Meyerozyma caribbica-adapted strains were able to ferment L-arabinose to ethanol in the presence of 3 g/L acetic acid at 35°C. Meyerozyma caribbica Mu 2.2f fermented L-arabinose to produce 3.0 g/L ethanol compared to the parental strain with 1.0 g/L ethanol in the absence of acetic acid. The adapted M. caribbica Mu 2.2f strain produced 3.6 and 0.8 g/L ethanol on L-arabinose and D-xylose, respectively, in the presence of acetic acid while the parental strain failed to grow. In a bioreactor, the adapted M. caribbica Mu 2.2f strain produced 5.7 g/L ethanol in the presence of 3 g/L acetic acid with an ethanol yield and productivity of 0.338 g/g and 0.158 g/L/h, respectively, at a KLa value of 3.3 h−1. The adapted strain produced 26.7 g/L L-arabitol with a yield of 0.900 g/g at a KLa value of 4.9 h−1.Efficient conversion of pentose sugars to ethanol is important for an economically viable lignocellulosic bioethanol process. Ten yeasts fermenting both D-xylose and L-arabinose were subjected to an adaptation process with L-arabinose as carbon source in a medium containing acetic acid. Four  Biofuels are increasingly becoming a renewable alternative to fossil fuels, and it is estimated that by 2035 approximately one-quarter of the world's energy will be generated from plant biomass . First-gLignocellulose is composed of three polymeric components, namely, cellulose, hemicellulose, and lignin. Cellulose is an unbranched polysaccharide consisting of glucose units, while hemicellulose is a branched polysaccharide consisting of hexose  and pentose (mainly D-xylose or L-arabinose) sugars residues, which often also has acetyl groups . HemicelD-xylose and L-arabinose released from hemicellulose generally constitute a significant fraction  of lignocellulosic biomass. Therefore, their conversion to bioethanol is essential for an economically feasible production process , 10. FerThree hundred and ninety yeasts, previously isolated from banana waste, the gut of dung beetles, herbal concoctions, and marula wine were streaked on D-xylose or L-arabinose agar plates , and 0.2 g/L chloramphenicol) and incubated at 30°C for five days. Yeast isolates were inoculated on freshly inoculated yeast malt agar (YM) slants  and incubated at 30°C for 24 hours before moving to 4°C for long term storage [2PO4, 1 g/L MgSO4, and 0.2 g/L chloramphenicol. The fermentation test tubes were incubated at 30°C for five days. Sugar fermentation was indicated by the presence of a gas bubble in the Durham tube. Scheffersomyces stipitis NRRL Y-7124 was used as a positive control for both D-xylose and L-arabinose fermentation. Experiments were performed in duplicate.All yeasts capable of growing on both D-xylose and L-arabinose agar plates were inoculated into test tubes with a Durham tube. These test tubes contained modified fermentation media as described by Silva et al.  consistiTM Kit (Zymo Research) was used for genomic DNA extraction, according to the instructions of the manufacturer. The ITS1 region of all selected yeasts was amplified using the PCR primers ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [µl reactions using the EconoTaq Plus Green Master Mix (Lucigen). The following PCR conditions were used: an initial denaturation at 95°C for 2 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min. A final extension at 72°C for 10 min was followed by holding at 4°C. The D1/D2 domain of the 26S rDNA region was also amplified for all yeast isolates using primers NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′) as described above. The DNA sequencing was done with ABI V3.1 BigDye according to the manufacturer's instructions on the ABI 3500 XL Instrument by Inqaba Biotechnical Industries (Pty) Ltd (South Africa).All yeast isolates capable of fermenting D-xylose and L-arabinose were identified using DNA sequencing as described by Makhuvele et al. . The ZR T GC-3′) . Amplifihttp://www.ncbi.nlm.nih.gov/BLAST/) using the Basic Local Alignment Search Tool (BLAST). The sequences obtained were deposited in GenBank.Sequence data were cleaned using Chromas software  followed by BioEdit  to produ600nm) of 0.1. The