AR-13503 ; (iii) Y-27632 vs. AR-13503 ; (iv) AR-13324 (1 μM) vs. AR-13503 ; and (v) AR-13324 (0.1 μM) vs. AR-13503 . Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; *  KIP1, preventing the CECs from proliferating [The corneal endothelium (CE) plays a critical role in corneal transparency through a balance between the leaky cellular barrier and the active ionic pump mechanism that maintains corneal deturgescence ,2. Humanferating ,5. Corneferating . The current treatment to restore vision involves the replacement of the diseased CE through a corneal transplant of a healthy donor corneal graft . HoweverIn conditions leading to massive endothelial damage or advanced FECD with widespread endothelial dysfunction, a cell-based therapy approach is needed, where the isolation of healthy CECs from donor corneas, with or without cellular expansion, is required prior to delivery into the anterior chamber ,16. DeliThe Rho-associated coiled-coil protein kinase is part of the serine/threonine family and acts as an effector of the Rho signaling pathway. With 65% of overall identity, two isoforms are present in the human body: ROCK-1 and ROCK-2 . They arIn the cornea, reports have shown that the use of ROCK inhibitors enhanced endothelial wound closure , as wellIn this study, we investigated various classes of ROCK inhibitors for their effects on primary CECs cultured using an established dual media culture system . With re2 that were deemed unsuitable for transplantation , or Saving Sight , each with written consent from the next of kin, and adhering to the principles outlined in the Declaration of Helsinki. For in vitro optimization studies, procured donor corneas were from younger donors ranging from 4 to 35 years old ; whereas for ex vivo studies, older donors (>50 years old) were procured. All cornea pairs had endothelial cell density (ECD) of at least 2000 cells/mmantation . CorneosTo investigate and compare the effects of different ROCK inhibitors on human CECs, various ROCKi compounds were obtained. The compound Y-27632 was purchased from Miltenyi Biotec GmbH . Research grade standard of the FDA-approved ophthalmic compound known as Netasurdil (AR-13324), along with its active compound, AR-13503, and Verosudil (AR-12286)  were obtStructures of ROCK-1 and ROCK-2 were obtained from the Protein Data Bank (PDB) according to the highest resolution found for either protein, using a cluster cut-off of 0.51. Redundant structures were excluded based on clustering the co-crystallized ligands by their structural similarity. In silico docking of several ROCK inhibitor molecules from different classes  and Y27632 was performed with the Glide application  within t4 cells per cm2 onto surfaces pre-coated with FNC coating mixture for further expansion. All cell cultures were propagated within a humidified atmosphere at 37 °C with 5% CO2. Primary human CECs were isolated using a two-step “peel and digest” approach  and prop4 cells/cm2 in each well of the E-Plate that had been pre-coated with FNC coating mix . The CECs were left to be stabilized over 24 h in M5-Endo without any ROCKi. On the following day, the CECs were exposed independently to M5-Endo medium containing the respective ROCKi . These experiments involved the use of E-Plates  with gold-microelectrodes fused to their culture surface. As the CECs adhered, presence of cells at the electrode–solution interface impedes electron flow. The impedance is measured by the integrated software and is converted into arbitrary cell indices for comparative analysis. Here, the negative controls in each experiment were its respective donor-matched primary CECs in M5-Endo media without any ROCKi supplementation, whereas the positive control group used in the analysis was M5-Endo media containing 10 µM Y-27632 . PrimaryOCKi see  for 24 hOCKi see A. ElectrOCKi see A and at OCKi see A.3 cells per cm2 and maintained in M5-Endo for 24 h (Day 1). On the second day (Day 2), the medium was switched to each respective condition, and cells were cultured for another 24 h. On the third day, cells were incubated in M4-F99 containing 10 mM of EdU for 24 h. Subsequently, samples were rinsed once with PBS before they were fixed in freshly prepared 4% PFA for 15 min at room temperature. Next, Samples were rinsed twice with 3% BSA in PBS and were incubated in 0.5% Triton X-100 in PBS for 20 min at room temperature for blocking and permeabilization. Incorporated EdU was detected by fluorescent-azide-coupling Click-iT reaction where samples were incubated for 30 min in the dark with a reaction mixture containing Click-iT EdU reaction buffer, CuSO4, azide-conjugated Alexa Fluor 488 dye, and reaction buffer additive. Following that, samples were rinsed with 3% BSA before incubating in 5 µg/mL Hoechst 33,342 for 10 min at room temperature in the dark. Finally, samples were washed twice in PBS and mounted in Vectashield containing 4,6-diamidino-2-phenylindole (DAPI), . Labelled proliferative cells were examined under a Zeiss Axioplan 2 fluorescence microscope . At least 250 nuclei were analyzed for each experimental condition.The proliferation rates of primary CECs were assessed using the EdU incorporation Click-iT cell proliferation assay  as per the manufacturer’s instructions. Two ROCK inhibitors, AR-13324 and AR-13503, were assessed for their capacity to enhance proliferation of CECs, with two concentrations (100 nM or 1 µM for AR-13324 and 1 µM or 10 µM for AR-13503). Donor-matched CECs with no ROCKi added served as negative control, whereas CECs with Y-27632 added served as positive control. Briefly, cultured CECs, passaged using TS, were seeded onto FNC-coated glass slides at a density of 5 × 10To assess corneal endothelial wound closure after ROCKi exposure, we chose a previously described approach with two types of wounds created in ex vivo cultured corneas: (1) DM-stripped to expose bare posterior stroma (peeled wound) and (2) CEC-denuded DM (scratched wound) . BrieflyFollowing wound creation, donor corneas were maintained in an ex vivo culture setup in M5-Endo medium supplemented with Y-27632  or AR-13324  throughout the course of the three-week study as previously described . The culSpecimens were imaged immediately following initial wound creation and every week until Day 21. In order to enhance the visualization of the created wounds, each cornea was briefly incubated for 30 s in a 0.2% Trypan Blue solution  as described previously . FollowiDonor-matched corneas (n = 3) were randomly selected for Trypan Blue/Alizarin red staining following the end of ex vivo culture. Alizarin red solution (0.5%) was freshly prepared on the day of staining. The Alizarin red powder  was dissolved in distilled water. The pH was titrated to 4.5 before filtering using a syringe pump filter. Each of these corneas was first stained for 3 min in buffered 0.2% TBS, and the cornea was then immediately stained in freshly prepared Alizarin red solution. The specimen was then washed for 60 s in a wash buffer prior to wet mounting and examination with a Zeiss Axioplan 2 microscope for wide-field images and a Zeiss Confocal Microscope , for high-magnification images.p < 0.05 was achieved. Prism 6.0  was used for data analysis; comparisons in cellular proliferation were carried out using Tukey’s Test for multiple comparisons. Kruskal–Wallis with Dunn post hoc corrections for multiple comparisons were applied to detect intergroup differences in endothelial migration. The Friedman test was used to analyze the endothelial wound recovery rate within each wound. All numerical data obtained were expressed as mean ± standard deviation (SD) unless otherwise stated. The results were deemed statistically significant when p < 0.0001). All ROCKi classes presented a stronger mean docking score than Y-27632 (p < 0.0001). The frequency of compounds presenting highest docking score was higher in the isoquinoline, aminofurazan, and benzodiazepine classes for ROCK-I; and in isoquinolines and amides for ROCK-II , 34 presented a significantly higher docking score for ROCK-1, when compared to Y-27632  and 16.31% ± 9.92% (p < 0.001) for the AR-13324 group; and 62.14% ± 40.75% (p < 0.05) and 18.42% ± 21.04% (p < 0.001) for the Y-27632 group. The weekly mean wound healing rate (WHR) was significantly higher for scraped compared to peeled wounds in both groups at Day 7 , and it progressively decreased for both groups as the corneal endothelial sheet became more confluent . Conversely, the donor age strongly correlated with delayed wound healing for peeled wounds of both Y-27632 and AR-13324 .There was an apparent association between increasing age and decreased maximum and daily endothelial migration rates for peeled and scratched wounds. This trend was similar with both ROCK inhibitors and far more prominent for peeled compared to scraped wounds. Interestingly, the donor age had a stronger correlation with a delay in the wound healing for scraped wounds in corneas treated with Y-27632 than ones supplemented with AR-13324, although this observation was not statistically significant  covered a smaller area than the recovery of cells in the scrapped areas , and were mostly restricted to the adjacent to the wound edge. The endothelial mosaic near the acellular zones were heterogeneous, with cells that were relatively larger and less hexagonal when compared to cells in the non-wounded areas . Alizarin red staining showed that exposure to AR-13324 A is comped areas A§,B§§, aed areas A‡,B‡‡. Rho-kinase proteins play a role in many cellular events in different cell types . With reThe incorporation of ROCK inhibitors in the cultivation of primary human CECs or as promoter of corneal endothelial wound healing is relatively new. It should be noted that the majority of the studies that described the effectiveness in the usage of ROCK inhibitors for primary human CECs have been conducted using the compound Y-27632 ,39,45,59p = 0.93; p = 0.17; Conversely, Netarsudil (AR-13324) has been FDA approved, and with the availability of GMP culture formulation for the growth of primary human CECs , it coulp < 0.001); 10 µM Y-27632 (p < 0.01); 1 µM AR-13324 (p < 0.01); and 0.1 µM AR-13324 (p < 0.05) were made. At the concentration of 1 µM of AR-13503, statistical significance was achieved when the proliferation rates were compared against untreated CECs (p < 0.05). A recent study reported a lower dose of AR-13503 to be effective at greater than 1000 times than Y-27632, and suggested the efficacy of this lower dosage to be highly specific for the effects against ROCK [In this study, in addition to comparing Y-27632 and Netarsudil in a series of donor-matched studies, we also evaluated the active metabolite of Netarsudil known as AR-13503 , and assnst ROCK ; it is cFor the endothelial wound recovery study, we have selected only corneas above 60 years old, which encompass the majority of the patients eligible for DM removal and adjunct with ROCKi treatment for FECD . This deThe donor age also seemed to impact the endothelial healing dynamics for both types of wound. Previously, Soh et al. demonstrated that Y-27632 slowed the decline of wound healing significantly in corneas older than 50 years . SimilarIt should be noted that the ex vivo experiment is limited by a confounding factor that must not be ignored. Unlike the endothelial wound healing experiments conducted on animals, TGF-ß is absent from both the ex vivo culture system and the in vitro experimental studies. TGF-ß is a constituent of the aqueous humor, which is a known inhibitor of the proliferation of primary CECs . MoreoveThe initial outcomes with ROCK inhibitors in the field of tissue engineering and regenerative medicine are exciting and promising. While tissue engineering of primary CEC has recently reached a breakthrough, DSO is yet controversial. Nevertheless, additional randomized controlled clinical trials similar to those of Kinoshita et al.  and MacsIn conclusion, we were able to demonstrate that additional ROCK inhibitor classes and compounds other than Ripasudil and Y-27632 hold promise in the treatment of corneal endothelial dysfunction with tissue engineering. Furthermore, the comparison between Y-27632 with the FDA-approved GMP-compliant ROCK inhibitor, AR-13324 (Netarsudil), showed similar outcomes with an in vitro cell culture system and an ex vivo wound regeneration model. Hence, this drug could be a candidate for cellular therapeutics or as an adjunct drug in regenerative treatments for Fuchs endothelial corneal dystrophy in humans.


