All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arβ were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes ( Testosttor (AR) Figure 1tor (AR) , 3. Althtor (AR) . Therefooduction . SRD5A1 oduction . In factehaviors –12, and ehaviors .srd5a genes are present in various teleost species , member of the 11-oxygenated class of androgens, is a major active androgen mainly produced in gonads , 15. It  species , 32, 33, species , 34–36.g for 5 minutes. Other plasma samples were purchased from AllCells  and ProMedDX . All plasma samples were collected under IRB-approved collection protocols and subject informed consent. The donors were 5 men (aged 32.2 ± 5.5 y) and 5 women (aged 29.2 ± 6.3y).The research protocol using human materials was approved at the ethical committee of Asahikawa Medical University and University of Fukui. Blood samples were collected in collection tubes containing heparin from the median cubital vein of 2 healthy women volunteers (obtaining informed consent) at University of Fukui Hospital in 2007. Plasma was separated by centrifugation at 1000g for 5 minutes.Animal experiments were performed using protocols approved by the Animal Care and Use Committee of Kumamoto University . All experiments were performed in accordance with the relevant guidelines and regulations. The sexes of juvenile eels were artificially directed by feeding a commercial diet supplemented with or without E2 . Male an2/95% air at 37 °C. Hepa-E1 cells were cultured in E-RDF with 5% FBS at 28 °C. Cells were dispensed into 24-well plates at 5 × 104 cells per well 24 h before transfection. HEK293 and CV-1 cells were transfected using HilyMax  according to the manufacturer’s instructions. Hepa-E1 cells were transfected using Lipofectamine LTX  according to the manufacturer’s instructions. At 24 h or 2 days post-transfection, the cells were treated with vehicle (EtOH) or androgens. Luciferase activity was determined using a dual luciferase reporter assay system (Renilla luciferase assay. Firefly luciferase activities (relative light units) were normalized by Renilla luciferase activities. Each data point represents the mean of at least three independent experiments.HEK293 and CV-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% COy system . Measurevia real-time PCR using the LightCycler 480 . β-actin (human) and ef1 [eel . Cloning of ORF sequences were performed by PCR-based methods using ovary and testis cDNAs.Putative nucleotide sequences encoding eel srd5a1, srd5a2a and srd5a2b were provided from the database, JPEEL2016 , human SRD5A2 (ABQ59050.1), mouse Srd5a2 (NP_444418.1), chicken Srd5a2 (XP_001235447.1), Japanese quail Srd5a1 (XP_015709859.1), Japanese quail Srd5a2 (XP_015713214.1), three-toed box turtle Srd5a1 (XP_024056462.1), three-toed box turtle Srd5a2 (XP_026505351.1), zebrafish srd5a1 (AAI64429.1), zebrafish srd5a2 (XP_005157051.1), Atlantic salmon srda5a1 (XP_014033435.1), Atlantic salmon srd5a2 (NP_001134686.1), rainbow trout srd5a1 (XP_021428075.1). rainbow trout srd5a2 (XP_021413035.1) and elephant shark srd5a1 (NP_001279361.1).The pQCXIP expressing human SRD5A1, human SRD5A2, eel srd5a1, eel srd5a2a and eel srd5a2b were generated by cloning the open reading frame of each gene into a pQCXIP vector (Invitrogen). The pcDNA3 expressing eel arα and arβ were generated by cloning the open reading frame of each gene into a pcDNA vector . A Slp-ARU/Luc reporter, pQCXIP/green fluorescent protein (GFP) and pQCXIP/human AR were prepared as described .tert-butyl ether (MTBE). After the MTBE layer was evaporated to dryness, the extract was dissolved in 0.5 mL of methanol and diluted with 1 ml of distilled water. The sample was applied to OASIS MAX cartridge which had been successively conditioned with 3 ml of methanol and 3 ml of distilled water. After the cartridge was washed with 1 ml of distilled water, 1 ml of methanol/distilled water/acetic acid , and 1 ml of 1% pyridine solution, the steroids were eluted with 1 ml of methanol/pyridine . After evaporation, the residue was reacted with 50 μl of mixed solution  for 30 min at room temperature. After the reaction, the sample was dissolved in 0.5 ml of ethyl acetate/hexane/acetic acid  and the mixture was applied to HyperSep Silica cartridge (Thermo Fisher Scientific) which had been successively conditioned with 3 mL of acetone and 3 ml of hexane. The cartridge was washed with 1 mL of hexane, and 2 mL of ethyl acetate/hexane . T, DHT, 11-KDHT and 11-KT were eluted with 2.5 ml of acetone/hexane . After evaporation, the residue was dissolved in 0.1 ml of acetonitrile/distilled water  and the solution was subjected to a LC-MS/MS.Quantification of T, 11-KT, DHT and 11-KDHT in plasma and culture media by LC-MS/MS are based on methods as described [, ASKA PhP< 0.05) were assessed by the Student’s t-test, one-way ANOVA followed by Tukey’s multiple comparison tests and two-way ANOVA followed by Tukey’s multiple comparison tests using SigmaPlot 14  and EZR  which is a graphical user interface for R  as described  genes were isolated in eel  and mammalian AR, we compared the androgen-dependent transcriptional activities of DHT and 11KDHT with the activities of T and 11KT using the luciferase reporter system in fish and mammalian cell lines Figure 6This study demonstrated that DHT and 11KDHT are potential important androgens in eels. In contrast, 11KDHT was undetectable in healthy human plasma, although DHT was present.SRD5A2 gene cause 46, XY disorders of sex development, resulting from low DHT production , whereas it was over several hundred-fold higher in Hepa-E1 cells, at levels similar to that of human AR in CV-1 cells. A similar phenomenon was noted in measurements of flounder estrogen receptor-mediated transactivation by estrogens and estrogenic compounds (The 5α-reduced androgens, DHT and 11KDHT more strongly activated human AR and eel arβ than T and 11KT did, whereas such remarkable differences were not detected for eel arα. In teleosts, the two ar genes have been formed by duplication of an ancestral AR gene during a teleost specific genome duplication event. Based on the phylogenic analyses, Ogino and colleagues proposed that teleost arβ preserves the ancestral AR functions, whereas arα has acquired new properties during more rapid evolution . Our resompounds . BecauseIn summary, we demonstrated that DHT and 11KDHT are potent endogenous androgens in fish. The properties of these androgens could provide useful insights into elucidating ambiguous ar-mediated phenomena reported in fish. In contrast, 11KDHT is undetectable in healthy humans. Nevertheless, it has been often reported that the profiles of 11-oxygenated androgens are markedly changed under pathological conditions , 55–59. ‘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 ethical committee of Asahikawa Medical University and University of Fukui. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Animal Care and Use Committee of Kumamoto University.TY, YI, TSe, JU, ST, NS, AU and TK obtained funding and designed the study. TY, HI, YI, TSe, JU, MI, MO, DM, TI, TSa, YM and TK performed the experiments and collected the data. TY, HI, YI, MK and TK wrote the manuscript. All authors contributed to the article and approved the submitted version.Author YM was employed by the company ASKA Pharmaceutical Medical Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effect of CaO concentration and the sintering temperature on phase composition and mechanical characteristics of CaO-stabilized zirconia ceramic made of baddeleyite is investigated.Wet high-energy milling and uniaxial pressing are used to fabricate CaO-stabilized tetragonal zirconia polycrystalline ceramic (Ca-TZP) with decent mechanical characteristics, i.e., a hardness of 11.5 GPa, Young’s modulus of 230 GPa, and fracture toughness of 13 MPa·m Pure zirconia is monoclinic from ambient temperature to 1170 °C. In the range of 1170–2370 °C, it is tetragonal, and from 2370 °C to the melting point, it is cubic [2→m-ZrO2, a transition occurs at 950 °C which is accompanied with an increase in volume by about 4.5% [2→m-ZrO2 transition and to keep t-ZrO2 at room temperature [2 grains, are called tetragonal zirconia polycrystals (TZP). TZP exists in the certain range of the dopant concentrations.Zirconia can exist in three allotropic forms: monoclinic m-ZrO, tetrago2 in the range of 96.5–98.5 wt%. Baddeleyite is much cheaper than zirconia synthesized from zircon, but chemical ways of stabilizing are not efficient for baddeleyite. In [2 nanopowder. CaO was chosen as an inexpensive alternative to Y2O3 and CeO2 which commonly used for producing engineering TZP ceramics.TZP ceramics are attractive because of their excellent room-temperature mechanical properties . Mainly yite. In , high-en2→m-ZrO2 transformation throughout the material volume. Below a certain grain size, local t-ZrO2→m-ZrO2 transformation induced by mechanical impact, giving rise to TZP ceramics toughening, does not occur, resulting in reduced TZP ceramics fracture toughness. The increase in sintering temperature