.The Institutional Review Board approved this project. For a prior prospective study assessing the diagnostic value of CCTA using a 320-detector CT scanner, 100 patients with previously placed coronary stents were recruited from a single tertiary medical hospital to undergo both CCTA and ICA within a 4-day time interval. Informed consent forms were obtained from all subjects. The exclusion criteria comprised the following: previous history of cardiac bypass surgery; adverse reactions to iodinated contrast media; renal insufficiency with glomerular filtration rates of less than 45 mL/min/1.73 mAll patients underwent both non-enhanced CT and CCTA on a 320-detector CT scanner  with 320 detector rows, each 0.5 mm wide, and a gantry rotation time of 0.35 s with z-axial coverage of up to 160 mm. Calcium scores were measured on non-enhanced CT images, assessed and recorded on a separate workstation . Slice thickness was set at 3 mm for Agatston scoring and 1 mm for CCTA, with the coverage area extending from the pulmonary trunk to the diaphragm. The CT images were obtained based on a standard protocol ,29,30.Tube voltage (kV) and current (mA) were selected according to the patients’ BMI. Kernel settings FC03 and FC05 were used. For vascular enhancement, a bolus of contrast medium  was administered intravenously, with a volume of 60 to 70 mL at a rate of 5 mL/s through the antecubital vein using a power injector , followed by 40 mL of saline chase at a rate of 5 mL/s. CT scanning was started manually when contrast medium reached peak attenuation in the left ventricle.Before the scan, a β-blocker Inderal  was administered orally to keep the heart rate below 65 bpm for the one-beat scan. A short-acting cardio-selective β-blocker, Esmolol  was administered intravenously if there were any contraindications to Inderal, such as asthma, atrioventricular conduction block, and Raynaud syndrome. For dilatation and optimal visualization of the coronary arteries, 0.6 mg of Nitrostat  was administered sublingually 3–5 min before the scan.All ICA was performed within 4 days after CCTA. All images were recorded at 15 frames/s by a monoplane X-ray angiogram  at a resolution of 512 × 512 pixels. The ICA studies were performed and analyzed by two experienced cardiologists blinded to the CCTA data. Minimal lumen diameters were quantitatively measured in projections showing the most severe narrowing, using a quantitative analysis software package , as shown in CT volume data converted from original DICOM  images were transferred to a separate workstation equipped with the software Analyze V 11.0  for image post-processing and generation of 3D VIE images. Post-processing of CT data was performed with a CT number thresholding technique ,28. GeneThe minimal luminal diameters were quantitatively measured on curved planar reformatted CCTA images, in views showing the greatest degree of stenosis in the left anterior descending (LAD), left circumflex (LCX), and right coronary (RCA) arteries. All vessels greater than 1.5 mm in diameter were evaluated . A stenoVIE interpretation was performed jointly by 2 observers  who were blinded to the results of ICA, but not CCTA, because CCTA images are required to generate the VIE images and locate the vessel segments. Thirty patients’ CCTA and ICA images were used to train both observers to construct and interpret VIE images, as well as to determine the inter- and intra-observer variability, before proceeding with the main study. During this training session, the observers learned to choose appropriate CT thresholds in order to generate VIE images of optimal quality, so that the degree of stenosis in each vessel matched their appearance on CCTA images under most circumstances . The LAD, LCX, and RCA were each divided into proximal, middle, and distal segments for VIE analysis. The left main coronary artery, posterior descending artery, and other branches  were included only if they had stents or suspected CAD on CCTA. Vessel segments were excluded if they could not be evaluated on VIE due to hypoplasia or a location distal to totally occluded segments.The VIE images were interpreted qualitatively, categorizing each vessel segment as either negative or positive for CAD or ISR; vessel segments were considered positive if the lumen appeared to be more than 50% obstructed, either by filling defects or circumferential narrowing. If the degree of stenosis was visually equivocal (more or less 50%), the diagnosis of VIE generally followed that of CCTA. The diagnostic value of CCTA alone is compared with that of CCTA-based VIE , using ICA results as the gold standard.t-test. Categorical variables were expressed as numbers and percentages and analyzed by Chi-square or Fisher’s Exact test. Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value for original CCTA and VIE images in detecting stenosis in coronary arteries with stents or calcifications were calculated from χ2 tests of contingency. Kappa values were used to assess inter- and intra-observer variability, and to estimate the degree of agreement between CCTA, VIE and ICA in diagnosing coronary stenosis. A p value of less than 0.05 was considered significant for all statistical evaluations.All quantitative variables were expressed as mean ± standard deviation and compared by Student’s Between November 2010 and July 2012, one hundred patients with coronary stents agreed to undergo both CCTA and ICA examinations. All subjects except one underwent both CCTA and ICA within a one-day interval; one subject underwent ICA four days after CCTA. Of these 100 subjects , 91 were males. All patients had at least one coronary stent placed in the main coronary arteries. The average total Agatson’s calcium score was 249.5 (range 0 to 2928).All vessel segments with either stents or suspected CAD seen on CCTA were analyzed using VIE. In total, 902 vessel segments, including 193 stented vessel segments and 231 plaque segments, including 115 calcified plaque segments, 85 mixed plaque segments, and 31 non-calcified plaque segments, were analyzed. Some examples of CCTA, VIE, and ICA images in corresponding vessel segments are shown in The intra-observer agreement was weak for both observers  when comparing the results from the first 30 training cases with the second-round interpretation. Inter-observer agreement was moderate after the training round was completed (kappa = 0.726).p = 0.025), accuracy (p = 0.046), and positive predictive value (p = 0.047). The kappa value for CCTA agreement with ICA in diagnosing ISR or CAD showed an overall improvement from 0.779 to 0.851 following the addition of VIE interpretation, although this change did not reach statistical significance (p = 0.112). Overall, out of a total of 902 analyzed vessel segments , 82  were proven to have ISR by ICA. CCTA had a sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 95.7, 93.5, 93.8, 66.7, and 99.4, respectively, in diagnosing ISR. With VIE, the corresponding values (shown in %) were 87.0, 97.6, 96.9, 83.3, and 98.2, respectively, for diagnosing ISR . The speOf the 115 vessel segments with calcified plaques, four (3.5%) were proven to have CAD by ICA. For these segments, CCTA/VIE had sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 75.0/50.0, 88.3/95.5, 87.8/93.9, 18.8/28.6, and 99.0/98.1, respectively, in diagnosing CAD . The speOf the 85 vessel segments with mixed plaques, 34 (40.0%) were proven to have CAD by ICA. For these segments, CCTA/VIE had sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 91.2/91.2, 88.2/92.2, 89.4/91.8, 83.8/88.6, and 93.8/94.0, respectively, in diagnosing CAD . There wOf the 31 vessel segments with non-calcified plaques, 21 (67.7%) were proven to have CAD by ICA. For these segments, CCTA/VIE had sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 100.0/100.0, 70.0/80.0, 94.3/93.6, 87.5/91.3, and 100.0/100.0, respectively, in diagnosing CAD, without any statistically significant difference .p = 0.011 and p = 0.047, respectively).Overall, for the combined 424 vessel segments with stents and plaques, CCTA/VIE had sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (shown in %) of 93.9/90.2, 90.4/95.6, 91.0/94.6, 70.0/83.1, and 98.4/97.6, respectively, in diagnosing CAD . The spep = 0.001), VIE (p < 0.001), and ICA (p < 0.001). Smaller stent diameters also resulted in significantly decreased diagnostic accuracy for both CCTA (p = 0.004) and VIE (p < 0.001), although there was no significant difference in this effect between CCTA and VIE (p = 0.547). Stent types  had no significant effect on ICA results (p = 0.822).Stent details were available for 190 out of the 193 stented vessel segments, including 144 drug-eluting stents and 46 bare-metal stents. The stents ranged from 1.3 mm to 5 mm in diameter. Smaller stent diameters were significantly associated with ISR in CCTA . Of the 902 vessel segments analyzed in this study, CCTA had 33 false positive results, among which VIE was able to correctly identify 25 (75.8%) as true negative results ; in such instances, the threshold needs to be set higher. With some practice, observers can become adequately adept at choosing the appropriate threshold and constructing quality VIE images for interpretation. In our study, there were seven vessel segments  that appeared to be ISR or CAD on VIE images, but were diagnosed as only intimal hyperplasia (IH) or mild stenosis on both CCTA and ICA  are required for optimal VIE image generation. Assessing all of the coronary artery segments in a patient without referring to CCTA images is time-consuming and may require well over an hour per patient. Since CCTA already has a high negative predictive value , a moreIn addition to the limitations of VIE, this study has some limitations of its own. This is a single-center study, with only 100 subjects and 902 evaluated vessel segments—in particular, the number of positive vessel segments with calcified plaques is small, only four. Based on our results, a larger scale study might be expected to produce more statistically significant results. Second, the learning curve of the VIE software may have affected the interpreters differently as