Surgical techniques and outcomes of minimally invasive anatomic liver resection (AR) for hepatocellular carcinoma (HCC) are undefined. In 327 HCC patients undergoing 185 open (OAR) and 142 minimally invasive  ARs, perioperative and long-term outcomes were compared, using propensity score matching. After matching (91:91), compared to OAR, MIAR was significantly associated with longer operative time; less blood loss; a lower transfusion rate; lower rates of 90-day major morbidity, bile leak or collection, and 90-day mortality; and shorter hospital stay. On the other hand, laparoscopic and robotic AR cohorts after matching (31:31) had comparable perioperative outcomes. Postoperative overall and recurrence-free survivals of newly developed HCC were comparable between OAR and MIAR or between laparoscopic and robotic cases. MIAR was technically standardized using the extrahepatic Glissonian approach. MIAR was safe, feasible, and oncologically acceptable and would be the first choice of AR in selected HCC patients.p = 0.028); less blood loss ; a lower transfusion rate ; lower rates of major 90-day morbidity , bile leak or collection , and 90-day mortality ; and shorter hospital stay . On the other hand, laparoscopic and robotic AR cohorts after matching (31:31) had comparable perioperative outcomes. Overall and recurrence-free survivals after AR for newly developed HCC were comparable between OAR and MIAR, with potentially improved survivals in MIAR. The survivals were comparable between laparoscopic and robotic AR. MIAR was technically standardized using the extrahepatic Glissonian approach. MIAR was safe, feasible, and oncologically acceptable and would be the first choice of AR in selected HCC patients.Surgical techniques and outcomes of minimally invasive anatomic liver resection (AR) using the extrahepatic Glissonian approach for hepatocellular carcinoma (HCC) are undefined. In 327 HCC cases undergoing 185 open (OAR) and 142 minimally invasive  ARs, perioperative and long-term outcomes were compared between the approaches, using propensity score matching. After matching (91:91), compared to OAR, MIAR was significantly associated with longer operative time (643 vs. 579 min,  Anatomic liver resection (AR) is a hepatectomy procedure, where an anatomically determined liver territory that is supplied by the arbitrary Glissonian or portal pedicles (GPs) is completely and optimally resected. Therefore, AR includes both major liver resection of three or more Couinaud’s segments as well as parenchyma-preserving resection represented by mono- or bi- (sub)segmentectomy and monosectionectomy, and their combinatory resection. AR is expected to achieve both high curability and functional safety in liver resection for malignancy. In particular, parenchyma-sparing AR (PSAR), such as isolated segmentectomy and sectionectomy, is considered to confer benefits for resection of hepatocellular carcinoma (HCC), which is characterized by intra-portal vein tumor spread and accompanying impaired hepatic functional reserve ,2,3.The safety and feasibility of minimally invasive AR (MIAR) has been partly established in previous studies ,6,7,8,9,In this single-center study on 327 consecutive AR cases of HCC, including 185 OAR and 142 MIAR cases, we present our standardized surgical techniques for MIAR and compare perioperative outcomes between OAR and MIAR, as well as between laparoscopic and robotic AR, using propensity score matching (PSM) analyses. In addition, long-term outcomes after AR for newly developed HCC were compared between OAR and MIAR, as well as between laparoscopic and robotic AR. Based on the results, we discuss the technical, surgical, and oncologic aspects of MIAR in surgical management of HCC.The terminology for liver anatomy and procedures of hepatectomy was primarily based on the Brisbane 2000 Terminology of Liver Anatomy and Resections  and CouiAt our institution, AR was the first choice of hepatectomy procedure for HCC, if appropriate and applicable, irrespective of the surgical approaches; open, laparoscopic, or robotic. Selection of AR and the type of hepatectomy were based on the location, number, and size of tumors as well as patients’ hepatic functional reserve, mostly according to the so-called Makuuchi criteria, and physical status .Selection of OAR or MIAR was based on the operative difficulties depending on the tumor and patient characteristics (Ban’s criteria)  and the Selection of the surgical approaches was greatly affected by the medical paying system. Until 2015, before the start of national insurance coverage of MIAR in our country, the open approach was the first choice for AR in most patients, while from 2016, the laparoscopic approach had the priority, if indicated. Until March 2022, robotic liver resection was a practice at patients’ own expense in Japan, which significantly affected selection of the laparoscopic or robotic approach. After starting reimbursement of robotic liver resection from April 2022, robotic AR was selected at the surgeons’ or patients’ preferences as well as depending on the machine availability in the hospital.Patient, tumor, and surgical baseline data were retrospectively collected from the patients’ medical charts. The patient baseline data included age, sex, body mass index (BMI), American Society of Anesthesiology—Performance Status (ASA) score, presence of diabetes, serum biomarkers, etiology of background liver disease (hepatitis B (HBV), hepatitis C (HCV) or non-B and non-C (NBNC)), indocyanine green (ICG) retention rate at 15 min (ICGR15), Child–Pugh grade, and histologically proven cirrhosis . Tumor characteristics included location, number and size of the tumors, tumor stages according to the classification of the Liver Cancer Study Group of Japan , and serThe surgical techniques of AR described below were consistently used either in OAR or MIAR and in any types of AR, irrespective of location of the target anatomic liver territory ,17,18,19Then, the optimal amount of parenchyma was resected along the demarcation line or the intersegmental plane that was visualized by macroscopic discoloration or by the ICG negative staining method ,6,17. DuSeveral examples of the extrahepatic Glissonian approach to isolate the target GPs for MIAR are described in ®) was used in open and laparoscopic cases, and a clamp-crushing method and ultrasonic coagulating shears were used in robotic cases. Pringle maneuver was not used routinely but was applied on demand. Parenchyma was dissected in the cranial-to-caudal direction, and AR was completed , use of the Pringle maneuver, and open conversion (in MIAR). Postoperative outcomes were evaluated by the serum maximum levels of maximum total bilirubin (TB), aspartate aminotransferase (AST) and C-reactive protein (CRP), the serum minimum level of prothrombin time (PT), morbidity graded according to the Clavien–Dindo (C–D) classification , mortaliContinuous data were expressed as median with range (baseline data) or interquartile range (perioperative data) and compared using the Kruskal–Wallis test. Categorical data were compared using the Pearson’s chi-square test. In some comparative studies, 1:1 PSM was conducted to reduce biases. In comparison of perioperative outcomes between the OAR and MIAR cohorts, the following 11 variables were matched for PSM: age, sex, ASA class (I or II/≥III), ICGR15 (<13.0%/≥13.0%), etiology of underlying liver disease (HBV/HCV/NBNC), tumor number (single/multiple), tumor size , tumor location (AL/PS), tumor stage (I or II/≥III), types of resection , and previous hepatectomy (yes/no). In comparison of perioperative outcomes between the laparoscopic and robotic cohorts, age, sex, presence of cirrhosis (yes/no), tumor number (single/multiple), tumor size , types of resection, and previous hepatectomy (yes/no) were matched. In analyses of long-term outcomes, we examined newly developed HCC cases only.In comparison of long-term outcomes between the OAR and MIAR cohorts, 11 variables, including age, sex, ASA class, etiology, presence of cirrhosis, ICGR15 (<13.0%/≥13.0%), tumor number (single/multiple), tumor size , tumor stage, pathological tumor differentiation, and types of resection, were matched to reduce biases. Long-term outcomes were also compared between the unmatched and matched laparoscopic and robotic AR cohorts. In this analysis, age, sex, tumor size , tumor number (single/multiple), and presence of histology-proven cirrhosis (yes/no) were matched.p <0.050 was considered statistically significant. Statistical analyses were performed using JMP® software ver. 14.0 .The PSM method was the nearest neighborhood method with a caliper width of 0.20. The standard mean deviation (SMD) was calculated for all studied variables, and an SMD <0.20 was confirmed for all matched variables, which indicated appropriate matching. The postoperative overall survival (OS) and recurrence-free survival (RFS) were analyzed only in patients with newly developed HCC, using the Kaplan–Meier method. Between 2010 and October 2022, we performed 667 liver resections for HCC, including 306 open, 279 laparoscopic, and 82 robotic resections, at our institution. Among these 667 cases, we retrospectively reviewed the consecutive 327 AR cases, consisting of 185 OAR and 142 MIAR (102 laparoscopic and 40 robotic) cases, where AR using the extrahepatic Glissonian approach was performed without biliary or vascular reconstruction or concomitant resection of extrahepatic organs.Patient and Tumor Baseline DataPatient and tumor baseline data were compared between OAR and MIAR . Before Comparative perioperative outcomes are shown in p = 0.028), less blood loss , a lower transfusion rate , a lower maximum TB , higher maximum AST , and lower maximum CRP  levels. Furthermore, MIAR was significantly associated with lower rates of overall  and major  morbidity, a lower rate of bile leak or collection , and shorter LOS .After 1:1 PSM (91:91), compared to OAR, MIAR was significantly associated with longer operative time , the higher maximum AST level , and the lower minimum PT level  in the robotic AR group. After PSM, both groups had comparable perioperative outcomes.Perioperative outcomes are shown in In the next set of analyses, we studied long-term outcomes after AR in 276 patients with newly