@article{ludwig_abraham_mckinney_freund_stewart_garman_barbas_sudan_gonzalez_2023, title={45: Comparison of the Effects of Normothermic Machine Perfusion and Cold Storage Preservation on Porcine Intestinal Allograft Regenerative Potential and Viability}, volume={107}, ISSN={0041-1337}, url={http://dx.doi.org/10.1097/01.tp.0000945636.34372.db}, DOI={10.1097/01.tp.0000945636.34372.db}, abstractNote={Historically, intestinal transplantation (IT) has been reserved as the last treatment option for patients with irreversible intestinal failure who are unable to tolerate total parenteral nutrition. Successful IT is reliant upon graft health at the time of donation, minimizing graft injury that may occur during procurement, storage, and IT, and the ability of the graft to heal following insult. Unfortunately, the intestine is easily damaged by ischemia-reperfusion injury (IRI). IRI induces intestinal epithelial cell apoptosis and damages the mucosal barrier, which can result in bacterial translocation and activation of the local and systemic immune and inflammatory response, ultimately contributing to graft failure, rejection, and decreased recipient survival. The current, preferred method of intestinal preservation prior to IT is static cold storage (CS), however the prolonged hypothermic ischemia of CS causes cell injury and intensifies the IRI that occurs during transplantation. Furthermore, IRI to the epithelial crypt region diminishes the intestine’s ability to heal by inducing loss of the highly proliferative intestinal stem cells (ISCs) that are responsible for maintenance, regeneration, and repair of the epithelium, critical to graft health. Thus, the investigation of alternative organ preservation techniques that reduce IRI, cellular damage, and graft injury are warranted to overall improve IT success. Normothermic machine perfusion (NMP) is a preservation method that reduces inflammation and promotes graft regeneration in other organs by preventing CS-associated IRI. However, NMP has not been described for intestine. We hypothesized that, compared to CS, intestinal NMP will induce less epithelial injury and better protect ISC regenerative potential and viability. 15 porcine intestines were flushed with UW solution, stored at 4°C (CS), or perfused with 34°C perfusate (NMP) for 6hr, and transplanted (n=9). Recipient pigs were recovered from anesthesia. Jejunal and ileal segments were collected immediately after flushing, serving as control tissue (CO), after 6hr of CS or NMP, and after 1hr of reperfusion post-IT. Histologic injury was assessed. Crypts isolated after flushing (CO), 6hr CS or NMP, and 1hr of reperfusion post-IT were cultured. Spheroid number, size, and EdU staining quantified ISC viability and proliferation. Expression of ISC and cellular proliferation genes and proteins were measured. Histologically, NMP tissue had mild epithelial erosion and increased columnar cell attenuation and expression of ISC and proliferation genes/proteins was observed. NMP spheroid areas and proliferating cell numbers were significantly larger than control and CS. Apoptotic cells were increased following CS. Post-graft reperfusion, CS had increased injury compared to uninjured control and NMP tissue. Compared to CS, NMP may improve graft regenerative potential, resulting in transplantation of healthier bowel and superior recipient survival.}, number={7S}, journal={Transplantation}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Ludwig, Elsa and Abraham, Nader and McKinney, Caroline and Freund, John and Stewart, Amy and Garman, Katherine and Barbas, Andrew and Sudan, Debra and Gonzalez, Liara}, year={2023}, month={Jun}, pages={25–25} }
 @article{schaaf_polkoff_carter_stewart_sheahan_freund_ginzel_snyder_roper_piedrahita_et al._2023, title={A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease}, volume={37}, ISSN={["1530-6860"]}, DOI={10.1096/fj.202300223R}, abstractNote={AbstractIntestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein‐Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence‐activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5‐H2B‐GFP and wild‐type pigs. Ileum and colon LGR5‐H2B‐GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5‐H2B‐GFP colonoids. Crypt‐base, green fluorescent protein (GFP) expressing cells co‐localized with ISC biomarkers. LGR5‐H2B‐GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5‐H2B‐GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5‐H2B‐GFP pig crypt‐base cells. LGR5‐H2B‐GFP/APCnull colonoids had cystic growth in WNT/R‐spondin‐depleted media and significantly upregulated WNT/β‐catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5‐H2B‐GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt‐base FISH, underscore the significance of this novel LGR5‐H2B‐GFP pig to translational ISC research.}, number={6}, journal={FASEB JOURNAL}, author={Schaaf, Cecilia R. and Polkoff, Kathryn M. and Carter, Amber and Stewart, Amy S. and Sheahan, Breanna and Freund, John and Ginzel, Joshua and Snyder, Joshua C. and Roper, Jatin and Piedrahita, Jorge A. and et al.}, year={2023}, month={Jun} }