flasks were then incubated as above for 96 hours. Two-millilitre samples were withdrawn at 24, 48, 72, and 96 hours to determine the ethanol concentration using gas chromatography (GC). Duplicate cultures were prepared for each yeast.Precultures were prepared in 250 ml Erlenmeyer flasks containing 25 ml of modified fermentation media as described earlier with D-xylose or L-arabinose as carbon source. The flasks were inoculated with yeasts able to ferment both D-xylose and L-arabinose and incubated at 30°C with shaking at 200 rpm for 48 hours. The cultures were used to inoculate 250 ml Erlenmeyer flasks containing 100 ml of the same media to a starting optical density  to determine the maximum growth temperature. Different concentrations of acetic acid  were added in the same media  to determine the ability of the yeasts to grow in the presence of acetic acid during incubation at 30°C. Yeasts with acetic acid tolerance and thermotolerance were selected for evolutionary engineering. These experiments were done in duplicate.Yeast strains able to ferment both pentose sugars in the presence of 3 g/L acetic acid were inoculated onto agar plates containing 6.7 g/L YNB supplemented with 30 g/L L-arabinose and 3 g/L acetic acid and incubated at 35°C for 24 hours at pH 5.0. Colonies were restreaked onto the same media and incubated at 35°C for 24 hours, and the process was repeated 50 times. The process was then repeated 50 times with incubation at 37°C followed by 40°C .4, 3 g/L KH4PO2, and 0.2 g/L chloramphenicol) with 3 g/L acetic acid. Each flask contained L-arabinose as carbon source, and sampling was done every 24 hours for 5 days.The best-adapted yeast strains (ability to ferment in the presence of acetic acid at elevated temperatures) were screened for ethanol production at different temperatures  in 250 ml Erlenmeyer flasks containing modified fermentation medium  was used to determine D-xylose, L-arabinose, L-arabitol, and D-xylitol concentrations, with gas chromatography (GC) used for ethanol determination.KLa) values . The experiments were conducted in triplicate for a period of 120 hours. Sampling was done regularly to determine biomass, ethanol, L-arabinose, and L-arabitol concentrations in the bioreactor. Biomass determinations were done using dry weight in grams, whereas sugar and ethanol concentrations were determined using HPLC and GC, respectively.The best-adapted yeast strain was further evaluated for ethanol production in a BioFlo New Brunswick Bioreactor using a three-litre fermenting vessel containing one litre of media. The same fermenting media as indicated earlier was used with L-arabinose as carbon source. Fermentation in the bioreactor was conducted at a fixed pH of 5.0 by adding 3M HCl to prevent an increase in media pH at 35°C. Aeration and agitation were used to maintain different fixed volumetric oxygen transfer coefficient  values. In this method, the oxygen concentration in the uninoculated medium was reduced to zero by gassing in nitrogen gas. The deoxygenated medium was reaerated and agitated at a fixed agitation speed and aeration rate using a calibrated polarographic oxygen sensor to measure dissolved oxygen in the medium. The polarographic oxygen sensor was previously calibrated at atmospheric pressure according to the instructions of the manufacturer. The concentration of dissolved oxygen in the medium was monitored using the following equation:Various aeration rates and agitation speeds were used to determine the effect of oxygen on ethanol production by the selected adapted yeast strain. The dynamic gassing-out method was applied to determine the different KLa values were calculated using ln(C∗L − CL) versus time, where CL is the concentration of dissolved oxygen in the fermentation broth and C∗L is the saturated dissolved oxygen concentration in the fermentation medium [KLa values tested during fermentation of L-arabinose were 2.3 h−1, 3.3 h−1, and 4.9 h−1 with air that was introduced into the bioreactor at 0.1 vvm for all KLa values with the agitation speed at 100 rpm for 2.3 h−1, 150 rpm for 3.3 h−1, and 200 rpm for 4.9 h−1. The range