developed HCC, who underwent 163 OARs and 113 MIARs (89 laparoscopic and 24 robotic ARs), respectively, and compared OS and RFS between OAR and MIAR and between laparoscopic and robotic AR.Baseline data were compared between 163 patients undergoing OAR and 113 patients undergoing MIAR for newly developed HCC . Before p < 0.0001; 5-year rate: 84.4% vs. 50.0%) and longer RFS  than OAR patients. For the matched cohorts after PSM, compared to OAR (n = 76), MIAR patients (n = 76) were associated with statistically longer OS  and longer RFS .Comparing survival rates between the unmatched patients undergoing MIAR (n = 113) and OAR (n = 163), MIAR patients had statistically longer OS (p = 0.006) and a lower rate of tumors ≥4.0 cm . One-to-one PSM identified 22 laparoscopic and 22 robotic matched cases. After PSM, all evaluated patient and tumor characteristics were comparable between the laparoscopic and robotic AR cohorts.Patient and tumor baseline data were compared between the unmatched 89 laparoscopic AR and 24 robotic AR cases . Before Comparison between the unmatched cohorts of laparoscopic (n = 89) and robotic (n = 24) approaches showed comparable OS A and RFSp < 0.0001). Between the matched OAR and MIAR cohorts (76:76), the recurrence rate was still significantly higher in OAR  than in MIAR  (p = 0.033).In the study period, tumor recurrence was observed in 162 (58.7%) of the entire 276 patients, including 111 OAR and 51 MIAR patients; the recurrence rate was significantly lower in the unmatched MIAR than in the OAR cohorts  and a significantly lower rate of the first recurrence within 1 postoperative year . Pathologic tumor stages were more advanced in recurrent cases in the unmatched OAR cohort (p = 0.008), while they were comparable between the matched OAR and MIAR cohorts. Pathologic tumor differentiations were comparable between the recurrent cases in the unmatched and matched cohorts. Perioperative morbidity, which has been suggested to affect tumor recurrence in previous studies [The details of recurrence and its treatments are shown in  studies ,22, was p = 0.094) and repeat hepatectomy . Systemic pharmacological treatment using molecular targeted agents (MTAs), such as sorafenib and lenvatinib, or immunotherapy using immune checkpoint inhibitors (ICIs), such as atezolizumab, were performed at a similar rate between the OAR and MIAR cohorts with recurrence (17.7% vs. 17.1%). On the other hand, in the matched cohorts, patterns of recurrence, RFS, timing of the first recurrence, and the rate of resection or systemic pharmacological treatment for recurrent tumors were comparable between OAR and MIAR . Further, when the study period was divided into the early (2010–2015) and late (2016–2022) periods, MIAR was significantly more frequently performed than OAR in the late period in a comparison between the unmatched (n = 95 (84.1%) vs. n = 40 (24.5%), p < 0.0001) and matched (n = 61 (80.3%) vs. n = 14 (18.4%), p < 0.0001) cohorts.To further investigate factors that potentially affect the long-term outcomes after resection of newly developed HCC, we studied the times or era of surgery because significant changes occurred socially in the strategies of HCC treatment as well as in the control of HCV using direct acting antivirals (DAAs) during the study period. We first examined the trends of application of OAR or MIAR throughout the study period . As showp = 0.001). Furthermore, RFS in MIAR, with a 5-year rate of 47.4% and the median time of 40.4 months, was significantly longer than RFS in OAR, with a 5-year rate of 21.8% and a median time of 7.6 months (p = 0.004).At our institution, eradication of HCV using DAAs became active from around 2016, which was coincidental with a remarkable change in the surgical approach from OAR to MIAR. To exclude the impacts of HCV eradication on postoperative recurrence and hepatic functional status and to study the simple impact of different approaches on survival, we examined the long-term outcomes in 184 AR cases without associated HCV infection, i.e., 55 HBV and 129 NBNC cases. One-to-one PSM identified the 42 OAR and 42 MIAR matched cases from the 107 OAR and 77 MIAR unmatched cases. The survival data showed that OS in MIAR, with a 5-year rate of 85.2% and not-reached median survival, was significantly longer than OS in OAR, with a 5-year rate of 43.3% and the median survival time of 48.9 months -based analyses showed that compared to OAR, MIAR was significantly associated with less blood loss, a lower transfusion rate, lower postoperative TB and CRP levels, lower rates of 90-day overall and major morbidity and bile leak or intraabdominal collection, a lower rate of 90-day mortality, and shorter LOS. On the other hand, a significantly higher postoperative AST level was observed in the MIAR compared to the OAR group. Next, we compared perioperative outcomes between matched laparoscopic and robotic AR cohorts (46:46), which showed comparable results. Finally, we studied the long-term outcomes after AR for newly developed HCC. PSM-based analyses matching patient and tumor characteristics and types of resection showed comparable or potentially superior OS and RFS in the MIAR cohort compared to those in the OAR cohort, as well as comparable outcomes between the laparoscopic and robotic approaches.As for technical aspects of MIAR, we believe, from the current and previous results, that we can standardize MIAR by using the extrahepatic Glissonian approach and HV root-at first one-way parenchymal dissection ,18,19,23Previous studies comparing open and minimally invasive liver resections have shown less blood loss, decreased morbidity, and shorter LOS in the latter ,27,28,29Several studies have shown comparable long-term outcomes after resection of HCC between the open and minimally invasive approaches ,28. OnlyOn the other hand, it is worth noting that even the matched MIAR cohort after PSM had significantly longer OS and RFS than the OAR cohort. To seek the scenarios behind such potentially better survivals in MIAR, we addressed several relevant points. First, we examined the details of recurrence and found that the patterns of recurrence, the incidence of extrahepatic metastasis and pathologic tumor differentiation, and stages were comparable between the cohorts . Second,Other factors potentially contributing to the more favorable RFS in MIAR include theoretical immunological advantages conferred by its lower invasiveness. The advantages can stem from factors including less physical damage, less disturbance in the perioperative metabolic and nutritional status, and the resultant early recovery after surgery. In our results, such lower invasiveness in MIAR was evidenced by less blood loss, the lower levels of postoperative TB and CRP, and shorter LOS, compared to OAR . FurtherAnother point to be taken into consideration is that in most previous studies comparing open and minimally invasive hepatectomy, both AR and NAR were included. Since NAR tended to be performed for HCC patients with impaired hepatic functional reserve, inclusion of both AR and NAR cases in the study cohorts and the case number ratio of each cohort may have affected the entire comparative survival data on which both oncologic and hepatic functional factors had significant impacts.Collectively, our data were not enough to explain the differences in the long-term outcomes between the matched OAR and MIAR cohorts, and in the first place, the relatively small sample size in each cohort precludes definite conclusions. Nonetheless, our results suggest that the long-term outcomes after AR for newly developed HCC were at least comparable between OAR and MIAR. Larger studies incorporating more evaluation facets, such as post-recurrence treatment modalities, patient nutritional status and immunity characteristics and tumor genetic information, are necessary to investigate the impact of a minimally invasive approach to AR on long-term outcomes of resected HCC.In view of our results showing mostly better perioperative outcomes in MIAR than in OAR, as well as comparable or potentially more favorable long-term outcomes in MIAR, it is reasonable to suggest that MIAR would be the first choice for a surgical approach to HCC, at least by an expert. On the other hand, despite the expected functional merits of robotics providing surgical dexterity, the robotic approach did not show advantages in surgical outcomes over the laparoscopic approach to AR in this study. However, the sample size was small, particularly in the robotic group, and techniques of robotic liver surgery are still developing. Nonetheless, the robotic platform may have potential advantages in perioperative outcomes in complex anatomic resection, such as less blood loss, decreased open conversion rate, and decreased morbidity, as suggested in other study settings ,32,33. LThere are several limitations to this study. First, this is a retrospective, observational, single-center study, though PSM was conducted to reduce biases. Second, the sample sizes in each PSM-matched cohort are relatively small. Third, long-term outcomes should be carefully interpreted because of the abovementioned small sample size, small number of matched variables in PSM-based analyses, and potential biases from ‘difficult-to-match’ factors, including selection of approach, learning curve of techniques, patient immunological status, era-dependent development of DAAs for HCV eradication, and advances in post-recurrence pharmacological therapy for HCC.Although MIAR is technically demanding, particularly for HCC, because of potential underlying liver dysfunction, it can be technically standardized by the extrahepatic Glissonian approach and HV root-at first one-way parenchymal dissection. Furthermore, MIAR for HCC was safe, feasible, and oncologically acceptable, with perioperative outcomes mostly superior to those in OAR and with comparable or potentially more favorable long-term outcomes. A laparoscopic or robotic approach would be the first choice for AR in selected surgical HCC patients, at least by experts. Further larger studies are warranted to investigate potential advantages of MIAR for HCC in terms of long-term outcomes, as well as perioperative and long-term advantages of the robotic or laparoscopic approach over the counterpart.