they gradually gained experience evaluating the one hundred subjects’ CCTA, VIE, and ICA images, as reflected in the intra- and inter-observer variability. Furthermore, in our study, the stents’ sizes had a significant impact on the diagnostic accuracy for both CCTA and VIE, which was expected since smaller diameter stents are known to affect the diagnostic accuracy of CCTA . Future Our study showed that VIE can be a helpful tool when evaluating coronary arteries with stents and plaques using CCTA, significantly improving the specificity, accuracy, and positive predictive value. This advantage may ultimately benefit patients by improving the diagnostic accuracy of CCTA and reducing unnecessary invasive procedures. Thus, CCTA-generated VIE could be incorporated into standard CCTA during the diagnostic assessment of coronary stents or plaques.


Molecularly imprinted polymer (MIP) computational design is expected to become a routine technique prior to synthesis to produce polymers with high affinity and selectivity towards target molecules. Furthermore, using these simulations reduces the cost of optimizing polymerization composition. There are several computational methods used in MIP fabrication and each requires a comprehensive study in order to select a process with results that are most similar to properties exhibited by polymers synthesized through laboratory experiments. Until now, no review has linked computational strategies with experimental results, which are needed to determine the method that is most appropriate for use in designing MIP with high molecular recognition. This review will present an update of the computational approaches started from 2016 until now on quantum mechanics, molecular mechanics and molecular dynamics that have been widely used. It will also discuss the linear correlation between computational results and the polymer performance tests through laboratory experiments to examine to what extent these methods can be relied upon to obtain polymers with high molecular recognition. Based on the literature search, density functional theory (DFT) with various hybrid functions and basis sets is most often used as a theoretical method to provide a shorter MIP manufacturing process as well as good analytical performance as recognition material. A molecularly imprinted polymer (MIP) is a synthetic material that has molecular recognition ability with high affinity and selectivity for a particular molecule (template) through the formation of active sites with the shape, size and pattern of functional groups that are complementary to the template used during synthesis ,2,3. As The computational approach to design MIPs generally employs molecular modeling (quantum mechanics), molecular mechanics or molecular dynamics . QuantumApart from using quantum mechanics, some researchers have also used molecular mechanics ,37 and mThe last major review on the computational approach of MIP design was published by Cowen et al. . The focThe formation of a complex between a template and a functional monomer is the first step in MIP preparation. Thus, identifying functional monomers that have a strong interaction with the template is crucial for the success of MIP synthesis . The binQuantum mechanics is also known as the electronic structure method; it is the most popular MIP design and evaluation technique . In addiBoth prior to and after 2016, quantum mechanics methods used to design MIPs have employed ab initio, semiempirical and DFT approaches. The latter is the most frequently used method to design MIPs, in particular to find the best functional monomer and optimal template to functional monomer ratio. A DFT approach provides harmony between accuracy and computational expense. The difference in DFT methods employed before and after 2016 lies in the hybrid functions that are chosen and the basis function. The ab initio approach has been used to calculate the template–functional monomer complex binding energy to select the best functional monomer with a strong interaction with template or to determine the optimal template to functional monomer ratio. Prior to 2016 and until now, the ab initio approach to design MIPs has used HF/6-31G(d) and HF/3-21G. These approaches have been used mainly to select the best functional monomer based on calculating the monomer–template complex binding energy with various functional monomers; the most negative value indicates the strongest interaction. Besides, the method has also been used to determine the best template to functional monomer ratio, determined from the binding energies considering several ratios. The ab initio approach with the HF method with the 3-21G basis set has been used by Hasanah et al. ,52 and LHasanah et al.  used theFurthermore, several studies have optimized the solvent besides the functional monomer in calculations to obtain more accurate prepolymerization simulation results. Luo et al.  used HF/Saad et al.  evaluateHosny et al.  used theTo determine which ab initio method is best to design MIPs, we must consider the agreement between