and time leads to larger grain size [Mechanical properties of TZP ceramics strongly depend on its grain size . Above aain size .The aim of this work is to define CaO concentration and the sintering regime which gives rise to a Ca-TZP ceramic originating from baddeleyite which possesses competitive mechanical properties.2 powders by wet high-energy co-milling of CaO powder  and the baddeleyite concentrate powder with zirconia content of 99.3%  using the planetary mill Pulverisette 7 Premium Line  in the same way as described in [We produced 1–5 wt% CaO-stabilized ZrOribed in . The sizThe obtained powders were uniaxially pressed under 560 MPa into pellets of a 10 mm diameter and a 2 mm thickness. The fabricated pellets were sintered at the temperature range from 1100 to 1400 °C for 4 h in air atmosphere in a muffle furnace. The sintering temperature was 500 °C and the cooling rate was 5 °C/min. The samples were cooled naturally from 500 °C to room temperature.2 and c-ZrO2 was calculated in the TOPAS software  by applying the Scherrer equation to the characteristic peaks of the tetragonal and cubic phases located in the 20–80° 2θ range.Phase composition analysis of sintered ceramics was performed with the help of an X-ray diffractometer (XRD) D2 Phaser  at room temperature. The XRD patterns were recorded in the 20–80° 2θ range and assigned using the PDF-2 Diffraction Database File compiled by the International Centre for Diffraction. Phase content was determined from the XRD patterns by the Rietveld method in the TOPAS software . The average grain size of t-ZrOCK) was calculated from length of radial cracks starting from the corners of the indents with the Anstis equation [H is Vickers hardness, E is Young’s modulus, P is indentation load to produce cracks, and C is the crack length (the distance between the center of the indent and crack tip). The Vickers indentation fracture toughness tests were performed on a hardness tester Duramin A300  using loads of 196.2 and 294.3 N. Cracks lengths were calculated using optical microscope Axio Observer.A1m . Before tests, all the samples were polished with diamond-containing slurries. All mechanical characteristics measurements were carried out at room temperature. Sintered ceramics containing m-ZrO2 are not tested because of numerous cracks already present in the samples before measurements.Young’s modulus of the samples was measured on a nanoindentometer G200  equipped with a Berkovich diamond indenter. Young’s modulus of the samples was calculated from the load-penetration depth curves obtained under peak load of 5 N using the Oliver–Pharr method . Hardnesequation :(1)KC=0.2 phase dominates in the ceramic if 1-wt% CaO is used. The characteristic peaks of m-ZrO2 at 24.1°, 28.2°, 31.5°, 34.2°, 49.3°, 50.6°, and 55.5° are observed in the XRD pattern. The most intensive characteristic peak of t-ZrO2 at 30.2° is very weak compared to m-ZrO2 reflections. Phase composition of ceramic dramatically changes at 2 wt% CaO. The peaks of m-ZrO2 disappear and only the peaks of t-ZrO2 at 30.2°, 34.6°, 35.2°, 50.2°, 50.7°, 59.3°, and 60.2° are observed in the XRD pattern. This indicates that a 2-wt% CaO-ZrO2 ceramic is a TZP ceramic. A further increase in CaO concentration from 3 to 5 wt% leads to an increase in c-ZrO2 content from 7 to 23 wt% and, respectively, t-ZrO2 content reduction from 93 to 77 wt%. The intensity of the c-ZrO2 peaks increase with the rise in c-ZrO2 content. The characteristic peaks of c-ZrO2 at 35.0° and 59.7° are clearly observed in the XRD pattern of a 5-wt% CaO-ZrO2 ceramic. Other characteristic peaks of c-ZrO2 at 30.1° and 50.2° overlap with the neighboring peaks of t-ZrO2. With the rise in CaO concentration from 2 to 5 wt%, the average grain size of t-ZrO2 decreases from 93 to 63 nm and the average grain size of c-ZrO2, on the contrary, increases to 83 nm.The m-ZrO2 ceramic is sensitive to the sintering temperature higher than 1300 °C despite CaO concentration (2 becomes to dominate. The t-ZrO2 is practically absent in a ceramic and c-ZrO2 content increases with the rise in CaO concentration as evidenced by an increase in the intensity of the characteristic peaks of c-ZrO2.It is revealed that phase composition of CaO-ZrOntration . If the 2 ceramic consists only of t-ZrO2 if sintering is performed at 1300 °C and lower temperatures. An increase in the sintering temperature induces zirconia grains growth. The average grain size of t-ZrO2 increases from 79 to 93 nm with the rise in the sintering temperature from 1100 to 1300 °C. Phase composition of the 2-wt% CaO-ZrO2 ceramic dramatically changes if sintering temperature of 1350 °C is used. The TZP ceramic transforms into one mainly composes of m-ZrO2. The radical change in phase composition of the 2-wt% CaO-ZrO2 ceramic with the rise in the sintering temperature from 1300 to 1350 °C is related to critical t-ZrO2 grain size. In [2 grain size cannot exceed 100 nm. The Ca-TZP ceramic examined in [2 is 93 nm if the Ca-TZP ceramic is sintered at 1300 °C. For comparison, in Y2O3-stabilized TZP ceramics, t-ZrO2 grains up to 1 μm in size can exist [2O3-stabilized TZP ceramics.It is found that the 2-wt% CaO-ZrOsize. In , it was mined in  was prod2 containing ceramics (Peaks’ broadening in the XRD patterns of m-ZrOceramics  may be dMechanical characteristics of CaO-containing zirconia ceramics made from baddeleyite were examined. It was found that an increase in CaO concentration from 2 to 5 wt% leads to a rise in hardness by 5.5% and a decrease in fracture toughness by 29.9% . Wherein2 ceramic being TZP ceramic has the best combination of mechanical characteristics and the highest fracture toughness value. In terms of hardness and Young’s modulus, the fabricated 2-wt% CaO-ZrO2 ceramic corresponds to engineering Y-TZP ceramics made of chemically synthesized zirconia stabilized with Y2O3, and surpasses them in terms of fracture toughness [2→m-ZrO2 transformation which causes volume expansion and shear strains. It applies compressive stress at the crack tip to prevent crack propagation. As a result, further crack growth is suppressed and strength of a ceramic increases. The rise in a dopant concentration leads to a decrease in the fraction of transformable t-ZrO2 in favor of c-ZrO2 incapable of transformation. It explains fracture toughness reduction in the fabricated zirconia ceramic with an increase in CaO concentration (2 ceramic compared to the t-ZrO2 one. Indeed, hardness measurements induce t-ZrO2→m-ZrO2 transformation under an indenter tip. Thus, resulting hardness includes hardness of t-ZrO2 and m-ZrO2 phases. A decrease in the fraction of transformable t-ZrO2 negatively affects mechanically-induced t-ZrO2→m-ZrO2 transformation and respectively reduces m-ZrO2 contribution to the resulting hardness. To confirm this assumption, we tested a 1-wt% CaO-ZrO2 ceramic, containing more than 90 wt% m-ZrO2, using a nanoindentometer due to numerous cracks in the sample. Hardness was calculated from the obtained load–penetration depth curves using the Oliver–Pharr method. The prints size was significantly smaller than the analyzed region bounded by the cracks. A 5-wt% CaO-ZrO2 ceramic, mainly composed of t-ZrO2 with a reduced ability to t-ZrO2→m-ZrO2 transformation, was tested on a nanoindentometer too. A nanoindentation by a Berkovich diamond indenter with a tip of a 20-nm radius showed that hardness of a 1-wt% CaO-ZrO2 ceramic is significantly lower than the hardness of a 5-wt% CaO-ZrO2 ceramic, namely, 8.24 ± 0.37 GPa versus 12.63 ± 0.41 GPa.It can be seen from oughness . High froughness . The strntration . It can 2 ceramic hardness with the rise in CaO concentration cannot be explained in terms of the empirical Hall-Petch relationship [H is the measured hardness, H0 is the intrinsic hardness related to the resistance of lattice to dislocation motion, k is the material-specific strengthening coefficient, and D is the average grain size.It should be noted that an increase in CaO-ZrOtionship , which d2 ceramic hardness on the inversed square root of the effective grain size is not a line as required by the Hall–Petch relationship (2 ceramic was calculated for each CaO concentration from the range of 2–5 wt% knowing the content of t-ZrO2 and c-ZrO2 as well as the average grain size of t-ZrO2 and c-ZrO2 (The observed dependence of CaO-ZrOtionship . The effd c-ZrO2 .2 ceramic (It is revealed that the rise in the sintering temperature in the range of 1100–1200 °C results in simultaneous increase in hardness, fracture toughness, and Young’s modulus of a 2-wt% CaO-ZrO ceramic  despite  ceramic . It allo2 ceramic sintered at temperatures of 1100–1300 °C measured by the Archimedes method with distilled water as the immersing medium is presented in Indeed, the sintering temperature of 1100 °C is lower than the Tammann temperature of zirconia ; therefo2O3-stabilized TZP ceramic densification, which in turn led to the rise in Young’s modulus, bending strength and fracture toughness of the given ceramic.According to the data in 2 ceramic is 35.5% higher