 @article{veerasammy_gonzalez_báez‐ramos_schaaf_stewart_ludwig_mckinney‐aguirre_freund_robertson_gonzalez_2023, title={Changes in equine intestinal stem/progenitor cell number at resection margins in cases of small intestinal strangulation}, volume={55}, ISSN={0425-1644 2042-3306}, url={http://dx.doi.org/10.1111/evj.13927}, DOI={10.1111/evj.13927}, abstractNote={AbstractBackgroundIntestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression.ObjectivesIntestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post‐operative complications, or case outcome.Study designRetrospective cohort study.MethodsIntestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post‐operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one‐way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post‐operative complications negatively related to outcome were determined using linear regression modelling.ResultsThirty‐six cases of SIS were evaluated. Ki67+ cell counts were decreased in the proximal (mean = 15.45 cells; 95% CI = 10.27–20.63; SD = 4.17; p = 0.02) and distal resection margins (mean = 15.05; 95% CI = 8.46–21.64; SD = 4.141; p = 0.03) in horses euthanised postoperatively compared to control (mean = 23.62 cells; 95% CI = 19.42–27.83; SD = 5.883). In the distal resection margin, an increase in SOX9+Ki67+ cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236–1.123; p = 0.008, SE = 0.1393).Main limitationsSmall population size.ConclusionsProliferating cell and ISC numbers may be associated with case outcome.}, number={6}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Veerasammy, Brittany and Gonzalez, Gabriel and Báez‐Ramos, Patricia and Schaaf, Cecilia R. and Stewart, Amy Stieler and Ludwig, Elsa K. and McKinney‐Aguirre, Caroline and Freund, John and Robertson, James and Gonzalez, Liara M.}, year={2023}, month={Feb}, pages={995–1002} }
 @article{ludwig_abraham_schaaf_mckinney_freund_stewart_veerasammy_thomas_cardona_garman_et al._2023, title={Comparison of the effects of normothermic machine perfusion and cold storage preservation on porcine intestinal allograft regenerative potential and viability}, volume={24}, ISSN={1600-6135}, url={http://dx.doi.org/10.1016/j.ajt.2023.10.026}, DOI={10.1016/j.ajt.2023.10.026}, abstractNote={<h2>Abstract</h2> Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.}, number={4}, journal={American Journal of Transplantation}, publisher={Elsevier BV}, author={Ludwig, Elsa K. and Abraham, Nader and Schaaf, Cecilia R. and McKinney, Caroline A. and Freund, John and Stewart, Amy S. and Veerasammy, Brittany A. and Thomas, Mallory and Cardona, Diana M. and Garman, Katherine and et al.}, year={2023}, month={Oct}, pages={564–576} }
 @article{stewart_schaaf_veerasammy_freund_gonzalez_2022, title={Culture of equine intestinal epithelial stem cells after delayed tissue storage for future applications}, volume={18}, ISSN={["1746-6148"]}, DOI={10.1186/s12917-022-03552-6}, abstractNote={Abstract
                Background
                Equine intestinal epithelial stem cells (ISCs) serve as potential targets to treat horses with severe intestinal injury. The ability to isolate and store ISCs from intestinal biopsies creates an opportunity for both in vitro experiments to study ISC dynamics in a variety of intestinal diseases, and, in the future, utilize these cells as a possible therapy. If biopsies could be successfully stored prior to processing for ISCs, this would increase the availability of sample repositories for future experimental and therapeutic use. However, delayed culture of equine ISCs following prolonged sample storage has not been described. The objective of this study was to describe the isolation and culture of equine ISCs following delayed tissue storage. Small intestinal full thickness biopsies were collected post euthanasia. Fresh tissue was immediately processed or stored at 4 °C for 24, 48 and 72 h (H) before processing. Intestinal stem cells (crypts) were dissociated and cultured. Size, growth efficiency and proliferation potential were compared between resultant enteroids (“mini-guts”) derived from each storage timepoint. In a separate study, growth efficiency of cryopreserved crypts was compared to cryopreserved enteroid fragments to investigate prolonged storage techniques.
              