Before starting surgery for the resection of an intracranial tumor, its outlines are typically marked on the skin of the patient. This allows for the planning of the optimal skin incision, craniotomy, and angle of approach. Conventionally, the surgeon determines tumor borders using neuronavigation with a tracked pointer. However, interpretation errors can lead to important deviations, especially for deep-seated tumors, potentially resulting in a suboptimal approach with incomplete exposure. Augmented reality (AR) allows displaying of the tumor and critical structures directly on the patient, which can simplify and improve surgical preparation.We developed an AR-based workflow for intracranial tumor resection planning deployed on the Microsoft HoloLens II, which exploits the built-in infrared-camera for tracking the patient. We initially performed a phantom study to assess the accuracy of the registration and tracking. Following this, we evaluated the AR-based planning step in a prospective clinical study for patients undergoing resection of a brain tumor. This planning step was performed by 12 surgeons and trainees with varying degrees of experience. After patient registration, tumor outlines were marked on the patient's skin by different investigators, consecutively using a conventional neuronavigation system and an AR-based system. Their performance in both registration and delineation was measured in terms of accuracy and duration and compared.p < 0.001), with an average time reduction of 39%.During phantom testing, registration errors remained below 2.0 mm and 2.0° for both AR-based navigation and conventional neuronavigation, with no significant difference between both systems. In the prospective clinical trial, 20 patients underwent tumor resection planning. Registration accuracy was independent of user experience for both AR-based navigation and the commercial neuronavigation system. AR-guided tumor delineation was deemed superior in 65% of cases, equally good in 30% of cases, and inferior in 5% of cases when compared to the conventional navigation system. The overall planning time  was significantly reduced through the adoption of the AR workflow (By providing a more intuitive visualization of relevant data to the surgeon, AR navigation provides an accurate method for tumor resection planning that is quicker and more intuitive than conventional neuronavigation. Further research should focus on intraoperative implementations. Prior to the resection of intracranial tumors, careful planning and preparation are required. This entails the review of preoperative medical imaging data as well as the preparation of the computer-assisted surgical navigation hardware , allowing the surgeon to plan an optimal approach to the tumor. The plan consists of an entry point and a target; the entry point defines the center of approach around which the craniotomy will be made, the target being the whole tumor. Often, this plan is refined immediately prior to the intervention, after patient registration, when the tumor outline is drawn on the patient's skin to serve as a guide for incision and craniotomy.A neuronavigation system is often used to define a virtual three-dimensional (3D) coordinate system around a fixed patient and spatially align any prior medical imaging data to the patient within that coordinate system. This process is known as registration and can be achieved through accurate tracking of a hand-held stylus within the virtual coordinate system. The stylus allows the surgeon to identify points on the patient's face or localize fiducials. These data serve as the target for aligning imaging data. Following this, the tracked stylus is then used to localize tumor borders, which serve as a basis for the plan. The drawbacks of this approach are inherent limitations of current state-of-the-art neuronavigation systems. First, visualization of the imaging data used to guide the surgeon is typically carried out on secondary displays, resulting in a disconnect between the surgeon and the patient. Second, the 3D imaging data are often represented as two-dimensional (2D) projections, either orthogonal multiplanar reconstructions or oblique views in line with the stylus. This implies that the user performs a mental transformation between the 2D space on the monitor and the 3D space of the patient, often without the ability to focus on both spaces concurrently. Although 3D visualization techniques such as volume rendering or segmented surface models aid in the interpretation of the imaging data, the disconnection in space and the cognitive burden of the transformation still remain . The conAugmented reality (AR) has the potential to address these limitations, by providing a means to view relevant 3D medical imaging data superimposed onto the patient. This provides a more intuitive data representation as it keeps the surgeon's focus on the patient and reduces the mental load of transforming information , 2. IvanTo address the current limitations of conventional neuronavigation systems and off-the-shelf AR hardware, we developed an AR-based navigation system deployed on the Microsoft HoloLens II , which ehttp://slicer.org, version 4.10.2) to produce a 3D “skin” model of the phantom head (http://openigtlink.org). A ground truth registration transform was defined through least-square-fit between the infrared-reflecting sphere coordinates in CT space and their corresponding coordinates in local tracker space. The latter was established through caliper measurements (Mitutoyo 530–112). The registration quality was calculated as the relative transformation to the ground truth.Registration performance of the AR-based navigation system was evaluated through a phantom study. A phantom consisting of an Alderson Radiation Therapy Phantom (slices 1–9)  rigidly affixed to a reference star with four infrared-reflecting spheres  was CT scanned . This sctom head . The 3D tom head , while 1A prospective clinical pilot study was designed to evaluate registration and tracking accuracy as well as the practical advantages of the developed AR navigation system. The primary endpoint was the validation of the registration and delineation accuracy in a direct comparison with a conventional neuronavigation system, and the secondary endpoint was the time it took to perform the preoperative surgical planning. Adult patients at the University Hospital of Brussels  diagnosed with an intracerebral tumor and planned for resection surgery were eligible for inclusion in the study. Exclusion criteria included patients with extra-axial tumors and those requiring prone positioning during the intervention. The study protocol was approved by the Ethics Committee of UZ Brussel and validated by the Belgian Federal Agency of Medicines and Health Products (FAGG/AFMPS). All patients provided written informed consent.A total of 12 surgeons and trainees with varying degrees of experience were involved in this study. Resection planning was performed preoperatively with the patient positioned on the operating table, the head rigidly fixed in a Mayfield clamp. At the start of the experiment, the expert neurosurgeon performing the surgery defined the angle of approach for the surgery by marking the central entry point. This point served as the reference for tumor delineation for both the conventional neuronavigation and the AR system. During the experiment, the planning was performed in two steps: first the registration step by using a tracing method with a tracked pointer, and second the delineation step indicating the tumor outlines, projected on the patient's skin from the chosen approach angle, with a marker. The registration step and delineation step were both timed. After registration, the person performing the tasks could gather the necessary equipment and proceed to the delineation step without being timed.A flow diagram of the experiment for a single case is shown in Conventional navigation was performed using the Brainlab Curve system, and the included infrared tracked stylus was used to register preoperative medical imaging data to the fixed patient. Following the device protocol, we sequentially identified three patient landmarks  using the stylus to define an initial alignment. Next, a sparse point cloud was sampled from the patient's skin, which the system used to refine the registration. Following this, the investigator verified the registration by identifying recognizable landmarks on the patient's skin and checking for correspondences on the navigation system.Upon completion of the registration, the investigator proceeded to the tumor delineation. From the predefined central entry point, the investigator identified the anterior, posterior, medial, and lateral borders of the tumor using the tracked pointer and inspecting the inline views of the imaging data on the external screen and outlined these on the patient's skin. When the investigator was satisfied with the result, completion was confirmed.Preoperatively, surface meshes were generated based on the patient's preoperative medical imaging data using Brainlab Elements software. This resulted in a skin, cerebrum, and tumor model. The surface meshes were post-processed to reduce vertex count using quadric-edge-collapse decimation, with a target vertex count of approximately 10,000 each. In addition, we logged four 3D landmarks defining the patient's left/right lateral canthus, nasion, and tip-of-nose. These models and 3D landmark files were loaded into the AR application prior to the procedure.The application to be deployed on the HoloLens II AR-HMD was developed using the Unity game development environment (version 2019.4.28f1) and incorporated inside-out infrared tracking . The styTo be able to compare results between techniques but also to minimize any learning curve, the workflow of the application was designed to mimic the workflow of the neuronavigation system and consisted of three key stages. The first is the definition of a 3D coordinate system around the patient. This is performed by defining a coordinate system around the Brainlab reference star, whose tracked pose served as a basis for all data processing. The application advances to the next stage only after establishing a stable star-defined coordinate system.The next stage entails the registration between medical imaging data and the patient . Using tThe third and final stage is planning. For this, we display an orthographic projection of the tumor (originating from its center of mass) which follows the stylus' tip position . The tumFor each clinical case, the planning using either guidance technique was performed by two different investigators in order to attenuate learning effects resulting from the first technique. Following the above protocols, both trainee and expert surgeons performed clinical trials using both devices. This provided insight into operator variance for each system at varying skill levels. To average out interindividual differences in planning accuracy and duration, the investigators were rotated over both guidance techniques. Tumor delineation accuracy was evaluated by direct comparison between the delineation resulting from either the AR or the neuronavigation workflow and a delineation of the expert surgeon using neuronavigation. For this purpose, photographs from a fixed camera viewpoint were used. The investigator delineation was assessed by the expert neurosurgeon and scored in terms of resemblance to the expert delineation and detail of delineation. Workflow duration, including registration and delineation steps, for both the neuronavigation system and AR system, was timed using a stopwatch.t-test. The comparison of registration variance between both techniques was assessed through a one-way f-test. We considered p-values of < 0.05 to be statistically significant.Statistical analysis was performed in Python (3.9) with the SciPy statistics library (1.7.0). Comparison between workflow duration data for both the AR and conventional techniques was assessed through Welch's p = 0.57) from the conventional navigation system: 1.17 ± 1.46 mm and 1.05 ± 1.37°. However, with outlier data removed  from conventional neuronavigation registration data, the registration error was reduced to 0.39 ± 0.14 mm and 0.38 ± 0.19°, which was statistically better than the registration performed on the AR device (p = 0.014). The root mean square point-to-plane distances (istances  between p = 0.14) when compared to the traditional navigation system (n = 13), equally good in 30% of cases (n = 6), and inferior in 5% of cases (n = 1) when compared to the conventional navigation system  (p < 0.001), though a significant reduction in registration time between the techniques was also seen (p = 0.014). Registration and tumor delineation using AR navigation was subjectively assessed to be more intuitive by all test subjects.Overall planning time  was significantly reduced when using AR navigation (< 0.001) . In 90% We performed a prospective clinical pilot study investigating the accuracy and efficacy of an AR solution toward tumor resection planning. Our primary goal was to demonstrate that the registration and delineation accuracy of the AR approach met the accuracy performance of a conventional neuronavigation system. The evaluation on a phantom head indicated that the registration workflow of the AR system was accurate and could perform adequately in an operating room environment. During our clinical trial in the operating room, we demonstrated that AR assistance was quick and intuitive, and allowed accurate tumor resection planning in a neurosurgical setting. The proposed system closely matched the registration quality of the state-of-the-art neuronavigation system, while improving the accuracy and reducing the duration of the resection planning.Other research groups have focused on the use of off-the-shelf AR-HMDs for surgical navigation, as these devices often contain much of the hardware required for computer-aided navigation, i.e., computational power, display, and tracking. However, since the current consumer-grade HMDs have a tracking error of approximately 2 cm and offer no direct tracking of the patient, nor a quantifiable registration method, a dedicated tracking optimization needs to be developed before surgical use is possible . VariousIn terms of registration accuracy, the calculated ground truth registration resulted in a mean point-to-point transformation error of 0.39 mm between the estimated local infrared-reflecting sphere coordinates of the Brainlab star and their corresponding position in CT space, indicating an acceptable ground truth to compare against. The registration performance obtained on the AR device (1.42 ± 0.42 mm and 0.95 ± 0.36°) surpasses prior state-of-the-art AR-HMD registration quality of 3.9 ± 1.8 mm and 4.9 ± 2.4° and is nearing parity with commercial systems . The prep < 0.001) (p < 0.001), highlighting the increased intuitiveness for tumor delineation using the AR visualization. This intuitiveness is corroborated by the significant reduction in registration time (p = 0.014) in the absence of a significant difference in registration variability compared to the conventional navigation system. The abovementioned results, in addition to our previous findings on intracranial drain placement, lead us to hypothesize that AR has the ability to flatten the learning curve and increase surgeon confidence for tumor resection planning as well, by making the planning step more visual in nature  , with ann nature . HoweverFrom a technical standpoint, there were two prominent limitations. The first was the lack of a known ground-truth local geometry of the Brainlab reference star, necessitating an estimation. This limited the quality of a ground truth registration transform when quantifying the performance of the Brainlab system. Second, the handheld stylus used in the proposed AR solution was pre-calibrated, and the tooltip location was taken as a constant throughout the trial. It is possible that this could have been corrupted through mechanical means, resulting in inaccurate data sampling. This could also explain the AP registration error observed during phantom trials . With reFurther research should focus on expanding and evaluating the use of high-accuracy AR-HMD guidance intraoperatively, where experienced neurosurgeons use the AR system not only during the planning step but also throughout later steps of the surgery while still using the conventional neuronavigation system as the principal source of surgical guidance. Since magnification is often not possible on these HMDs, the implementation of an exoscope as a source of magnification, in combination with an AR or VR headset, should be explored and compared to an AR infusion already available in the microscope.We developed an AR navigation system, deployed on the HoloLens II AR-HMD, that matches the registration accuracy of a state-of-the-art neuronavigation system. By providing a more intuitive visualization of relevant data to the surgeon, the AR navigation workflow provides an accurate method for tumor resection planning that resulted in more detailed tumor delineations and reduced preoperative planning time when compared to the conventional neuronavigation system. Further research should focus on intraoperative implementations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of UZ Brussel and validated by the Belgian Federal Agency of Medicines and Health Products (FAGG/AFMPS). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FV, TF, WG, MB, BJ, TS, JV, and JD: research design. FV, TF, FB, WG, QN, MB, and JD: acquisition of data. FV, TF, FB, JV, and JD: analysis and interpretation of data. FV, TF, FB, WG, TS, JV, and JD: drafted the manuscript. All authors: critically revised the manuscript. All listed authors have made substantial contributions to the presented study, read, and approved the final submitted manuscript.