if sintering occurs at 1300 °C and not at 1200 °C. It can be related to the over-stabilization effect [2 grains become smaller and their ability to t-ZrO2→m-ZrO2 transformation reduces. An increase in the sintering temperature induces observed t-ZrO2 grains growth and thus facilitates t-ZrO2→m-ZrO2 transformation following the introduction of a crack.Fracture toughness of a 2-wt% CaO-ZrO2 ceramic was examined too. It was found that sintering time reduction from 4 to 1 h has no effect on hardness, fracture toughness, and Young’s modulus of a 2-wt% CaO-ZrO2 ceramic within the measurements error. The 2-wt% CaO-ZrO2 ceramic sintered at 1300 °C for 1 h is characterized by the following mechanical characteristics: hardness of 11.52 ± 0.05 GPa, Young’s modulus of 226 ± 8 GPa, and fracture toughness of 12.83 ± 0.41 MPa·m0.5.To reduce energy consumption, the effect of sintering time on mechanical characteristics of a 2-wt% CaO-ZrO2, SO3, P2O5, TiO2, Fe2O3, CaO, and MgO [2 ceramics made of the baddeleite concentrate and chemically synthesized monoclinic zirconia. According to 2 ceramics sintered at 1300 °C were chosen for testing. It was found that both ceramics made of chemically synthesized monoclinic zirconia and the baddeleyite concentrate are TZP ceramics. Their mechanical characteristics are given in The used baddeleyite concentrate contains impurities in the amount of 0.7 wt%. The dominating ones are SiO and MgO . To inveIt can be seen from 2 ceramic sintered at 1300 °C for 1 h possesses hardness ~11.5 GPa, Young’s modulus ~230 GPa, and fracture toughness ~13 MPa·m0.5. In terms of hardness and Young’s modulus, the fabricated 2-wt% CaO-ZrO2 ceramic corresponds to engineering Y-TZP ceramics made of chemically synthesized zirconia stabilized with Y2O3, and surpasses them in terms of fracture toughness. It is revealed that the rise in the CaO concentration from 2 to 5 wt% stimulates c-ZrO2 content increase and a Ca-TZP ceramic transforms into a ceramic, consisting of both t-ZrO2 and c-ZrO2. An increase in CaO concentration leads to the rise in hardness and a decrease in fracture toughness of a CaO-ZrO2 ceramic wherein its Young’s modulus remains unchanged. Furthermore, it is found that Ca-TZP ceramic cannot be produced if the sintering temperature of 1350 °C or higher is used.Optimal dopant concentration and the sintering regime to obtain Ca-TZP ceramic originating from baddeleyite with competitive mechanical characteristics are defined. The 2-wt% CaO-ZrO


With SVFMO in a porous structure (simulating the porous anode layer), its electronic conductivity can also reach 70 S cm−1 when testing at 800 °C in an H2 atmosphere; (3) SVFMO with more oxygen vacancies achieves higher catalytic ability for fuel gas, as an SOFCs anode layer demonstrates 720 mW cm−2 at 850 °C.Perovskite oxides using solid oxide fuel cells (SOFCs) anodes should possess high chemical stability, adequate electronic conductivity and excellent catalytic oxidation for fuel gas. In this work, the medium-entropy SrV The molerance . Tao et ss steam . Sr2MgMo2CoMoO6 as SOFCs anode has revealed high catalytic ability, itself showing apparent phase decomposition under reducing atmospheres such as the creation of secondary phase Sr2MoO4 [0.8Sr0.2Cr0.95Ru0.05O3 as SOFCs anode shows apparent phase decomposition in a wet H2 atmosphere [3 doping with Nb exhibits good chemical stability (and high conductivity: 120 S cm−1), but shows poor electrocatalytic activity toward H2 oxidation [0.75Sr0.25Cr0.5Mn0.5O3 and Sr2MgMoO6 ceramics are only about 1 S cm−1 and 10 S cm−1 [As SOFC anode materials, the general requirements include high electronic conductivity, excellent catalytic activity towards electro-oxidation of fuels, suitable porosity to allow the flow of fuel molecules and reaction products, adequate durability while exposing in fuel gases and low cost ,2. Accor Sr2MoO4 ,12. La0.mosphere . SrTiO3 xidation . Another0 S cm−1 . More im2FeMoO6  and Sr2VMoO6-based perovskite oxides can be fabricated by sintering in reducing atmosphere (5% H2/Ar mixture) [−1 or higher at operational temperature [5 S cm−1 . For porous Ni-YSZ composite anodes, there extrinsic total conductivities are still much higher than 100 S cm−1 at 1000 °C [2FeMoO6 (SFMO) can be used as SOFCs anode with high catalytic activity and sufficient ionic conductivity [0.5Mo0.5O3 (SVMO). It is predictable that the conductivity will further decrease with SFMO anode in suitable porosity. SVMO in reducing atmosphere (such as in H2) demonstrated super high electronic conductivity, for example, as high as 2500 S cm−1 at 300 °C. However, SVMO as anode material demonstrated moderate catalytic ability for fuel gas due to its low oxide-ion conductivity [However, a lot of works have proved that the fabrication environment for a small number of perovskite oxides must be in reducing environments. For example, Amixture) ,17. Thesperature . For Ni- 1000 °C . In our uctivity . However1/3Fe1/3Mo1/3O3 (SVFMO) with V, Fe and Mo as B-site cations was fabricated by reducing 5% H2/Ar atmosphere. As SOFCs anode material, SVFMO demonstrated high reduction catalytic ability for fuel gas. In an H2 atmosphere, its bulk conductivity can reach as high as 200~500 S cm−1 from 30 to 800 °C. With SVFMO in a porous structure (simulating the anode structure), its conductivity can also reach to about 134–70 S cm−1 from 30 to 800 °C in H2 atmosphere, which is comparable to the conventional Ni-YSZ based anode catalyst. At last, by introducing SVMO and SFMO as contrast materials, SVFMO as SOFCs anode (La0.9Sr0.1Ga0.8Mg0.2O3−x (LSGM) as supporting electrolyte and La0.8Sr0.2Co0.8Fe0.2O3−x (LSCF) as cathode) demonstrated the maximum power output, such as 720 mW cm−2 at 850 °C, and excellent long-term stability. The superhigh electronic conductivity, excellent catalytic ability and structural stability in reducing environments indicate that SVFMO has great application potential for use as SOFCs anode material.It is urgently need that we develop a new anode with high catalytic activity, high conductivity and excellent structural stability when working in a reducing anode environment. Here, in order to make use of the high catalytic ability and high ion conductivity of perovskite-based oxides, the drawbacks such as phase decomposition and low bulk conductivity must be overcome. Based on the higher structural stability of SFMO and SVMO in a reducing atmosphere, we try to further unite the high catalytic ability of SFMO and the high electronic conductivity of SVMO. In this work, single perovskite SrV3, V2O5, Fe2O3 and MoO3 were used as raw materials , and the final fabricating condition was calcination at 1200 °C in 5% H2/Ar for 10 h. SFMO powders were synthesized via sol-gel method with stoichiometric Sr(NO3)2, Fe(NO3)3·9H2O, (NH4)6Mo7O24·4H2O and citric acid as starting materials . The final calcination for pure-phase Sr2FeMoO6 was at 1150 °C in 5% H2/Ar for 10 h. LSGM pellets were synthesized by solid-state reaction with La2O3, SrCO3, Ga2O3, and MgO as starting materials  and sintered at 1450 °C for 20 h. The LSGM electrolyte synthesized by solid phase method was fixed in a thickness of about 250–300 μm. Ce0.8Sm0.2O2 (SDC) powders were synthesized via sol-gel method with stoichiometric Sm(NO3)3, Ce(NO3)3·6H2O, and citric acid as starting materials . The final calcination temperature was 800 °C in air for 10 h to obtain the pure phase SDC. La0.8Sr0.2Co0.8Fe0.2O3−x (LSCF) cathode was also synthesized via sol-gel method with stoichiometric La(NO3)3·6H2O, Sr(NO3)2, Co(NO3)3·6H2O, Fe(NO3)3·9H2O, and citric acid as starting materials . The final calcination temperature was 1050 °C in air for 10 h.SVMO and SVFMO powders were synthesized by a conventional solid-state reaction. Stoichiometric ratio of SrCO2/Ar 5 h to obtain symmetrical cells. To fabricate single cells, the electrode powders  were grinded with the electrode adhesive to form the electrode slurry. The SDC slurry was deposited onto one side of the LSGM electrolyte by silk-screen printing and then heated at 1300 °C for 1 h to obtain the buffer layer. The cathode slurry (LSCF) was deposited onto another side of the LSGM by silk-screen printing and heated at 1100 °C in air for 5 h. Then, the anode slurry was deposited onto the SDC buffer layer by silk-screen printing and heated to 950 °C in N2 for 5 h.First, the electrode adhesive was prepared by mixing ethyl cellulose and terpinol  at a mass ratio of 1:9. To fabricate a symmetric cell, the slurry prepared by mixing SDC synthesized by sol-gel method and electrode adhesive was deposited onto both sides of the LSGM electrolyte by silk-screen printing and then heated at 1300 °C for 1h to obtain the buffer layer. The anode slurry was deposited onto both sides of the SDC by screen printing and then heated at 950 °C for 5 h in 5% H−1. Thermogravimetric (TG) curves were obtained in a thermal analyzer  for the thermal analysis. The thermogravimetric sample had a heating rate of 10 °C min−1. Thermal expansion coefficient (TEC) was measured with a dilatometer  on the rod-shape