of different KLa values used was the same as described by Silva et al. [The n medium . The KLaa et al.  where D-µL was automatically injected onto the GC column using a split syringe AOC-20i + s. The ethanol in the samples was measured by comparing it with known ethanol standards [The ethanol content was determined with a GC-2010 Plus Shimadzu Gas Chromatograph. A ZB-WAX plus column was used at a starting temperature of 40°C and raised to 140°C after sample injection at a rate of 20°C/min. It was then raised to 200°C at a rate of 50°C/min and kept at this temperature for 2 min. Nitrogen was used as carrier gas at a flow rate of 17.6 mL/min and at a pressure of 100 kPa. The temperature of the detector was set at 255°C. For each sample, a volume of 1 H + (300 mm × 7 mm) column was used, and deionized water was used as the mobile phase. The temperature was set at 85°C, and the flow rate was 0.6 ml/min. A sample volume of 20 µl was injected onto the column using a SIL-20A autosampler. LC Solution Operation analysis was used to process sample data. Known standards of D-xylose, L-arabinose, L-arabitol, and D-xylitol were used to calculate the concentration unknowns [A Shimadzu prominence 20  HPLC instrument equipped with a RID 10A Refractive Index detector was used to quantify D-xylose, L-arabinose, L-arabitol, and D-xylitol. A Rezex RHM-Monosaccharide unknowns .Parabitol, ΔPethanol, ΔPxylitol) from the substrates  consumed. The ethanol productivity was calculated from the ratio between maximum ethanol concentration and fermentation time (h) at which the highest ethanol was observed. Cell concentrations were determined by correlating the optical density (OD) measurements spectrophotometrically at 600 nm with a standard curve of dry weight against optical density previously constructed. The biomass yield was determined by the ratio between cell concentration (g/L) and substrate utilized (g/L).Fermentation parameters, L-arabitol, cell biomass, ethanol and D-xylitol yield, ethanol productivity D-xylose, and L-arabinose consumption were determined as described below. L-arabitol (g/g), ethanol (g/g), and D-xylitol (g/g) yields were calculated as described by Cadete et al. , which cIn this study, a total of 390 yeasts, previously isolated from banana waste, the gut of dung beetles, marula wine, and herbal concoctions, were evaluated for the ability to grow and ferment both D-xylose and L-arabinose. Twenty-seven yeasts were able to grow on plates containing either D-xylose or L-arabinose, and 13 yeasts were able to ferment both sugars. Yeasts with preferred characteristics (growth at elevated temperatures in the presence of acetic acid) were selected for adaptation. The adapted yeasts were evaluated to select the best ethanol producing strain. The best-adapted yeast strain was evaluated in a bioreactor to determine the optimum aeration rate for ethanol production.Meyerozyma caribbica  followed by Cryptococcus terrestris  and Candida tropicalis (Kp34ey).Thirteen yeast isolates capable of D-xylose and L-arabinose fermentation were identified using ITS-5.8S and D1/D2 domain sequencing . Most ofMeyerozyma caribbica D14YE6 produced 3.9 g/L ethanol from L-arabinose, followed by M. caribbica D14W2 (1.9 g/L) and M. caribbica Mu 2.2f (0.7 g/L). As expected, the control strain S. stipitis NRRL Y-7124 produced the most ethanol (4.5 g/L) from D-xylose in 24 hours, followed by M. caribbica D14W2 and C. tropicalis Kp42ey with 1.2 g/L and 1.0 g/L of ethanol in 48 hours, respectively. The other yeasts produced less than 1 g/L of ethanol from D-xylose.Thirteen yeasts able to ferment both D-xylose and L-arabinose were evaluated for ethanol production. Maximum ethanol produced after a certain fermentation time is indicated in S. stipitis NRRL Y-7124. All yeasts belonging to Meyerozyma caribbica  were able to grow in the presence of 3 g/l acetic acid on agar slants with growth observed after two to four days of incubation at 30°C. The yeast strains C. tropicalis Kp42ey and S. stipitis NRRL Y-7124 were able to grow in the presence of 1 g/l acetic acid, while all the strains of C. terrestris  failed to