computational expense, accuracy of calculations and the level of compatibility between theoretical calculations and experimental performance. From the above-mentioned ab initio methods, HF/6-31G(d) presents better accuracy but needs more computational time, while HF/3-21G shows good agreement between the simulation and experimental results and offers a more efficient computational expense.Like the ab initio method, this approach has also been used to calculate binding energy. While the semiempirical approach calculates binding energy faster than the ab initio approach, the accuracy is dependent on the availability of parameters suitable for the molecule being analyzed. If the target molecule is similar to a molecule that exists in the database libraries used in the method parameterization, the result will be good. If the molecules differ significantly from the molecules used in the parameterization method, the answer may be very different from the experimental data . Before AM1 has been used to design MIPs by Ishak et al. , while PIshak et. al.  used AM1Li et al.  used PM3N,NI-dimethylacrylamide  was selected as the functional monomer due to its great binding energy ; it formed a stable complex with stigmasteryl methacrylate as a polymerizable template. To confirm the in silico modeling prediction, two MIP preparations were investigated, one with and the other without the functional comonomer N,NI-DMAAM. In addition, three different NIPs were prepared to examine the extent of nonspecific binding that contributes to the overall interaction performance, and in particular the role that the functional and cross-linking monomers play with respect to these interactions. Evaluation of the binding of stigmasterol to each MIP and NIP was conducted via static batch binding. These assays revealed that polymer containing N,NI-DMAAM comonomers exhibited a greater binding capacity (5.6 mg/g) and IF (7.1). This demonstrated the harmony between the computational simulation and the laboratory experiment results. Schwarz et al.  used PM3The use of the semiempirical approach in MIP design has shown a slight decline in recent years. However, PM3 is still used frequently to calculate the binding energy of template–functional monomer complexes to select the best functional monomer. Both AM1 and PM3 are efficient to use in MIP design because their computational costs are relatively inexpensive. Of note, PM3 is slightly more accurate in calculating binding energy than AM1.The DFT is the most commonly applied theoretical method because it often provides an excellent balance between computational cost and accuracy . Hybrid The DFT method with the ωB97XD hybrid function and the 6-31G basis set was employed to simulate binding sites, binding energy, the number of hydrogen bonds, the imprinted molar ratio, and the interaction mechanism to design a MIP for chloramphenicol (CAP) . The resp-dioxin (TCDD) [The DFT method with the M062X hybrid function has been used to design MIP for purifying tylosin, namely to choose the best functional monomer with the highest interaction energy; at the same time, the solvent was evaluated using the PCM . MAA wasn (TCDD) . The samn (TCDD) . In all GGA of the BLYP functional was employed to explore the intermolecular interactions and recognition properties of a MIP for deltamethrin (DM) . The resThe DFT combined with PM3 was used to optimize the stoichiometric ratio between template and functional monomer to design a MIP for chlorogenic acid (CGA) . The DFTMolecular mechanics methods are simpler options compared with the quantum mechanical approach. Furthermore, calculations are performed faster and more economically when using multicomponent systems to perform computations . The appPrior to 2016, molecular mechanics methods used to design MIPs included the CHARMM and AMBER force fields. Since 2016, the OPLS3 and MMFF94x force fields have become popular. In general, these force fields have been used for energy minimizations. There have been differences in the precision of the results, which depends on the level of accuracy expected. The use of the MMFF94x force field in the molecular operation environment (MOE) to minimize small molecules has produced satisfactory results in the evaluation of stability of hydrogen bonds as well as van der Waals adducts. The OPLS3 force field employs over an order of magnitude more reference data and associated parameter types relative to other commonly used small molecule force fields . As a consequence, OPLS3 achieves a high level of accuracy across performance benchmarks that assess small molecule conformational propensities and solvation. Sullivan et al.  investigAttallah et al.  used theMolecular dynamics use Newton’s equations of motion to simulate computationally the time evolution of a set of interacting atoms. This is a useful technique to determine conformational energies and noncovalent interactions of the complex in the molecular dynamics simulation . MoleculPrior to 2016, the molecular dynamics methods applied to design MIPs were the Tripos and COMPASS force