samples under a flow rate of about 50 mL min−1 5% H2/Ar. The elemental composition and valence electron states of the compound were further characterized by X-ray photoelectron spectroscopy (XPS) using the Thermo Fisher X-ray photoelectron spectrometer . The morphology and microstructure of the samples were characterized by scanning electron microscopy .The phase purity and crystal structure of the fabricated samples were identified by X-ray diffraction (XRD) using the Philips X’Pert PRO diffractometer . Temperature-programmed reduction (TPR) measurement was carried out to investigate the reducibility of the samples in a Vodo VDSORB-91x analyzer . The heating rate of the sample was 20 °C min2 atmosphere. The relative density of samples was measured via Archimedes drainage method. The as-prepared dense rectangular bars with a dimension of 25.00 × 5.00 × 1.50 mm3 were fabricated by uniaxial pressing at 300 MPa and sintering at 1200 °C (SVMO and SVFMO) and 1150 °C (SFMO) for 10 h in 5% H2/Ar. The porous bar samples were obtained by compressing the 30% starch + 70% sample (in weight ratio) and sintering at 950 °C for 10 h in 5% H2/Ar. The conductivity was subsequently measured using a four-probe method with the count’s measurement multimeter and DC power supply in H2 from 30 to 800 °C. Ag wires with a small amount of Ag paste in separate dots were used as current collectors. The area-specific resistance (ASR) of the anodes was measured with a two-electrode symmetrical cell configuration. Electrochemical impedance spectroscopy (EIS) of the cell was measured by an IM6 testing system over the frequency range 0.01–100 kHz. The cell polarization curve was tested using the liner scanning mode of the IM6 testing system at 10 mV s−1 from the open circuit voltage to 0 V.Dense bar (the relative density higher than 90%) and porous bar samples (the relative density about 65%) were prepared to test the conductivities as a function of temperature in H2/Ar atmosphere. From the enlarged figure of the XRD diffraction peak, we can see that only SFMO possess an apparent diffraction peak at 19.5°, which is the characteristic peak of double perovskite, meaning that SVMO and SVFMO are in a B-site disordered single perovskite structure [tructure . By the In single perovskite SVFMO, the cations in B positions are disordered. The disordered B-site surely will increase the entropy . To dist lattice . 2-TPR was tested to identify the released oxygen concentration. 2-TPR profiles testing between 400 and 850 °C. For SVMO, the apparent reduction peak appears between 750 and 850 °C, which is associated with the change in the oxidation states of V and Mo, such as from V5+ to V4+, V3+, or V2+ and Mo6+ to Mo5+ or Mo4+ [2 reduction. The weight loss of the three samples with increased temperature was tested by thermogravimetry (TG) to study the oxygen loss or the creation of oxygen vacancy. To compare the catalytic ability of the three samples, H or Mo4+ ,22. For 2 atmosphere. As shown in −1 at 30 °C and 279 S cm−1 at 800 °C. The conductivity for SVFMO is 508.4–207 S cm−1 for testing temperature from 30 to 800 °C. SFMO shows the lowest conductivity, demonstrating 315.3 to 114.2 S cm−1 from 30 to 800 °C. For comparison, the total conductivities for other reported typical perovskite anode materials, such as La0.75Sr0.25Cr0.5Mn0.5O3−δ [2MgMoO6 [2CoMoO6 [2 atmosphere [2/Ar for 5 h. As shown by the SEM in 2. As shown in −1 and at 800 °C are 78.8, 70.1 and 37.9 S cm−1, respectively. The high conductivities of the porous SVMO and SVFMO can match the demand of an anode layer as current collector. Conductivity is a critical factor for perovskite oxides using SOFCs electrode materials. The total conductivities of dense SVMO, SVFMO and SFMO bars are tested in the temperature range of 30–800 °C in an Hn0.5O3−δ , Sr2MgMo[2CoMoO6 , are als3+ 2p3/2 and Fe2+ 2p3/2 signals, demonstrating the coexistence of Fe3+ and Fe2+. As shown in 2+/Fe3+ is 50%: 50% in SVFMO and 43%: 57% in SFMO. As shown in 5+ increased apparently from SVMO (V5+ in 39%) to SVFMO (V5+ in 47%), which is mainly attributed to the co-doping of low-valence Fe2+ and Fe3+.The XPS were used to detect the possible valence states of V, Fe and Mo in the three samples. The mixed valence states of the elements were evaluated by fitting the XPS data. First, for the XPS of Fe 2p signals in SVFMO and SFMO , the pea4+, Mo5+ and Mo6+. It is worth noting that Mo4+ appears in SVMO and SVFMO, but not in SFMO. In 6+ proportion from SVMO  to SVFMO  and to SFMO . The increased proportion of Mo6+ is assigned to the decreased valence states from V5/4/3+ to co-existed V/Fe and then to Fe3/2+. The V, Fe and Mo with complicated valence states co-existing in the B-site of perovskite structure can form more small-polaron couples as electronic carriers (via the double-exchange mechanism), which is responsible for the high conductivity. For SVMO and SVFMO samples, the superhigh conductivities were attributed to the complicated valence states of V, Fe Mo cations. The increased conductivity was possibly associated with more V, Fe Mo elements in a lower oxidation state to form more small-polaron couples.In 2 atmosphere at 750, 800, and 850 °C. First, the three symmetric cells show almost the same ohmic resistance (ROhm). Then, the calculated interfacial polarization resistance (RP: the distance between the high and low frequency intercepts) was used to evaluate the catalytic ability of the anode materials. As shown in P decreases gradually with the increase of operating temperature, indicating the thermal activation process. The SVFMO anode shows the lowest RP among the three samples at the same temperature. To further analyze the anode catalytic ability, the impedances tested at 850 °C were fitted as shown in P1 was contributed to the charge-transfer process at the electrolyte/electrode interface and the electron transfer process accompanying the oxidation of fuel. RP2 is associated with the adsorption/diffusion of the fuel gas at the gas/anode interface and the surface diffusion of the intermediate species [P1 and RP2 were 0.06 and 0.75 Ω cm2. This means that the RP2 contribution is the main rate-limiting process, which corresponds to the adsorption/diffusion of the fuel gas at the gas/anode interface and the surface diffusion of the intermediate species. However, for the SFMO anode, RP1 and RP2 were 0.6 and 0.29 Ω cm2, suggesting the rate-limiting step was a charge transfer process. For the SVFMO anode, RP1 and RP2 were 0.16 and 0.36 Ω cm2, which means both the enhanced charge-transfer process and adsorption/diffusion for fuel gas. It is noteworthy that the medium-entropy SVFMO with V, Fe and Mo occupying the B-site can help to enhance electrochemical performance. P) are 0.825, 0.522 and 0.371 Ω cm2, respectively. The lower Rp for SVFMO anode indicates higher H2 catalytic ability and faster charge carrier transportation, which was attributed to the forming of more oxygen vacancies and more small-polaron couples. This is also confirmed by TG testing; SVFMO with more weight loss means more oxygen vacancy concentrations were created for hydrogen catalysis.To assess the electrochemical activity of SVMO, SVFMO and SFMO anodes, the symmetrical cells with anode│SDC│LSGM│SDC│anode configurations are used to test the EIS in an H species ,27. The −2 at 850, 800 and 750 °C, respectively. SVFMO as anode exhibits the highest power output of about 720, 484 and 267 mW cm−2 at 850, 800 and 750 °C in −2 at 850, 800 and 750 °C in 2 gas at 750 °C for 60 h. As shown in To evaluate the single-cell performance, LSGM (~250 μm) electrolyte-supported single cells with the configuration of anode|SDC|LSGM|LSCF were fabricated and tested in the temperature range of 750–850 °C. Although the electrolyte-supported single cell hinders us from obtaining the optimum fuel cell performance due to its high ohmic drop, it is useful for us to compare the relative performances of the various electrode materials. The current-voltage (I-V) and current-power (I-P) curves of the single cells are measured and shown in 2/Ar. First, as shown in −6 K−1, which provided good thermal expansion compatibility with typical SOFC electrolyte materials such as YSZ, LSGM and SDC (TEC: 10.4 × 10−6−12.9 × 10−6 K−1) [2-TPR results. As shown in For ceramic-based SOFCs, the thermal expansion coefficient of the ceramic components is a critical factor to influence the SOFCs compatibility, thermal stress and even the lifetime . In this0−6 K−1) ,33,34. MIn summary, medium-entropy SVFMO perovskite was fabricated in a reducing environment, demonstrating excellent chemical stability when used in fuel gas as SOFCs anode material. SVFMO with Fe, V and Mo co-existing in a B-site of the perovskite structure increased the configuration entropy and then benefited the oxygen ion migration in the lattice sites, which explained the high catalytic ability for fuel gas. At last, SVFMO with high conductivity, excellent long-term stability and good fuel catalytic activity was demonstrated as a promising SOFCs anode material.