By performing in-vacuo angle-resolved photoemission spectroscopy (ARPES) and ex-situ electrical transport measurements, we find that the superconducting transition temperature and the upper critical magnetic field are suppressed when the chemical potential approaches the Dirac point. We provide evidence to show that the observed interfacial superconductivity and its chemical potential dependence is the result of the competition between the Ruderman-Kittel-Kasuya-Yosida-type ferromagnetic coupling mediated by Dirac surface states and antiferromagnetic exchange couplings that generate the bicollinear antiferromagnetic order in the FeTe layer.Over the last decade, the possibility of realizing topological superconductivity (TSC) has generated much excitement. TSC can be created in electronic systems where the topological and superconducting orders coexist, motivating the continued exploration of candidate material platforms to this end. Here, we use molecular beam epitaxy (MBE) to synthesize heterostructures that host emergent interfacial superconductivity when a non-superconducting antiferromagnet (FeTe) is interfaced with a topological insulator (TI)  2Te3/FeTe bilayers, which feature emergent superconductivity at the interface with Tc ~ 12 K. Through angle-resolved photoemission spectroscopy and electrical transport measurements, they argue that the Dirac-fermion-mediated Ruderman-Kittel-Kasuya-Yosida-type interaction weakens antiferromagnetic order in FeTe layer, allowing for superconductivity.The authors study  This idea led to the fabrication of semiconductor nanowires covered with superconducting layers7 and magnetic metallic wires on superconducting substrates9. So far, several experiments have reported evidence of MZM in 1D semiconducting nanowire/superconductor devices, such as unusual zero-bias conductance peak6 and fractional AC Josephson effect7. However, there are serious concerns about the validity of such claims of MZMs in these devices12. These concerns provide new impetus to explore TSC in other systems.Decoherence of a quantum state causes errors during the process of quantum computation and thus is the major obstacle to the realization of practical quantum computers. Majorana zero modes (MZMs), which obey non-Abelian statistics, are arguably the most promising anyon platform towards fault-tolerant quantum computing since they are expected to be inherently robust against local perturbation and decoherence effects. MZMs are expected to be bound to vortex cores in the presence of topological superconductivity (TSC) and appear as a zero-energy state inside the superconducting gap. The TSC phase can be created in hybrid structures where an electronic system with spin-split bands caused by strong spin-orbit coupling (SOC) is proximally coupled to a conventional s-wave superconductor through the proximity effect, TSC is expected to emerge2. Compared to the Rashba bands (i.e. the strong SOC-induced momentum-dependent band splitting) in 1D semiconducting nanowire systems14, TI films have two advantages: (i) The helical Dirac surface states of TIs lift the spin degeneracy, thus bypassing the SOC-plus-Zeeman splitting control needed in 1D nanowire systems;4 (ii) The bandgap of canonical TIs such as the Bi-chalcogenides is typically a few hundred meV16 as compared to the magnetic field-induced Zeeman splitting of ~1 meV in 1D nanowire systems6. A large gap provides more flexibility for the realization of the TSC phase. Moreover, TI/superconductor heterostructures allow surface-sensitive probes such as angle-resolved photoemission spectroscopy (ARPES) and scanning tunneling microscopy and spectroscopy (STM/S) to directly probe the surface states. Such heterostructures also allow established transport techniques to be deployed for the exploration of the putative Majorana physics2. An ideal TI-based TSC platform would be comprised of an epitaxial heterostructure wherein both TI and superconductor layers are synthesized by molecular beam epitaxy (MBE), thus allowing precise engineering of the TI/superconductor interface and the band structure. However, there remains an important obstacle, specifically, the superconductivity in MBE-grown thin superconducting films usually disappears once a TI layer is grown on top, presumably due to the occurrence of charge transfer17.Since topological insulators (TIs) possess strong SOC-induced inherent topological nontrivial band structures with inverted bulk conduction and valence bands, TIs provide a natural platform to pursue TSC. When a TI is coupled to a conventional 2. Recently, interfacial superconductivity has been observed in Bi2Te3/FeTe and Sb2Te3/FeTe heterostructures with superconducting temperatures of Tc ~ 11.5 K and ~3.1 K, respectively19. In these two heterostructures, Bi2Te3 and Sb2Te3 are heavily electron- and hole-doped TI, respectively21, while FeTe is an antiferromagnetic iron chalcogenide that is non-superconducting without element doping23 and/or tensile stress24. Notably, a proximity effect-induced superconducting gap has been demonstrated on the top surface of the Bi2Te3/FeTe heterostructure, setting the stage for the potential TSC phase25. Compared to bulk iron-based superconductors with a band topology such as FeTe0.55Se0.45 (ref. 26) and LiFeAs (ref. 27), the 2Te3/FeTe heterostructures provide the tunability of chemical potential across a single Dirac cone, without resorting to introducing disorder or applying strain in the sample to tune for the TSC phase.An alternative approach is to exploit the emergent interfacial superconductivity that arises in certain TI/non-superconducting material heterostructures that fulfill the two essential ingredients of TSC, i.e. topological and superconducting orders2Te3/FeTe heterostructures with an atomically sharp interface, in which the chemical potential of the ternary TI 2Te3 can be tuned by varying the Bi/Sb ratio29. By performing in vacuo ARPES and ex situ electrical transport measurements, we find that interfacial superconductivity appears in all 2Te3/FeTe heterostructures that we studied and we find both the superconducting transition temperature and the upper critical magnetic field are suppressed when the chemical potential approaches the Dirac point. This observation indicates the correlation between massless Dirac electrons of the TI layer and the interfacial superconductivity in 2Te3/FeTe heterostructures. We show evidence that the chemical potential dependence of interfacial superconductivity is a result of the competition between bicollinear antiferromagnetic order in the FeTe layer and the Ruderman-Kittel-Kasuya-Yosida (RKKY) interaction mediated by surface Dirac electrons of the TI layer30.In this work, we use MBE to synthesize x1−Sbx)2Te3/FeTe (0 ≤ x ≤ 1) bilayer heterostructures are synthesized in an MBE chamber (ScientaOmicron), which is connected to an ARPES chamber. The MBE growth is monitored using in-situ reflection high-energy electron diffraction (RHEED) 2Te3/FeTe films are characterized by scanning transmission electron microscopy (STEM)  Fig. , and ARPEM) Fig. –11 measuEM) Fig.  with the2Te3 nor FeTe crystals by themselves are superconductors. The 2Te3 lattice is built from quintuple layer (QLs) containing two Bi layers and three Te layers29, while the FeTe unit cell (UC) consists of one Fe layer and two Te layers , the van der Waals nature of both materials allows for epitaxial growth of 2Te3/FeTe heterostructures. To evaluate the changes of in-plane lattice constant in 8QL (Bix1−Sbx)2Te3 layer on 50UC FeTe heterostructures, we extract the line intensity distribution curve from their RHEED patterns . With increasing x, the value of d expands, suggesting a gradual reduction of the lattice constant in the (Bix1-Sbx)2Te3 layer 2Te3/FeTe heterostructures. Moreover, we observe the twin domain feature with a rotation angle of ~30o in these heterostructures 2Te3 and FeTe, respectively. Moreover, a highly ordered and sharp interface is seen between 2Te3 and FeTe even with different lattice structures. Supplementary Figs. 2Te3/FeTe heterostructures.Neither ers Fig. . Althougin 8QL Bi−xSbx2Te32Te3/50 UC FeTe, and 8 QL Sb2Te3/50 UC FeTe heterostructures. The 50 UC FeTe sample shows a semiconducting-to-metallic transition at T ~ 54 K. Such behavior in the R-T curve of FeTe has been associated with the paramagnetic-to-antiferromagnetic phase transition19. This magnetic ordering temperature is known as the Néel temperature TN , the onset Tc,onset and the zero resistance Tc,0 are ~13.6 K and ~12.5 K, respectively , Tc,onset and Tc,0 are ~12.5 K and ~11.3 K, respectively 2Te3 layer, we perform in vacuo ARPES measurements on a series of (Bix1-Sbx)2Te3/FeTe heterostructures with different x 2Te3/FeTe heterostructures with 0 ≤ x ≤ 1, we observe linearly dispersed Dirac surface states at room temperature 2Te3/50 UC FeTe heterostructures. In addition, we find that with increasing x, one bulk valence band moves upwards, while the other one moves downwards 2Te3/50 UC FeTe heterostructures 2Te3/ FeTe heterostructures with different x under magnetic field μ0H = 0 T and μ0H = 9 T. As noted above, for the x = 0 sample, Tc,onset and Tc,0 are ~13.6 K and ~12.5 K, respectively  of the interfacial superconductivity in these (Bix1−Sbx)2Te3/FeTe heterostructures.To examine the role of the Dirac electrons in the development of the interfacial