fields. After 2016, other force fields have been used, namely the AMBER force field. In molecular dynamics, the force field determines the accuracy of a properly equilibrated molecular simulation to reproduce reality. In MIP design with molecular dynamics, researchers have faced an important choice of which force field is best suited to evaluate the monomer–template functional interaction. In general, molecular dynamics simulations used to design MIPs have been carried out in a canonical ensemble at a constant atom number, volume and temperature (NVT). The Tripos force field has been used in the SYBYL program combine with the LEAPFROG algorithm; this method can simultaneously screen many functional monomers in a virtual library. The COMPASS force field utilizes complex intermolecular interactions, which are quite complex compared with other force fields. Several systematic studies have demonstrated the excellent ability of the AMBER force field to reproduce very high-level data for hydrogen bonds and stacking interactions in the gas phase. Paredes-Ramos et al.  used molMadikizela et al.  employedMIP simulations using molecular dynamics have shown more realistic interactions between all MIP components. The use of the appropriate force field and software to execute the molecular dynamics process is fundamental for an accurate simulation. The COMPASS force field has shown good linearity between computational cost and the accuracy of the simulation result. Quantum mechanics methods, especially DFT approach, with various hybrid functions and basis sets are the most accurate to calculate and simulate interaction of few molecules . Less coComputational methods, including quantum and molecular mechanics as well as molecular dynamics, have been extensively used by researchers to design MIPs. These methods allow researchers to identify monomers exhibiting the best functions and interactions with templates. Of note, there has been excellent correlation between computations and experimental results associated with MIP analytical performance. Despite the possibility of employing numerous quantum mechanical approaches to achieve the best functional monomer through interaction with template molecules, several drawbacks exist. These include limitations in the MIP system, particularly the difficulty in handling the countless molecules required to obtain a comprehensive description of the prepolymerization system, for example, solvent involvement. Moreover, the duration required by this technique is protracted compared with molecular methods. The DFT quantum mechanical approach using various hybrid functionals and basis sets combined with the PCM is able to describe the interaction between functional monomers and template molecules in the presence of varying solvents, a method similar to the actual prepolymerization conditions.


ABSTRACT IMPACT: We present new programs aimed at training, retaining and preparing a diverse cadre of scientists to lead the field in transforming population health and advancing health equity OBJECTIVES/GOALS: To mitigate biases inherent to the R01 grant funding process, trainees from backgrounds underrepresented in medicine (URM) may benefit from enhanced mentorship and a longer ‘runway’ to funding. As such, we have deployed two synergistic programs that aim to support URM retention and advancement. METHODS/STUDY POPULATION: The URM Program for Advising in Research and Development (UPwARD) pairs URM trainees with 2 mentors: 1) an institutional leader from outside their discipline to serve as an internal advocate and 2) an external eminent scholar who will facilitate the scholar’s development and prominence within their discipline. Additionally, the KL2 Program to Launch URM Success (KL2 PLUS) offers URM trainees a third year of funding to focus on scholarship, grant writing and leadership development. Four specific training components of KL2 PLUS include: 1) PLUS II Seminar Series, 2) Faculty Success Program, 3) attendance at the AAMC Minority Faculty Leadership Conference, and 4) CTSI Committee Service. RESULTS/ANTICIPATED RESULTS: Along with measures of productivity , we will utilize social network analyses and measures of collaboration, retention, and future CTSI engagement to evaluate the programs “success’‘ as both are designed to enhance trainee scholarly development and expand their professional and social networks. UPwARD does so by supporting engagement with external mentors at professional meetings and travel to present work across institutions. PLUS writing accountability groups will enhance publication rates and grant submissions, while also building connections with other URM faculty. Trainees also serve on IN CTSI committees to groom talent for future IN CTSI leadership. DISCUSSION/SIGNIFICANCE OF FINDINGS: Systemic inequities underlie the ‘leaky pipeline’ challenge we face in cultivating a diverse cadre of senior scientists and independent investigators. With intentional programming and targeted investments, IN CTSI aims to advance more equitable funding outcomes and diverse leadership.