Salvia officinalis L.  possesses high activity against Trypanosoma brucei rhodesiense (Tbr), causative agent of East African Human Trypanosomiasis. We have now investigated in detail the antitrypanosomal constituents of this preparation. A variety of fractions were tested for antitrypanosomal activity and analyzed by UHPLC/+ESI QqTOF MS. The resulting data were used to generate a partial least squares (PLS) regression model that highlighted eight particular constituents that were likely to account for the major part of the bioactivity. These compounds were then purified and identified and their activity against the pathogen tested. All identified compounds (one flavonoid and eight diterpenes) displayed significant activity against Tbr, in some cases higher than that of the total tincture. From the overall results, it can be concluded that the antitrypanosomal activity of S. officinalis L. is, for the major part, caused by abietane-type diterpenes of the rosmanol/rosmaquinone group.As part of our studies on antiprotozoal activity of approved herbal medicinal products, we previously found that a commercial tincture from  Trypanosoma brucei subsp. Ref. + at m/z 290 was very low in abundance, but detectable.Compound e manool ,16, whice manool  . The range of biological activity of the isolated compounds was from 1.0 to 15 µg/mL, so that some compounds (1) and mixtures (3+4 and 6+7) displayed higher activity than the total extract. Of these, compounds 3 and 4 were not selected by the PLS model but, nevertheless, showed interesting activity (IC50 = 1.1 µg/mL and SI of 4.9). However, none of the tested substances displayed exceptionally high activity, so that the overall activity was probably a sum of their activities .As mentioned above, Tbr exhibited high sensitivity toward the total extract obtained from Sage tincture  displayed more promising antitrypanosomal activity, though to variable extent. Thus, the mixtures of 3 with 4 (rosmanol + rosmaquinone) and 6 with 7 (7-O-ethylrosmanol+ 7-O-ethylrosmaquinone) were the most active samples with IC50 values of 1.0 and 1.1 µg/mL, respectively, whereas compound 3 (7-O-methylrosmanol) was the least active compound of this group with an IC50 of about 8.3 µg/mL.The various diterpenoids with the common rosmanol skeleton  and, to a lesser extent, also peak G (a putative isomer of miltirone) were present in the later fractions , while those of the other diterpenes C–F and H abounded in the earlier fractions (FR17–FR20). This was reflected in the PLS model, where these two groups contributed to the different latent variables, PC2 and PC1, respectively, in 2, 5, 6–8) were present in the total extract in much higher quantities than B and G, so that it may safely be assumed that they accounted for the major part of the overall antitrypanosomal effect of Sage tincture. It is worth noting that, in spite of various reports on antiprotozoal activity of Salvia diterpenes from other species  was provided by Phytolab GmbH & Co KG, Vestenbergrsgreuth, Germany, and a sample of authentic miltirone was provided by Dr. S. Ślusarczyk, Wroclaw Medical University, Wrocław, Poland.The investigated Sage tincture was the same commercial product as investigated in our previous communication . A referColumn chromatography (CC) was performed at ambient pressure and room temperature using Sephadex LH-20 or Silica 60 as stationary phase in glass columns of appropriate size.Analytical thin-layer chromatography (TLC) was performed on silica 60 F 254 gel-coated aluminum sheets , usually developed over a distance of 8 cm and then observed under UV light (254 nm) followed by spraying with Anisaldehyde reagent (MeOH: acetic acid: sulphuric acid (85:10:5) + 0.5 mL Anisaldehyde . For analytical purposes, the sheets were cut into two 10-cm halves. The samples were applied in bands (width of 1 cm) and the plates developed over a distance of 8 cm with the eluent mixtures mentioned below.Preparative HPLC separations were performed on a Jasco  HPLC system consisting of a PU 2087 Plus Pump with DG2080-54 degasser, a AS2055 autosampler with 2000-µL injection loop and a C = 2060 column thermostat. The column used for the separations described below was a Nucleodur 250/10 C18 Htec 5 µm .3 or DMSO-d6 . Chemical shifts (δ) in ppm, the peak integral size, the multiplicity, and coupling constants (J) in Hz were analyzed for every sample. For 13C NMR solvent signals , the chemical shifts (δ) in ppm were analyzed.All NMR measurements were carried out at the Institute für Pharmazeutische und Medizinische Chemie der Universität Münster under the supervision and responsibility of Dr. Jens Köhler and staff. One- and two-dimensional NMR assays  were recorded on Agilent DD2 400 MHz or DD2 600 MHz spectrometers . The measuring frequencies are indicated for each compound in the respective table of NMR results. NMR spectra were acquired in CDCl2O , passed through a cotton filter, and subjected to CC on Sephadex ® LH-20 (400 g) packed and eluted isocratically with the same solvent system at a flow rate of 1 mL/min. The eluates were collected in portions of 15 mL. Eluates were collected and controlled by TLC . They were combined according to their TLC profiles into 38 fractions (FR1–FR38).The dried extract (5.1 g) obtained by evaporation of 300 mL of Sage tincture under reduced pressure was resuspended in 33 mL of MeOH-H2O (A): MeOH (B) with 50% B: 10 mL; 75% B: 10 mL; 100% B: 20 mL; re-conditioning with 10 mL 50% B. The eluates were combined according to their TLC profiles into nine subfractions (FR20-1–FR20-9). FR20-1–FR20-3 were eluted with 50% B, FR4–FR6 were eluted with 75% B, and FR20-7 and FR20-8 were eluted with 100% B, while FR20-9 was collected during re-equilibration.FR20 (230 mg) was further separated into four portions of 50–60 mg by CC on 5g Europrep C18-RP 20–45 µm (4.5 × 1.5 cm), with a gradient of H2  and a mixture of compounds 3+4  were isolated by semiprep. HPLC was done, using the following gradient of H2O (A): MeOH (B) with 65% B (0-9 min); 65–80% B (9–11 min); 80–100% B (12.2–20 min); 100–65% B (20–22 min); and re-conditioning was with 65% B from 22–26 min. Flow rate was 2.8 mL/min and column temperature was 40 °C.From FR20-5 (58 mg), compound 2O (A): MeOH (B) with 70% B (0–2.1 min); 70–80% B (2.1–4.1 min); 80–100% B (9.1–11.1 min); 100–70% B (15.2–18 min); and re-conditioning was with 65% B from 18–22 min. Flow rate was 2.8 mL/min, 30 °C, to yield 8 mg of compound 5, eluted with tR = 14.0 min under these conditions.FR20-6 (29 mg) was separated by semiprep. HPLC was with a gradient of H6+7 (tR = 17 min) was isolated by semiprep. HPLC was done using a gradient of H2O (A):MeOH (B) with 50–80% B (0–4 min); 80–100% B (4–10 min); 100–30% B (12.2–20 min), 30–50% B (20–24 min); and re-equilibration with 20% B from 24–25 min. Flow rate was 2.8 mL/min, 40 °C.From FR19 (39 mg), the mixture of compounds 9 was isolated from a separate workup of Sage tincture, in which 200 mL of the tincture were evaporated. The residue (3.4 g) was suspended in MeOH:H2O (2:3) and partitioned with 3 × 300 mL n-hexane. The residue after evaporation of the hexane partition (0.38 g) was subjected to CC on 150g, Silica 60 eluted isocratically with 100% n-hexane. Eluates were controlled by TLC and visualized under UV and by derivatization with anisaldehyde spray reagent. They were combined, according to the TLC profiles, into 37 fractions Hex1-Hex37. Fraction Hex6 represented a single constituent, compound 9 (3 mg).Compound Analytical data: +ESI QqTOF MS see All analyses were performed on a Bruker Daltonics  micrOTOF QII quadrupole/time-of-flight mass spectrometer with an Apollo electrospray ion source operated in positive ionization mode and coupled to a Dionex Ultimate 3000 RS Liquid Chromatography System  with a Dionex Acclaim RSLC 120, C18 column  using a binary gradient of water and acetonitrile, both with 0.1% formic acid. The LC system was additionally equipped with a Dionex Ultimate DAD-3000 RS (wavelength range of 200–400 nm) UV/Vis detector.m/z 100–1000 using the following instrument settings: nebulizer gas nitrogen, 4 bar; dry gas nitrogen, 9 L/min, 220 °C; capillary voltage, 4500 V; end plate offset, −500 V; transfer time, 100 µs; collision gas nitrogen. Collision energy and collision RF (Radio Frequency) settings were combined to each single spectrum of 1250 summations as follows: 624 summations with 80 eV collision energy and 130 Vpp + 313 summations with 16 eV collision energy and 130 Vpp + 313 summations with 16 eV collision energy and 130 Vpp. Internal data set calibration  mode) was performed for each analysis using the mass spectrum of a 10 mM solution of sodium formiate in 50% isopropanol that was infused during LC re-equilibration using a divert valve equipped with a 20-µL sample loop.The samples were dissolved in methanol at a concentration of 2 mg/mL. The injection volume was 10 µL. Separations were performed using a binary gradient of A: water, B: acetonitrile, both with 0.1% formic acid at a flow rate of 0.4 mL/min: from 0 to 0.2 min, isocratic at 5% B; 0.2 to 9.7 min, linear from 5% B to 100% B; 9.5 to 12.5 min, isocratic at 100% B; 12.5 to 12.6 min. linear from 100% B to 5% B; 12.6 to 15 min, isocratic at 5% B. The column was thermostatted to 40 °C. Eluted compounds were detected in UV/Vis over a wavelength range of 200–400 nm and by QTOF mass spectrometry in positive mode at 4 Hz over a mass range of 50 values, all performed in one coherent series, calibrated, processed, and converted to a descriptor table (“bucket table”) using the software ProfileAnalysis , applying the following parameters: Buckets were generated within a retention time range from 2 to 12 min and an m/z range of 100 to 800. Using the advanced bucketing function, individual buckets were built with a time window of 0.2 min and an m/z window of 25 mDa. They were only considered if present in at least three of the 17 analyses. The signal/noise threshold for recognition of molecular features was set to 5 with a correlation coefficient of 0.7 and a minimum compound length of 10 spectra. A smoothing width of 2 was applied. This resulted in a total of 230 bucket variables of the form <tR(min):m/z>, which were transformed to square root values for further analysis. The descriptor matrix  thus consisted of N = 17 observations x K = 230 descriptors. The table was exported to Microsoft Excel and the biological activity data expressed as the fractions‘ percentage of growth inhibition against Tbr at 10 µg/mL were added to constitute the Y matrix (dependent variable). The complete data table was then imported to The Unscrambler v 9.2 , which was used to calculate the PLS regression model. The variable data were used without normalization or scaling since it was found that this procedure yielded the best model in terms of statistical performance . It appeared that normalization was not necessary since all samples were analyzed in identical concentration, under identical conditions, and on the same day. Scaling the variables to unit variance is often performed prior to PLS modelling, in order to give all variables equal weight. However, in doing so, information on quantitative composition of the fractions would be discarded, which, however, turned out to be relevant in the present case since the unscaled data yielded a better model than after scaling. The resulting PLS model was validated with full cross validation . It consisted of two significant PLS components and showed the statistical parameters summarized in The X matrix was built from the LC/MS analyses of the 17 fractions with determined ICTrypanosoma brucei rhodesiense  and cytotoxicity tests against mammalian cells  were performed at the Swiss Tropical and Public Health Institute , according to established protocols and as previously described [In vitro assays for the bioactivity against escribed .S. officinalis L. Studies on the effect of combinations of such compounds are thus warranted.The considerable antitrypanosomal activity of Sage tincture reported previously can be attributed, at least for a major part, to its main diterpene constituents, abietane-lactones of the rosmanol type. It will be of particular interest to investigate, in further studies, the role of purified rosmaquinone congeners in this bioactivity. The result that different classes of constituents  account for the high activity of different fractions may indicate that they contribute in an additive or over-additive way to the overall anti-Tbr-activity of


The combination of 8.8 µM oxaliplatin (130 mg/m2) pre-treatment and a single −15 °C freeze resulted in a similar trend of increased PANC-1 cell death. In summary, these in vitro results suggest that freezing alone to temperatures in the range of −25 °C results in a high degree of PDAC destruction. Further, the data support a potential combinatorial chemo/cryo-therapeutic strategy for the treatment of PDAC. These results suggest that a reduction in chemotherapeutic dose may be possible when offered in combination with freezing for the treatment of PDAC.As the incidence of pancreatic ductal adenocarcinoma (PDAC) continues to grow, so does the need for new strategies for treatment. One such area being evaluated is cryoablation. While promising, studies remain limited and questions surrounding basic dosing  coupled with technological issues associated with accessing PDAC tumors and tumor proximity to vasculature and bile ducts, among others, have limited the use of cryoablation. Additionally, as chemotherapy remains the first-line of attack for PDAC, there is limited information on the impact of combining freezing with chemotherapy. As such, this study investigated the in vitro response of a PDAC cell line to freezing, chemotherapy, and the combination of chemotherapy pre-treatment and freezing. PANC-1 cells and PANC-1 tumor models were exposed to cryoablation (freezing insult) and compared to non-frozen controls. Additionally, PANC-1 cells were exposed to varying sub-clinical doses of gemcitabine or oxaliplatin alone and in combination with freezing. The results show that freezing to −10 °C did not affect viability, whereas −15 °C and −20 °C resulted in a reduction in 1 day post-freeze viability to 85% and 20%, respectively, though both recovered to controls by day 7. A complete cell loss was found following a single freeze below −25 °C. The combination of 100 nM gemcitabine (1.1 mg/m An estimated 60,430 new cases of pancreatic cancer (PaCa) will be diagnosed in 2021 in the US alone [While several treatment options exist for non-resectable pancreatic ductal adenocarcinoma (PDAC), they are all considered palliative. Palliative treatment options for non-resectable PDAC include chemotherapy , with the standard treatment for locally advanced PDAC being chemotherapy (FOLFIRINOX or Gem/Abraxane) with or without radiation , despiteCryoablation is typically performed percutaneously, whereby one or more cryoprobes are inserted and the target tissue is frozen. The freezing process is completely destructive to the tissue closest to the cryoprobe where temperatures are coldest ,21. As tCryoablation has primarily been used as palliative care in metastatic PaCa, but a small number of studies have found encouraging results, especially when combined with adjunctive techniques, including immunotherapy ,29. CryoFreezing has been shown to be as, or more, effective and safer than heat-based ablation in several indications ,34,35,36With cryoablation emerging as a treatment option for PaCa as well as the continued desire for improved therapeutic options, interest in the combination of cryoablation with other treatment modalities, such as anti-cancer agents, is growing. Studies by Xu et al. ,42 have 2 and up to 130 mg/m2, respectively [Given the promise for cryoablation to treat PaCa combined with a growing interest in adjunctive treatment strategies, in this study we investigated the freeze response of PaCa in vitro using the PANC-1 cell line in an effort to identify the lethal temperature as well as investigate the effect of the combination of cryoablation and chemotherapy for improving cancer destruction. PANC-1 cells are a well-accepted cell model for in vitro PaCa studies. PANC-1 cells are pleomorphic and are reported to express CK5.6, MNF-116, vimentin, chromogranin A, CD56, and SSTR2, but not E-cadherin, synaptophysin, or NTR1. PANC-1 cells are CD24−/+, CD44+, CD326−/+, and CD133/1− and have KRAS and TP53 mutations and homozygous deletions, including the first three exons of CDKN2A/p16INK4A, but no SMAD4/DPC4 mutations . Studiesectively ,74,75. Gectively . Regardl2 incubators and passaged at 80% confluence. Cells were plated into costar strip well plates at 15,000 cells per well and cultured for 3 days prior to freezing.PANC-1 cells ) were cultured in DMEM ) with 10% FBS ) and 1% penicillin/streptomycin . Cultures were maintained in T-75 flasks in 95% air/5% COw/v gel matrices as per SOP. Cells (0.75–1 × 106 cells/mL) were suspended in the liquid collagen solution within 3 mm × 40 mm tissue-engineered model (TEM) ring fixtures, then placed in 100 mm petri dishes and allowed to solidify for 30 min in a 37 °C hybridization oven as per Robilotto et al. and Baust et al. [Rat tail type I collagen solution  was used to form 0.2% t et al. ,76,77,782, respectively. In vivo doses typically range from 800 to 1000 mg/m2 in a single intravenous weekly treatment, with treatment cycles often occurring weekly for 8 weeks. Gemcitabine doses of 10, 50, and 100 nM were selected for freeze response combination studies. The drug was applied as described above for 48 h and removed 30 min prior to freezing.Gemcitabine  was prepared fresh in sterile water prior to each use and diluted to final sub clinical concentrations in media. The concentrations chosen for the dose response study were 10, 50, 100, 500, 1000, and 5000 nM gemcitabine for 48 h. The clinical equivalencies were 0.11, 0.56, 1.11, 5.57, 11.1, and 55.5 mg/m2, respectively, with a typical in vivo clinical dose of 130 mg/m2. Oxaliplatin doses of 3, 5, and 8.8 µM were selected for freeze response combination studies. The drug was applied as described above for 48 h and removed 30 min prior to freezing.Oxaliplatin  was prepared as a 5 mM stock solution in sterile water and stored at −20 °C. Stock solutions were thawed and diluted to final concentrations in media. Dose response study concentrations were 0.88, 1.5, 3, 5, 8.8, and 10 µM oxaliplatin with a 48-h exposure. The clinical equivalencies were 13, 22, 45, 73, 130, and 150 mg/mSamples in Costar 8-well strips (75 µL medium/well) were exposed to freezing temperatures of −15 °C in a refrigerated circulating bath  for 5 min. Thirty minutes prior to freezing, culture medium was aspirated and replaced with 75 µL per well of appropriate culture medium. Strips were placed into aluminum blocks, containing a thin coating of ethanol to facilitate complete contact and thermal exchange with each well, within the baths. Sample temperature was monitored in a cell-free well and ice nucleation was initiated at −2 °C using liquid nitrogen vapor to prevent supercooling. Sample temperature was recorded at 1-s intervals using a type T thermocouple . For freezing, samples were held for a total time of 5 min in the freezing bath, passively thawed at room temperature for 10 min under a laminar flow hood, and then placed at 37 °C for recovery and assessment. 