superconductivity, we perform electrical transport measurements on these 8 QL 2Te3/FeTe heterostructures, we perform electrical transport measurements under high perpendicular magnetic fields. Figure R-μ0H curves of six (Bix1−Sbx)2Te3/FeTe heterostructures at T = 1.5 K. We define the value of μ0Hc2,⊥ as the magnetic field at which R drops to half of the normal resistance. For the x = 0 sample, μ0Hc2,⊥ ~ 46.7 T, which is much larger than the value in a prior work18. With increasing x, the value of μ0Hc2,⊥ first decreases and then increases. Specifically, we find μ0Hc2,⊥ ~ 44.8 T, ~41.3 T, ~36.3 T, ~38.8 T, and 43 T for the x = 0.4, 0.7, 0.8, 0.95, and 1.0 samples, respectively. Therefore, μ0Hc2,⊥ has a minimum near x = 0.8 2Te3/FeTe heterostructures, we plot the values of kF, Tc,onset, Tc,0, and μ0Hc2,⊥ as a function of the Sb concentration x 2Te3/FeTe heterostructures.To reveal the link between Dirac surface states and the formation of the interfacial superconductivity in 2Te3/FeTe heterostructures. The dip observed in the phase diagram 2Te3 and FeTe layers, and (ii) the suppression of long-range antiferromagnetic order in the FeTe layer. First, both work functions in Bi2Te3 (~5.3 eV) and Sb2Te3 (~5.0 eV) are greater than that of FeTe (4.4 ~ 4.8 eV)34. This work function mismatch between (Bix1−Sbx)2Te3 and FeTe is expected to cause a charge transfer, which may lead to the different energy levels for the Dirac points on the top and bottom [i.e. the (Bix1−Sbx)2Te3/FeTe interface] surfaces of (Bix1−Sbx)2Te3. As a consequence, the minimum value of Tc is anticipated to be observed away from the Bi/Sb ratio at which the chemical potential crosses the Dirac point on the top surface of (Bix1−Sbx)2Te3. In our experiments, the chemical potential crosses the Dirac point at x = 0.8 2Te3 layer. Prior studies32 have claimed the occurrence of hole carrier transfer from Bi2Te3 to FeTe, which may give rise to interfacial superconductivity in the FeTe layer by screening out the strong Coulomb repulsion. However, in this doping effect hypothesis35, the x1−Sbx)2Te3/FeTe heterostructures is likely to be enhanced or suppressed monotonically by tuning x from 0 to 1. This assumption certainly cannot be the entire explanation in creating the interfacial superconductivity because it is inconsistent with the observed dip in our In the following, we discuss the possible origins of the interfacial superconductivity and its chemical potential dependence in 2Te3/FeTe heterostructures lies in the coupling with Dirac surface states that suppresses the long-range antiferromagnetic order of the FeTe layer. As noted above, our transport results indicate that the long-range antiferromagnetic order is weakened or even destroyed after the deposition of the (Bix1−Sbx)2Te3 layer 2Te3 layer, the local magnetic moments of Fe tend to be aligned to the spin direction of the Dirac surface states, leading to the RKKY interaction between different Fe magnetic moments. Prior studies39 have shown that the Dirac surface states mediated RKKY interaction consists of Heisenberg-like, Ising-like, and Dzyaloshinskii-Moriya (DM)-like terms, all of which are not compatible with the bi-collinear antiferromagnetic order and can lead to strong magnetic fluctuations. Therefore, the RKKY interaction, in addition to the doping effect32, may weaken or even destroy the bi-collinear antiferromagnetic order in the FeTe layer, so superconductivity is expected to emerge.To understand the yer Fig. . Besidesons Fig.  and are Tc observed in our experiments. As discussed in refs. 36, when the chemical potential approaches the Dirac point, the oscillation feature of RKKY interaction becomes weaker or even disappears as 43 have shown that that the dominant superconducting pairing is determined by the next nearest-neighbor exchange coupling 45. At the interface, x , the superconducting pairing shows a dip in the x1−Sbx)2Te3/FeTe heterostructures through the chemical potential tuning of spin-spin interaction across the interface.Next, we show that the RKKY interaction also explains the chemical potential dependence of n x Fig. , \documeram Fig. . Our exp38. A complete understanding of the influence of the complex RKKY interaction on the pairing symmetry and mechanism of the emergent interfacial superconductivity in (Bix1−Sbx)2Te3/FeTe heterostructures needs further theoretical and experimental studies.Besides the above direct correction to short-range 2Te3/FeTe heterostructures and find that interfacial superconductivity appears in these heterostructures. By performing ARPES and electrical transport measurements, we find the superconducting temperature and the upper critical magnetic fields are suppressed when the chemical potential approaches the Dirac point. These observations reveal the correlation between the interfacial superconductivity and the Dirac surface states in the TI layer, indicating the Dirac electrons in the TI layer must play a role in the formation of the interfacial superconductivity. The dip in the Tc ~ x phase diagram can be understood as a consequence of the complex competition between the RKKY interaction and antiferromagnetic exchange coupling. Our experiments provide evidence for Dirac-fermion-assisted interfacial superconductivity in 2Te3/FeTe heterostructures and thus provide strong motivation for using this as a model material system for exploring Majorana physics and topological quantum computation. We also envision that extending 2Te3/FeTe bilayers to multilayers could provide a rich platform for the pursuit of other emergent topological phenomena, including Weyl superconductors46.To summarize, we synthesize a series of x1−Sbx)2Te3/FeTe heterostructures are synthesized in a commercial MBE system (ScientaOmicron) with a vacuum better than 2 × 10−10 mbar. The insulating SrTiO3 (100) substrates are first soaked in ~80 °C deionized water for 1.5 h and then put in a diluted hydrochloric acid solution (~4.5% w/w) for 1 h. These SrTiO3 (100) substrates are then annealed at ~974 °C for 3 h in a tube furnace with flowing high-purity oxygen gas. Through the above heat treatments, the SrTiO3(100) substrate surface becomes passivated and atomically flat, which becomes suitable for the MBE growth of the FeTe films. We next load the heat-treated SrTiO3 (100) substrates into our MBE chamber and outgas them at 600 °C for ~1 h. High-purity Bi (99.9999%), Sb (99.9999%), Te (99.9999%), and Fe (99.995%) are evaporated from Knudsen effusion cells. The growth temperature is maintained at ~340 °C and ~210 °C for the MBE growth of the FeTe and 2Te3 layers, respectively. The growth rate is set to ~0.4 UC/min for the FeTe film, and ~0.2 QL/min for the 2Te3 layer, which is calibrated by measuring the thickness of the 2Te3/FeTe bilayer heterostructures using both STEM and atomic force microscopy measurements. Finally, to avoid possible contamination, a ~10 nm Te layer is deposited at room temperature on top of the bilayer heterostructures before their removal from the MBE chamber for ex situ electrical transport measurements.The 2Te3/FeTe heterostructures can be found in Supplementary Figs. The aberration-corrected ADF-STEM measurements are performed on an FEI Titanin-vacuo ARPES measurements in a chamber with a base vacuum better than 5 × 10−11 mbar. After finishing the MBE growth, the 2Te3/FeTe bilayer heterostructures are transferred from the MBE chamber to the ARPES chamber. The energy analyzer DA30L (ScientaOmicron) is used. We use a helium-discharge lamp with a photon energy of ~21.2 eV. The energy and angle resolutions are set to ~10 meV and ~0.1°, respectively. We note that He Iα light with an energy of ~21 eV is sensitive to the sample surface47.We perform 2Te3/FeTe bilayer heterostructures are scratched into a Hall bar geometry using a computer-controlled probe station. The Hall bar device size is ~1 mm × 0.5 mm 2Te3/FeTe heterostructures x1−Sbx)2Te3/FeTe heterostructure does not accurately reflect the carrier density  in the (Bix1−Sbx)2Te3 layer 2Te3 layer without the FeTe layer, a comprehensive analysis comparing kF derived from ARPES and Hall transport measurements has been carefully studied in our prior work28. Therefore, for the (Bix1−Sbx)2Te3/FeTe heterostructures used in this work, the kF value obtained from ARPES measurements is considered more reliable in reflecting the position of the chemical potential position of (Bix1−Sbx)2Te3.The Hall trace of the FeTe layer shows a nearly zero slope at low temperatures ] X-ray tube operating at ~45 kV and ~40 mA. The incident beam path includes a 2×Ge(220) asymmetric hybrid Kα1 monochromator and a 1/4° divergence slit. A PIXcel3D 1×1 detector operating in frame-based mode is used to measure the RSMs. The lower and upper levels of the pulse height distribution are set at ~4.02 and ~11.27 keV, respectively. The heterostructures are aligned using the SrTiO3(002) reflection and the azimuthal angle for off-axis peaks is aligned using the φ scans. The in-plane lattice constant is determined by measuring the off-axis peaks in the symmetric geometry after tilting the sample in χ because the reflections available for asymmetric scans had very low intensities. The 2θ positions for reflections are measured by fitting a Lorentzian function to the data.We perform XRD reciprocal space mapping (RSM) measurements on Bi2Te3/50 UC FeTe heterostructure, the