                Results
                Intestinal crypts were successfully isolated and cultured from all timepoints. At 72H post initial collection, the intestine was friable with epithelial sloughing; resultant dissociation yielded more partial crypts. Enteroids grown from crypts isolated at 72H were smaller with less proliferative potential (bud units, (median 6.5, 3.75–14.25)) than control (median 25, 15–28, p < 0.0001). No statistical differences were noted from tissues stored for 24H compared to control. Following cryopreservation, growth efficiency improved when cells were stored as enteroid fragments (median 81.6%, 66.2–109) compared to crypts (median 21.2%, 20–21.5, p = 0.01). The main limitations included a small sample size and lack of additional functional assays on enteroids.
              
                Conclusions
                Equine ISCs can be isolated and cultured after prolonged tissue storage. Resultant enteroids had minimal differences even after 24-48H of whole tissue storage. This suggests that ISCs could be isolated for several days from samples properly stored after procedures, including surgery or necropsy, and used to create ISC repositories for study or therapy of equine intestinal diseases.
              }, number={1}, journal={BMC VETERINARY RESEARCH}, author={Stewart, Amy Stieler and Schaaf, Cecilia R. and Veerasammy, Brittany and Freund, John M. and Gonzalez, Liara M.}, year={2022}, month={Dec} }
 @article{abraham_ludwig_schaaf_veerasammy_stewart_mckinney_freund_brassil_samy_gao_et al._2022, title={Orthotopic Transplantation of the Full-length Porcine Intestine After Normothermic Machine Perfusion}, volume={8}, ISSN={2373-8731}, url={http://dx.doi.org/10.1097/TXD.0000000000001390}, DOI={10.1097/TXD.0000000000001390}, abstractNote={
            Background.
            Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model.
          
          
            Methods.
            Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice.
          
          
            Results.
            In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function.
          
          
            Conclusions.
            This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.
          }, number={11}, journal={Transplantation Direct}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Abraham, Nader and Ludwig, Elsa K. and Schaaf, Cecilia R. and Veerasammy, Brittany and Stewart, Amy S. and McKinney, Caroline and Freund, John and Brassil, John and Samy, Kannan P. and Gao, Qimeng and et al.}, year={2022}, month={Oct}, pages={e1390} }
 @article{stewart_schaaf_luff_freund_becker_tufts_robertson_gonzalez_2021, title={HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia}, volume={321}, ISSN={["1522-1547"]}, url={https://doi.org/10.1152/ajpgi.00165.2021}, DOI={10.1152/ajpgi.00165.2021}, abstractNote={ This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets. }, number={5}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY}, publisher={American Physiological Society}, author={Stewart, Amy Stieler and Schaaf, Cecilia Renee and Luff, Jennifer A. and Freund, John M. and Becker, Thomas C. and Tufts, Sara R. and Robertson, James B. and Gonzalez, Liara M.}, year={2021}, month={Oct}, pages={G588–G602} }
 @article{singh_hung_shanahan_kanke_bonfini_dame_biraud_peck_oyesola_freund_et al._2020, title={Enteroendocrine Progenitor Cell-Enriched mir-7 Regulates Intestinal Epithelial Proliferation in an Xiap-Dependent Manner}, volume={9}, ISSN={["2352-345X"]}, DOI={10.1016/j.jcmgh.2019.11.001}, abstractNote={Background & AimsThe enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis.MethodsWe leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice.ResultsFirst, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling.ConclusionsThis study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling. The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis. We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice. First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling. This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.}, number={3}, journal={CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY}, author={Singh, Ajeet P. and Hung, Yu-Han and Shanahan, Michael T. and Kanke, Matt and Bonfini, Alessandro and Dame, Michael K. and Biraud, Mandy and Peck, Bailey C. E. and Oyesola, Oyebola O. and Freund, John M. and et al.}, year={2020}, pages={447–464} }
 @article{stieler stewart_freund_blikslager_gonzalez_2018, title={Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia}, volume={5}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/57647}, DOI={10.3791/57647}, abstractNote={Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberkühn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.}, number={135}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Stieler Stewart, Amy and Freund, John M and Blikslager, Anthony T and Gonzalez, Liara M}, year={2018}, month={May} }
 @article{stewart_freund_gonzalez_2017, title={Advanced three-dimensional culture of equine intestinal epithelial stem cells}, volume={50}, ISSN={0425-1644}, url={http://dx.doi.org/10.1111/evj.12734}, DOI={10.1111/evj.12734}, abstractNote={SummaryBackgroundIntestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine.ObjectivesTo describe the isolation and culture of small intestinal stem cells into three‐dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities.Study designDescriptive study.MethodsIntestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed.ResultsIntestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze‐thaw cycle.Main limitationsTissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time.ConclusionsThe successful culture of equine intestinal crypts into 3D “mini‐guts” allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).}, number={2}, journal={Equine Veterinary Journal}, publisher={Wiley}, author={Stewart, A. Stieler and Freund, J. M. and Gonzalez, L. M.}, year={2017}, month={Sep}, pages={241–248} }