2 pressure of 1500 psi [All tests were performed in a laminar flow hood to ensure sample sterility. Freezing was conducted using the PSN (Pressurized Sub-Cooled Nitrogen) Cryosystem  with an input N1500 psi . The cryIndividual cell-seeded TEMs were stacked into the 3-D configuration as detailed in Robilotto et al.  and submThe metabolic activity indicator alamarBlue  was utilized to assess cell viability. Stock alamarBlue was diluted 1:20 in Hank’s Balanced Salt Solution ) and applied to samples for 60 min (±1 min) at 37 °C. Raw fluorescent units were obtained using a TECAN Infinite plate reader  and analyzed using Microsoft Excel. Raw fluorescence units were converted to percentages based upon pre-freeze control values (±SEM). Assessments were conducted on day 1, 3, 5, and 7 of recovery. p < 0.01 was applied as the significance threshold.A minimum of 3 experimental repeats with an intra experimental repeat of 7 wells was performed in each condition (n ≥ 21). Statistical significance was determined by single-factor ANOVA where Following thawing, individual TEM layers were measured via calipers to determine the diameter of the frozen tissue  created following the freeze–thaw cycle. Ice ball radii were measured at cardinal locations around the probe surface to determine the symmetry of the freeze zone created. TEMs were then placed into culture to assess at 24 h post-freezing recovery. In situ sample viability assessment (live/dead assay) was performed to determine the extent of cell death (cryolesion) using the fluorescent probes calcein-AM and propidium iodide . Briefly, culture medium was decanted from the TEM samples and a working solution of 5 µg/mL calcein-AM  and 4 µg/mL propidium iodide  in 1× PBS (Corning) was added directly to each sample. Samples were incubated in the dark at 37 °C for 60 min (±1 min). Fluorescent staining was visualized using a Zeiss Axio Observer 7 with ZEN software . Panoramic digital images spanning the center of the freeze zone were acquired using a 1× objective and stitched together from a 6 × 30 set of overlapping images. Following acquisition, a scale bar was imprinted onto each of the images to enable direct image comparison. Diameters of the necrotic zones were then measured using the ZEN software measurement tool. All experiments were repeated a minimum of 3 times. Following testing, data were combined and averaged (±standard deviation) to determine mean ice ball size, isotherm distribution, and ablative diameter. Statistical significance was determined using single-factor ANOVA where noted.Samples were fixed in situ with methanol at day 1, 3, and 5 post-freeze. Briefly, media was aspirated and samples were rinsed with 1x PBS. Ice cold methanol was applied dropwise and samples were placed at −20 °C for 15 min. Methanol was removed and samples were rinsed three times with 1× PBS for 5 min each. Samples were exposed to blocking buffer  for 60 min at room temperature followed by incubation in primary antibody ) at 4 °C overnight. Secondary antibody ) was applied for 60 min at room temperature and samples were rinsed three times with 1× PBS for 5 min each. Samples were counterstained with Hoechst 33342  for 1 min prior to image acquisition. Samples were covered with 1× PBS and scanned using a CX5 CellInsight High Content Screening device (Thermo Fisher Scientific) with HCS studio software. Resultant images  were quantified using the Compartmental Analysis bioapplication with all reference levels set based on control samples, which allowed for identification of the subcellular location of active caspase-3. Immunofluorescent experiments were repeated a minimum of 3 times with an interexperimental analysis of a minimum of 3 wells/condition/experimental repeat. Data from 9 individual samples were combined and then converted to percentage (±SD) of cells positive for the nuclear localization of cleaved caspase-3 based on the total cell population present in condition.Acellular hydrogels are often utilized to assess the generation and spread of critical isotherms and the cooling power of cryoprobes. While useful, these models do not provide information on the response of cells or tissues to the freezing regime. To bridge this gap, we have previously reported on the use of in vitro, three-dimensional tissue constructs (TEM) as a clinically analogous test setup to provide for evaluation of the thermal performance of a cryoprobe as well as the assessment of the level of cell death associated with a given protocol ,80,81,82TEMs consisting of PANC-1 cells were prepared and then treated with a single freeze. TEMs were frozen using a 1.5 mm x 3 cm PSN cryoneedle (FrostBite) under a single 5-min freeze protocol in a circulating heat loaded model. The thermal profile within the TEM during the freeze procedure was measured in real-time at fixed positions radiating from the center of the cryoprobe freeze zone a. TEMs w3 (±0.3). The average size of the frozen areas measured on the micrographs corresponded to within 1 mm of those obtained with the thermal profile data collected during the freeze procedure. Following 24 h of recovery, TEMs had a zone of necrosis of 2.01 cm (±0.18) in diameter equating to a volume of 7.1 cm3 (±0.2 cm) after a single freeze. The area of complete destruction was found to be between the −20 °C and −25 °C isotherms and, when compared to the overall frozen volume, equated to −52.4% of the frozen mass being destroyed (necrotic). After 72 h of recovery, the necrotic diameter was found to be similar to 24 h post-freeze with a diameter of 1.99 cm (±0.18 cm) equating to 7.01 cm3 (±0.25 cm) or 53.2%. Following a single 5 min freeze, the frozen diameter was 2.56 cm (±0.12 cm) equating to a volume of 13.55 cmWith the identification of complete ablation below −25 °C and a zone of partial ablation between −20 °C and −10 °C in the TEM model, we compared our findings to those previously reported in the literature in various pancreatic models ,83,84,852, respectively. A typical clinical dose for gemcitabine for PaCa is 800–1000 mg/m2; as such, the evaluated concentrations represented a significantly lower dose than typically utilized clinically. A dose response study was conducted to determine the impact of a 48-h exposure of PANC-1 cells to gemcitabine at concentrations of 10, 50, 100, 500, 1000, and 5000 nM . The conAssessment of PANC-1 samples following 48 h exposure to 10, 50, 100, 500, 1000, and 5000 nM gemcitabine revealed sample viabilities of 97.0% (±1.8), 97.6% (±1.7), 97.8% (±1.4), 88.6% (±2.2), and 82.1% (±2.8) of untreated control samples (100% (±1.0)), respectively, at the time of drug removal (day 0) a. Assess2, respectively. A typical clinical dose for oxaliplatin for PaCa is 130 mg/m2; as such, several of the evaluated concentrations were significantly lower than a typical clinical dose. A dose response study was also conducted to determine the impact of a 48-h exposure of PANC-1 cells to oxaliplatin at concentrations of 0.88, 1.5, 3, 5, 8.8, and 10 µM . The conAssessment of PANC-1 samples following 48 h exposure to revealed sample viabilities of 91.5% (±2.3), 91.7% (±2.3), 93.7% (±1.8), 93.5% (±1.6), 92.7% (±1.5), and 81.9% (±2.2) of untreated control samples (100% (±1.1)) at the time of drug removal (day 0) b. AssessFollowing the confirmation of a significant level of cell survival following freezing to −15 °C and a precipitous drop in survival following freezing to −20 °C in the TEM model coupled with dose response studies showing a mild effect, a series of investigations into the impact of the combination of pre-treatment of PANC-1 cells with chemotherapeutic agents followed by freezing to −15 °C were conducted. These studies were conducted with the goal of obtaining complete PANC-1 cell death following freezing at −15 °C. Previous reports by our group as well as others have shown that this cryoablation/chemotherapy combination approach can significantly increase cell death following freezing to warmer sub-lethal temperatures ,70,71,72Given the observed minimal decrease in PANC-1 viability following exposure to 10 and 50 nM gemcitabine  as well as a significant level of PANC-1 survival following treatment with 100 nM (~25%), we explored the impact of combining gemcitabine pretreatment at these concentrations followed by freezing to −15 °C. The 10, 50, and 100 nM gemcitabine conditions were selected in an effort to evaluate the potential of reducing the drug concentration, thereby reducing the negative side effects experienced clinically while still yielding enhanced cell death when combined with freezing. Further, these doses were selected as none of them were completely lethal  and they covered a range of cell death from none (10 nM) to mild (50 nM = ~20% death) to extensive (100 nM = ~75% death) as determined in the dose response studies a. Similap < 0.01(%)). The surviving population rapidly recovered, reaching non-treated control levels by day 3.Accordingly, samples were pretreated with 10, 50, or 100 nM gemcitabine for 48 h and then frozen with a single 5-min freeze to −15 °C . PANC-1 p < 0.01(*)), 50 nM gemcitabine alone (97.6% (±1.7); p < 0.01(+)) or the −15 °C/10 nM gemcitabine combination condition (81.2% (±1.6); p < 0.01). Importantly, assessment of PANC-1 survival over the 7-day recovery period revealed a continued decline in the −15 °C/50 nM gemcitabine combination samples which differed from either condition alone. Specifically, PANC-1 cells exposed to 50 nM gemcitabine/−15 °C freeze combination resulted in a continued decrease in cell survival to 20.7% (±1.1), which was significant when compared to the day 7 viability of −15 °C freeze alone (123.7% (±1.6); p < 0.01(Ø)), 50 nM gemcitabine alone (85.2% (±3.5); p < 0.01($)), or the −15 °C/10 nM gemcitabine combination conditions (113.8% (±1.8); p < 0.01). This represented a ~3-fold increase in cell death versus 50 nM gemcitabine alone and a 4.5-fold increase compared to the −15 °C/10 nM gemcitabine combination condition.Given the observed high level of PANC-1 viability and subsequent recovery following exposure to −15 °C as well as varying responses to 10, 50, and 100 nM gemcitabine, we explored the impact of combining gemcitabine pretreatment at these concentrations followed by freezing on cell survival . The comp < 0.01(#)), 100 nM gemcitabine alone (97.8% (±1.4); p < 0.01), or the −15 °C/50 nM gemcitabine combination condition (72.3% (±2.7); p < 0.01). As with the 50 nM gemcitabine/−15 °C freeze combination, assessment of the PANC-1 survival over the 7-day recovery period revealed a continued decline in the −15 °C/100 nM gemcitabine combination sample survival of 5.6% (±0.3), which was significant when compared to the day 7 viability of −15 °C freeze alone (123.7% (±1.6); p < 0.01), 100 nM gemcitabine alone (25.7% (±1.3); p < 0.01(&)), or the −15 °C/50 nM gemcitabine combination condition (20.7% (±1.1); p < 0.01(@)). This represented a ~3.6-fold increase in cell death versus 100 nM gemcitabine alone and a 2.7-fold increase compared to the −15 °C/50 nM gemcitabine combination condition.When samples were treated with the combination of 100 nM gemcitabine pretreatment and freezing