out-of-plane Bi2Te3(0 0 15) and off-axis (0 1 5) reflections are measured  2Te3 layer grown on the FeTe layer implies no changes in the topological properties of (Bix1−Sbx)2Te3 (refs. 49). Moreover, the twin domains with a 30o rotation are further verified by φ scans of (Bix1−Sbx)2Te3/50 UC FeTe heterostructures . Therefore, we assume a comparable energy scale range of  meV for TN ~ 68 K)53. Therefore, it can significantly affect 2Te3/FeTe heterostructures.We employ the above method to calculate the strength of RKKY interaction in the Tc. Our theoretical calculations point out a possible macroscopic model to understand the superconducting Spin-singletwave superconductivity” below). In the context of the standard mean-field theory, the decomposition of the 40. The smaller Tc. Therefore, our experimental and theoretical studies suggest that the Dirac electrons participate in the formation of the interfacial superconductivity in 2Te3/FeTe heterostructures.As discussed in the main text, the ferromagnetic type RKKY interaction competes with the superconducting order near the TI/FeTe interface, which can lower ing Fig. 40. The ss-wave pairing symmetry in iron-based superconductors, which is induced by the 43. By solving the linearized gap equation, we will derive the corresponding mean-field 54.We employ the mean-field theory to study the Next, we consider the mean-field decomposition for the Tc dip observed in our experiments.After transforming the spin-spin interaction Hamiltonian to the electron-electron interaction Hamiltonian, we can perform the mean field decomposition to derive the linearized gap equation Supplementary InformationPeer Review File


Epidemiological data on Bell's palsy are vital for elucidating disease prevalence and enhancing therapeutic options. Our objective was to explore the prevalence and possible risk factors associated with Bell's palsy recurrence in the Clinical Center of the University of Debrecen service area. Secondary data analysis was performed using hospital discharge data, including patient information and comorbidities.Data was obtained from the Clinical Center of the University of Debrecen, on Bell's palsy patients who were treated at the hospital between January 1, 2015 and December 31, 2021. Multiple logistic regression analysis was used to examine the factors associated with Bell's palsy recurrence.Of the 613 patients analyzed, 5.87% had recurrent paralysis, and the median time interval between episodes was 315 days. Hypertension was significantly associated with Bell's palsy recurrence. Moreover, seasonal distribution analysis revealed that the number of Bell's palsy episodes was higher in colder seasons, with spring and winter having a significantly higher number of episodes than summer and autumn.This study provides insights into the prevalence and associated risk factors of Bell's palsy recurrence, which could aid in its management and help reduce the long-term consequences of the disease. Further research is necessary to determine the precise mechanisms underlying these findings. Despite the fact that Bell's palsy is a widespread condition, the epidemiological data on the disease are scarce . Bell's Infectious, immunological and ischemic processes are all implicated in the pathogenesis of Bell's palsy . The mosIn different groups, the yearly incidence of Bell's palsy has been found to range from 11 to 40/100,000 people , with a About 30% of patients are expected to experience long-term consequence, including incomplete eye closure, crocodile tears, oral incompetence during eating and drinking, articulation problems, muscular contracture, synkinesis, and facial discomfort. Facial palsy can negatively affect psychological wellbeing, quality of life and cause functional and aesthetic deficits . PatientThe variables linked to the occurrence of Bell's palsy, such as age, gender, season, pregnancy, and diabetes mellitus, have been subjected to much debate , 10, 14.The aim of this research was to explore the possible influencing factors and to estimate the prevalence of the reoccurrence of Bell's palsy in the service area of the Clinical Center of University of Debrecen. Moreover, we also aimed to describe the seasonality of the disease.Based on the information from the Clinical Center of University of Debrecen, secondary data analysis was carried out using hospital discharge data . Bell's palsy cases were identified by ICD-10 codes (G51.0). The data contained detailed information  on Bell's palsy patients treated at the institution between January 1, 2015, and December 31, 2021. To identify reoccurrence of cases, we defined a minimum 90 days interval between the last and the next admission of patients  were excluded, thus the final sample consisted of 613 patients with BP.The total sample consisted of 650 patients. Patients with missing information on the side of paralysis  codes based on free text analysis. Proportions with 95% confidence intervals were calculated for episodes in seasons. Multiple logistic regression model was used to explore the factors associated with the occurrence of Bell's palsy. Odds ratio (OR) with the corresponding 95% confidence intervals were calculated to determine the strength of association. Stata v17  was used for statistical analysis; The study was approved by the Regional Ethical Committee of University of Debrecen [5678-2021].n = 317) and 48.29% were female (n = 296). The age of the studied patients varied between 1 and 96 years, the mean age (±SD) was 43.02 ± 22.96 years.Of the 613 patients 51.71% were male  . Figure Thirty-six patients (5.87%) had recurrent paralysis 90 days after last admission. The median time interval between the last and next episode was 315 days.p = 0.095, 95% CI: 0.078–1.01). Gender was also not significant . However, hypertension was found to be a significant predictor of Bell's palsy recurrence . The analysis also revealed that the season was a significant predictor of Bell's palsy recurrence, where autumn compared with spring  and dyslipidemia  were significantly associated with higher odds of recurrence.In summary, age and gender were not found to be a significant factor in the model. In the present research, the mean age (±SD) was 43.02 ± 22.96 years, and 51.71% of the patients were male, which showed similar pattern to the existing literature regarding demographic composition of patients with Bell's palsy . AccordiIn this study, recurrent Bell's palsy was observed in 36 cases (5.87%), which was occurred mostly within 1 year after the last admission. The recurrent Bell's palsy, which can develop on either the ipsilateral or contralateral side of the first episode, is more likely to occur in the first 2 years from the onset . FindingOur results showed that some factors were found to be associated with the recurrence such as hyperlipidemia, hypertension and seasonal factors , while age, gender and NIDDM were not significant predictors.In our study, only hypertension showed significant association with recurrence. Patients with Bell's palsy had a higher rate of arterial hypertension . The delAccording to the literature, diabetes mellitus, as well as hypertension, dyslipidemia, and the combination of all three comorbidities  in a single patient, may affect the initial severity of blood pressure .The influence of diabetes mellitus, hypertension, and dyslipidemia on the result of the facial nerve in Bell's palsy is still the subject of an ongoing debate. Some authors have established in more recent research that there is no link between diabetes mellitus, hypertension, dyslipidemia, and eventual recovery from Bell's palsy .According to our results, Bell's palsy has been observed to occur more likely in spring and winter, while reoccurrence showed highest peak in autumn. Others also found that a higher incidence occurs during the cold seasons , 29. TheThe specific etiology of Bell's palsy needs to be determined in order to develop targeted treatment strategies . ImmediaThe strength of this study is that the database is representative to the Eastern Hungarian population, and we were able to investigate a time period. The study has some limitations. First, although the studied population is large, extrapolation of results should be made with caution, because our study is not representative for the whole Hungarian population. We used administrative database containing hospital discharge data, in which several lifestyle and socioeconomic factors could be underestimated; therefore, these factors were not involved in this secondary data analyses.Our findings highlight the importance of considering factors  when managing patients with Bell's palsy. Bell's palsy was more frequently observed throughout the winter and spring seasons, while the peak of reoccurrence was in autumn.There has been a lot of debate over whether underlying comorbidities like diabetes and hypertension have a role in the recurrence of Bell's palsy.Further research is needed to better understand the underlying mechanisms and etiology of disease to prevent recurrent Bell's palsy and improve the patients' quality of life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Study design, manuscript drafting, data collection, and analysis and interpretation: EV and AN. Critical revision of the manuscript: UB, IS, LH, and NK. All authors approval of the final version for publication.


Crystal violet staining and scanning electron microscopy revealed the synergistic action of colistin-FFA against biofilms. When used to treat murine RAW264.7 macrophages, this combination did not induce any adverse toxicity. Strikingly, the survival rates of bacterially infected Galleria mellonella larvae were improved by such combination treatment, which was also sufficient to reduce the measured bacterial loads in a murine thigh infection model. Mechanistic propidium iodide (PI) staining analysis further demonstrated the ability of these agents to alter bacterial permeability in a manner that enhanced the efficacy of colistin treatment. Together, these data thus demonstrate that colistin and FFA can be synergistically combined to combat the spread of COL-R Gram-negative bacteria, providing a promising therapeutic tool with the potential to protect against COL-R bacterial infections and improve patient outcomes.The continuous development of multidrug-resistant (MDR) Gram-negative bacteria poses a serious risk to public health on a worldwide scale. Colistin is used as the last-line antibiotic for the treatment of MDR pathogens, and colistin-resistant (COL-R) bacterial emergence thus has the potential to have a severe adverse impact on patient outcomes. In this study, synergistic activity was observed when colistin and flufenamic acid (FFA) were combined and used for the IMPORTANCE Colistin is a last-line antibiotic used for the treatment of MDR Gram-negative bacterial infections. However, increasing resistance to it has been observed during clinical treatment. In this work, we assessed the efficacy of the combination of colistin and FFA for the treatment of COL-R bacterial isolates, demonstrating that the combined treatment has effective antibacterial and antibiofilm activities. Due to its low cytotoxicity and good therapeutic effects in vitro, the colistin-FFA combination may be a potential candidate for research into a resistance-modifying agent to combat infections caused by COL-R Gram-negative bacteria. The rapid emergence of multidrug-resistant (MDR) bacteria due to the widespread and improper use of antibiotics poses a serious threat to global public health . The useBiofilms are communities of bacteria embedded in a three-dimensional (3D) matrix of extracellular polymeric substances that enable these microbes to persist in harsh environments . These bCandida albicans, Salmonella enterica serovar Typhimurium, and methicillin-resistant Staphylococcus aureus (MRSA) and to prevent the establishment of MRSA biofilms , a nonsteroidal anti-inflammatory drug (NSAID), is frequently used for the treatment of rheumatic conditions . Furtherbiofilms –14. To din vitro and in vivo analyses, thereby providing a foundation for future efforts to treat infections caused by COL-R pathogens.This study sought to examine whether colistin and FFA exhibit synergistic antimicrobial and antibiofilm activities when used to treat COL-R Gram-negative bacterial infections using multiple The MIC values of common clinical antibiotics used to treat specific bacterial isolates are summarized in P. aeruginosa TL1736, TL1744, and TL2967 , the FICI values for the combinations of colistin and FFA ranged from 0.0175 to 0.375 across all tested COL-R bacterial strains, consistent with robust synergistic activity such that FFA potentiates the activity of colistin. The majority of the isolates (22/31) regained colistin susceptibility in the presence of FFA.Checkerboard assays revealed that the MIC values of colistin were decreased by 4- to 512-fold across the tested bacterial isolates when combined with FFA . With thP. aeruginosa TL2314 and TL2917, E. coli DC5286 and DC7333, K. pneumoniae FK6556 and FK6663, and A. baumannii BM2370 and BM2431)  . Combina BM2431) . The comP < 0.05). The 595-nm absorbances of all eight strains were reduced by one-half, including that of P. aeruginosa, which has strong biofilm-forming abilities (P < 0.05). Altogether, the combination of colistin and FFA can thus interfere with the ability of COL-R Gram-negative bacteria to form biofilms while also enabling the eradication of mature biofilms.The effects of colistin and/or FFA treatment on biofilm establishment or the ability to eradicate established biofilms composed of COL-R Gram-negative bacteria were next assessed by a crystal violet staining approach. The combination of FFA and colistin at subinhibitory concentrations (sub-MICs) determined by the checkerboard assay were able to significantly suppress biofilm formation by the tested bacterial strains , doing sbilities . The combilities , compareP. aeruginosa TL2314 cells exhibited a thick biofilm covering the entire visual field and interwoven with regularly shaped bacteria, and a similar densely organized biofilm was observed after monotherapy with colistin (0.5 μg/mL) and FFA (32 μg/mL). Colistin-FFA treatment in combination, in contrast, led to significant reductions in cell numbers together with reduced biofilm numbers and densities and only a small amount of bacterial aggregation. High-magnification  imaging further confirmed the disruption of the original, complete, and tightly adherent biofilm, while visible bacterial cells showed morphological changes, including reductions in size and the release of cellular debris, together with the appearance of vesicles, bulges, and wrinkles.Scanning electron microscopy (SEM) analyses were next used to more thoroughly examine the effect of combination FFA-colistin treatment on biofilm formation . Untreatin vivo safety profile.RAW264.7 cells were next used as a model mammalian cell line to detect any potential FFA-associated cytotoxicity. However, no increases in RAW264.7 cell death were observed following treatment with high-dose FFA (128 μg/mL), relative to the dimethyl sulfoxide (DMSO) or control treatment . The combination regimen developed in this study thus offers an excellent Galleria mellonella larvae was next analyzed to gauge the in vivo synergistic benefits of FFA and colistin treatment in the context of COL-R bacterial infections. The TL2314, DC7333, FK6556, and BM2431 isolates were selected as targets for this experiment. While almost all untreated G. mellonella larvae died within 48 h following infection  such that the rates of survival of P. aeruginosa- and K. pneumoniae-infected larvae increased by 60% and those of E. coli- and A. baumannii-infected larvae increased to 80% after 168 h.The survival of nfection , the comP. aeruginosa TL2314 growth slightly 24 h after injection  yielded superior efficacies compared with the single treatments (P < 0.05). In vivo experiments showed that the combination of the two drugs had better antibacterial effects on COL-R P. aeruginosa TL2314 than single-agent treatments.A neutropenic mouse thigh infection model was also utilized to expand on these analyses. The administration of FFA (50 mg/kg of body weight) or colistin (7.5 mg/kg) alone inhibited njection . HoweverTo explore the mechanisms governing the observed synergistic efficacy of combination FFA-colistin treatment, propidium iodide (PI), which binds to nucleic acids in bacteria with damaged membranes, was used to test the integrity of the membranes of TL2314 cells after treatment with these antimicrobial agents. Membrane permeability, as measured by the fluorescent PI signal, was not affected by colistin alone  . HoweverP. aeruginosa, E. coli, K. pneumoniae, A. baumannii, and other bacterial strains has necessitated the use of colistin as a final bulwark against these MDR bacterial infections  upregulation and thereby protect against pulmonary damage induced by sepsis  of the tested bacterial isolates exhibited MDR phenotypes in susceptibility assays, emphasizing the need to closely monitor the emergence of colistin resistance in an effort to prevent the widespread dissemination of these resistant strains. Using checkerboard and time-kill assays, combination FFA-colistin treatment was found to synergistically eradicate all tested bacterial isolates other than the TL1736, TL1744, and TL2967 strains, potentially owing to strain-specific differences , which showed that FFA may induce damage to the bacterial membrane, thereby enhancing the antibacterial efficacy of colistin , E. coli (n = 8), K. pneumoniae (n = 8), and A. baumannii (n = 7) isolates. Bacterial identification was confirmed using a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)  approach, and isolates were stored in Luria-Bertani (LB) broth supplemented with 30% glycerol at −80°C. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 served as quality control isolates for this study.A total of 31 nonduplicate COL-R Gram-negative bacterial isolates were selected randomly from the First Affiliated Hospital of Wenzhou Medical University, including COL-R FFA was obtained from Shanghai Yuanye Biotechnology Co., Ltd. , and stock solutions of 10 mg/mL were prepared by initially dissolving FFA in dimethyl sulfoxide (DMSO) and further diluting it with distilled deionized water to the concentration required for subsequent experiments . ColistiA broth microdilution strategy was utilized to compute the MIC values of FFA and a range of common antibiotics when used to treat the 31 selected clinical COL-R bacterial isolates, as described in a previous report . Brieflyin vitro synergistic efficacy of colistin and FFA was examined using a checkerboard assay approach using a modified version of a protocol employed in a previous report  (from 128 μg/mL to 0.125 μg/mL), with the y axis representing 2-fold serial dilutions of FFA (drug B) (from 512 μg/mL to 8 μg/mL), to form a matrix. Individual bacterial suspensions were diluted with sterile saline to a 0.5 McFarland standard following culture overnight, and these suspensions were then diluted 1:100 using CAMHB. The resultant 100-μL bacterial suspension was added to the prepared 96-well plates containing 50 μL each of FFA and colistin to give a final bacterial concentration of 7.5 × 105 CFU/mL. Results were examined after an incubation period of 16 to 20 h at 37°C.The s report . BrieflyFractional inhibitory concentration (FIC) index values were calculated as follows: FIC(drug A) = (MIC of drug A in the combination)/, FIC(drug B) = (MIC of drug B in the combination)/, and FICI = FIC(drug A) + FIC(drug B). FICI values of ≤0.5, 0.5 to 4, and >4 indicate synergistic, indifferent, and antagonistic interactions, respectively . All anaP. aeruginosa, E. coli, K. pneumoniae, and A. baumannii strains (n = 2 each) selected at random to assess COL-R Gram-negative bacterial growth kinetics using a modified version of a previously reported protocol  was measured on a microplate reader (Multiskan FC). The experiment was repeated three times.Crystal violet staining was used to assess the inhibition of biofilm formation based on a modified version of a previously reported protocol . The bacA slightly modified version of a previously reported protocol was used to test the ability of colistin and/or FFA to eradicate established bacterial biofilms . BrieflyP. aeruginosa TL2314 were observed by scanning electron microscopy to facilitate in-depth analyses of the effects of colistin and/or FFA, using a slightly modified version of a previously reported protocol (Biofilms of protocol . These b2 incubator at 37°C. Cells were harvested with trypsin and transferred into 96-well plates (1 × 105 cells/well in 100 μL). After a 24-h incubation, 10-μL serial dilutions of FFA  or combinations of FFA and colistin were added to individual wells. After an additional 24-h incubation, 10 μL of cell counting kit 8 (CCK-8) reagent  was added to each well. Following a 1-h incubation at 37°C in the dark, the OD450 was measured on a microplate reader (Multiskan FC). FBS containing DMSO (the solvent used for FFA) as a vehicle control was used as a negative control  were cultured to confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a CO control . Three dG. mellonella infection model system was used to assess the in vivo synergistic efficacy of FFA and colistin (5 CFU/mL. Treatment groups in this assay included control (untreated), colistin , FFA , and combination colistin-FFA groups (n = 10/group). A sterile saline solution was used to treat control larvae. Larvae (250 to 350 mg) were randomly assigned to individual groups, and a 10-μL volume of the prepared bacterial solution was injected with a microinjector into the rear left proleg of each G. mellonella specimen, with 10 μL of appropriate antibiotic treatments being injected 2 h after bacterial inoculation. G. mellonella survival rates were recorded after 24, 48, 72, 96, 120, 144, and 168 h. When larvae persistently failed to react to physical stimulation, they were regarded as dead. Survival rates were assessed with Kaplan-Meier curves and log rank tests. All analyses were repeated in triplicate.A modified version of a previously reported colistin . Brieflyin vivo efficacy of colistin and FFA. Female BALB/c mice  were utilized in a manner consistent with Laboratory animal - Requirements of environment and housing facilities (GB 14925-2010)  . The Zhe25-2010) .3), mice were intraperitoneally injected with cyclophosphamide  4 days (150 mg/kg) and 1 day (100 mg/kg) prior to the establishment of a thigh infection model. P. aeruginosa TL2314 was selected at random as a model COL-R strain, and a 100-μL suspension of these bacteria in the exponential phase of growth (1.5 × 107 CFU/mL) was injected into the posterior thigh muscle of each mouse. Two hours following bacterial injection, mice were intraperitoneally administered colistin (7.5 mg/kg) alone or together with FFA (50 mg/kg) (10 CFU per gram from the thigh muscle homogenates. Groups of 3 mice (6 thigh infections) were included for each dosing regimen.To induce neutropenia (≤100 neutrophils per mm0 mg/kg) . After 2Membrane permeability was assessed using a modified version of a previously reported protocol . Brieflyt tests using GraphPad Prism 8.0. Statistical significance is indicated by asterisks in the figures .Data are means ± standard deviations (SD) from three or more replicate trials. Data were compared with two-sample