to −15 °C, a further decrease in cell viability was noted compared to either condition alone as well as the 50 nM combination samples. Specifically, PANC-1 cells exposed to 100 nM gemcitabine for 48 h followed by a single freeze at −15 °C resulted in a decrease in cell survival to 60.9% (±1.8), which was significant when compared to −15 °C freeze alone (93.9.0% (±1.4); 2, respectively). Dose response studies revealed this range also yielded a varying level of impact from minimal cell death and full recovery (3 µM) to continual decline with moderate cell survival (8.8 µM = 59.9% (±2.0)) following 7 days of recovery (p < 0.01(%)) or 3 µM oxaliplatin alone (93.7% (±1.8); p < 0.01). Continued assessment of PANC-1 survival over the 7-day recovery period revealed a slow regrowth of the surviving population reaching 84.9% (±2.2) of the pretreated controls. When samples were treated with the combination of 5 µM oxaliplatin pretreatment and freezing to −15 °C, a similar decrease in initial cell viability was noted at day 1 as with 3 µM oxaliplatin/−15 °C samples (77.3% (±2.1) vs. 75.5% (±1.2), respectively; p= 0.089). While not statistically different from the 3 µM oxaliplatin/−15 °C samples, this again was significant from either 5 µM oxaliplatin or −15 °C freezing alone (p < 0.01(*)). In contrast to the 3 µM oxaliplatin/−15 °C samples, assessment of sample viability following the 7-day recovery interval revealed a continued decline in overall viability to 68.0% (±2.4(+)) (p < 0.01) versus the recovery noted in the 3 µM oxaliplatin/−15 °C condition. In addition to gemcitabine pretreatment, investigation of the combination of low-dose oxaliplatin pretreatment and mild freezing was also conducted. The 3, 5, and 8.8 µM conditions were selected as, while none of them were completely lethal, they covered a range of clinical dose equivalents of ~1/3, 2/3, and clinical ) and 68.0% (±2.4) for the 5 µM oxaliplatin/−15 °C samples (p < 0.01(@)). This represented a ~68% increase in cell death versus 8.8 µM oxaliplatin alone and a 90% increase compared to the 5 µM oxaliplatin/−15 °C combination condition.When samples were treated with 8.8 µM oxaliplatin and then frozen to −15 °C, a similar decrease in day 1 cell viability was again observed as with the 3 and 5 µM oxaliplatin/−15 °C samples (68.0% (±2.1) vs. 75.5% (±1.2) and 77.3% (±2.1), respectively). As with the 5 µM oxaliplatin/−15 °C samples, analysis over the 7-day recovery interval revealed a continued decline in sample viability to 35.7% (±1.8) in the 8.8 µM oxaliplatin/−15 °C samples. This decline resulted in a significant decrease in overall survival compared to the 3 and 5 µM oxaliplatin/−15 °C conditions as well as the freeze and 8.8 µM oxaliplatin alone conditions. Specifically, overall viability at day 7 for the 8.8 µM oxaliplatin/−15 °C samples was 35.7% (±1.8) compared to 59.9% (±2.0) in 8.8 µM oxaliplatin alone (p = 0.01(+)). Following freezing to −15° C alone, a slight increase in the number of cells with active cleaved caspase-3 within the nucleus was observed (9.3% (±1.1) vs. 4.5% (±1), respectively (= 0.03)); however, the number of cells with active caspase-3 was observed to decrease to controls by day 5. Interestingly, in PaCa samples treated with gemcitabine followed by freezing to −15 °C, a significant increase in the percent of cells with active caspase-3 within the nucleus compared to all other conditions was noted. Specifically, in the combination treated samples, 38.1% (±4.5)) of the remaining cell population was found to have caspase-3 activity within the nucleus. The percent of cells with nuclear caspase-3 activity was further observed to remain elevated compared to the other conditions throughout the 5-day recovery interval (day 3: 35.2% (±3) and day 5: 27.2% (±2.6)). This was significant compared to gemcitabine and −15 °C alone samples )Based on the significant increase in PaCa cell death observed following the combination of mild freezing and low-dose gemcitabine compared to either freezing or gemcitabine alone (day 1: Gem/−15 °C: 72.3% (±2.7) vs. −15 °C: 93.0% (±1.4) vs. 50 nM gemcitabine: 97.6% (±1.7)) coupled with the continued decline over the 7-day assessment interval, studies were conducted to determine if the combination treatment may be activating an enhanced apoptotic response resulting in the increased PaCa cell death. To this end, we examined the level of active caspase-3 (cleaved caspase-3) within the nuclease of PaCa cells following freezing to −15 °C, 50 nM gemcitabine, or combination treatment . AnalysiThere is little question that new approaches and technologies are needed for the treatment of PaCa. The standard treatment for PaCa patients is surgical resection or chemotherapy followed by chemo-radiation therapy, resulting in a median survival of six to twelve months ,3. Yet, In this study, we investigated the survival of a PaCa cell line (PANC-1) following freezing in an effort to identify the minimal lethal temperature (dose) necessary for complete cell destruction. PANC-1 cells have been reported to be more resistant to freezing and heating injury as well as chemotherapeutic treatment than other PaCa cell lines  ,61,62,63Initial freeze response studies examined PANC-1 cell survival following exposure to a single freeze event applied using a cryoprobe in a TEM. Studies revealed exposure to ≥−15 °C resulted in minimal cell death whereas exposure to temperatures below −15 °C resulted in increasing cell death as the temperature decreased until <−25 °C was reached, wherein complete cell death was observed . These f2) in combination with a single freezing event was found to increase the minimal lethal temperature to −15 °C, as illustrated by a significant increase in cell death on day 1 and complete cell death by day 7 ) 1 day following treatment and a 145% increase 5 days post treatment. This increase in caspase-3 activity was even greater when compared to freeze alone samples (D1 = 3.2-fold and D5 = 3.5-fold increase). The prolonged increase in active caspase-3 within the nucleus in combination samples is believed to be responsible for the observed increased cell death following combination treatment. Moreover, the observed prolonged increased caspase-3 activity is further believed to be responsible for the continued cell death observed in combination samples over the assessment interval.With the observed increase in cell death following combination treatment with −15 °C and 50 nM gemcitabine, studies were conducted to determine that the increased cell death was a result of increased apoptosis. To this end, samples were analyzed for the presence of active caspase-3 (cleaved caspase-3) following treatment. Our analysis focused on the assessment of cleaved caspase-3 within the nucleus of cells remaining following treatment. Assessment of nuclear cleaved caspase-3 was elected given caspase-3 is an executioner enzyme responsible for DNA cleavage in the terminal stage of apoptosis. Pro-caspase-3 (inactive form) is present in the cytoplasm and must be activated and transported into the nucleus as part of the apoptotic caspase cascade in order for caspase-mediated apoptosis to be completed. As such, assessment of cleaved caspase-3 within the nucleus is a key indicator of the involvement and manifestation of apoptotic cell death. Analysis of nuclear cleaved caspase-3 in samples following treatment revealed a mild increase in the percentage of cells with active caspase-3 in gemcitabine and −15 °C alone samples 1 day following treatment compared to controls . In compAlthough the treatment of PANC-1 cells with gemcitabine or oxaliplatin at 37 °C resulted in a gradual decline in viability over the 7-day assessment period at each concentration evaluated, the combination of drug pretreatment and freezing significantly accelerated the rate of decline and, under select conditions (doses), complete cell death was achieved following the combination of low-dose agent pre-exposure and a single freeze at −15 °C . This isp < 0.01 ) and a further decrease by day 7 to 20.7% (±1.1). Importantly, pretreatment with 100 nM gemcitabine in combination with freezing to −15 °C resulted in a significant increase in cell death on day 1 and day 7 compared to 50 nM (p < 0.01 (@)) with near-complete destruction (viability = 5.6% (±0.3)) by day 7 . As such, these findings need to be further explored in vivo. Further, the in vitro nature of this study provides a near-optimal environment for cell recovery following treatment. In vivo, following freezing and thawing, additional stressors, including prolonged tissue ischemia, inflammatory response, and the activation of apoptosis, provide additional destructive events, thereby increasing the level of cell death. These factors may further increase the minimal lethal temperature. Another limitation is that the freeze interval was limited to a single 5-min freeze. Clinically, 10-min freeze procedures have been traditionally utilized for the treatment of other cancers. Studies have suggested that attainment of the minimal lethal temperature for 30 s to 1 min can achieve cell death and that longer holds do not yield increased cell death ,79,80. AIn conclusion, our findings suggest that the minimal lethal temperature for PaCa following cryoablation is between −20 °C and −25 °C. Combination studies demonstrated that pretreatment with sub-clinical doses of gemcitabine (<1/10) followed by freezing elevates the lethal temperature to −15 °C, wherein complete cell destruction can be obtained. Similar results were found when combining one-half clinical and clinical doses of oxaliplatin with freezing at −15 °C. The data suggest that this combinatorial approach yielded an elevation of the minimum lethal temperature for PaCa from −25 °C to the −15 °C range. Extrapolating these in vitro findings to an in vivo scenario, the data suggest that both freezing alone and in combination with gemcitabine or oxaliplatin has the potential to improve outcome while reducing comorbidities associated with freezing and/or chemotherapy and may provide a new strategy for the treatment of PaCa. While promising, these in vitro findings need to be examined in vivo prior to clinical utilization.