grow in the presence of acetic acid. Meyerozyma caribbica D28L3, D14W2, D28L4, D14YE6, and Mu 2.2f were able to grow at a maximum temperature of 40°C, with M. caribbica D14YE1 and D14YE2 growing at 37°C. Candida tropicalis Kp42ey and S. stipitis NRRL Y-7124 were able to grow at 35°C, while all four strains belonging to C. terrestris  grew at a maximum of 30°C along with M. caribbica D4WPO1.The thirteen yeast isolates were tested for the ability to grow at elevated temperatures and in the presence of acetic acid with L-arabinose as carbon source . D-xylosC. tropicalis Kp42ey; M. caribbica D28L3, D14W2, D28L4, D14YE6, Mu 2.2f, D14YE1, D14YE2, D4WPO1; and S. stipitis NRRL Y-7124) were selected for adaptation. For the initial step of adaptation, yeasts were grown on agar plates containing 3 g/L acetic acid at 35°C. Yeast strains that grew during the first stage of adaptation were adapted further to 3 g/L acetic acid at 37°C and subsequently on agar plates with 3 g/L acetic acid at 40°C. Candida tropicalis Kp42ey and S. stipitis NRRL Y-7124 failed to grow on agar plates containing 3 g/L acetic acid when incubated at 35°C, while M. caribbica D14YE2, D4WPO1, D14W2, and D28L4 failed to grow on agar plates with 3 g/L acetic acid at 40°C. Only M. caribbica D28L3, D14YE1, D14YE6, and Mu 2.2f were able to adapt to the most stringent conditions.Yeast strains with the ability to grow in the presence of acetic acid and at temperatures above 30°C were adapted on YNB agar plates containing L-arabinose. Ten yeast strains  were screened for L-arabinose (30 g/L) fermentation at 35 and 37°C. Although these yeasts were capable of growth on agar plates at 40°C, none of them were able to grow in liquid medium at 40°C. At 35°C, L-arabinose containing liquid medium with 3 g/L acetic acid, M. caribbica Mu 2.2f, and M. caribbica D14YE1 produced 4.3 g/L and 1.2 g/L ethanol in 24 hours, respectively  to 1.7 g/L in the adapted strain (with acetic acid). The adapted strain of M. caribbica Mu 2.2f produced ethanol at a yield and productivity of 0.221 g/g and 0.047 g/L/h, respectively, in L-arabinose with acetic acid. The yeast M. caribbica D28L3 consumed most of the L-arabinose  and converted it mostly to L-arabitol (19.2 g/l) rather than ethanol (0.6 g/L). This strain had an L-arabitol yield of 0.792 g/g compared to M. caribbica D14YE6 (second best L-arabitol producer) producing 6.6 g/L L-arabitol with a yield of 0.299 g/g. It was decided that M. caribbica Mu 2.2f should be investigated further, since it produced the most ethanol in the presence of acetic acid at 35 and 37°C.Ethanol production, yield, and productivity of the adapted yeasts grown on fermentation medium containing acetic acid at 37°C are presented in M. caribbica Mu 2.2f were evaluated for their ability to ferment pentose sugars in the presence and absence of 3 g/L acetic acid  than from D-xylose (0.8 g/L) after 36 hours of fermentation . The ethM. caribbica Mu 2.2f was evaluated at different volumetric oxygen transfer coefficients  in a bioreactor in order to determine the ideal aeration strategy for maximum ethanol production and L-arabinose consumption. The temperature of the bioreactor was controlled at 35°C and the pH kept at 5.0 with 30 g/L of L-arabinose as carbon source. The adapted strain produced the highest ethanol concentration (5.7 g/L) at a KLa value of 3.3 h−1 after 36 hours compared to 4.2 g/L for KLa 4.9 h−1 and 3.3 g/L for KLa 2.3 h−1 (La 3.3 h−1 (0.338 g/g). The ethanol productivity at KLa 4.9 h−1 was 0.175 g/L/h, compared to 0.158 g/L/h for KLa 3.3 h−1 and 0.106 g/L for 2.3 h−1. The maximum ethanol produced at KLa 4.9 h−1 was obtained after 24 hours, which contributed to the higher ethanol productivity, while at KLa 2.3 h−1 and 3.3 h−1 the maximum ethanol production was observed after 36 hours.The adapted strain of  2.3 h−1 , Table 7KLa value of 4.9 h−1, 18.2 g/L at KLa 3.3, and 9.9 g/L at KLa 2.3 h−1. Similarly, the L-arabitol yield was 0.9 g/g at KLa 4.9 h−1, compared to 0.66 g/g for 3.3 h−1 and 0.37 g/g for KLa 2.3 h−1. Furthermore, L-arabinose present in the fermentation medium was fully consumed at a KLa value of 4.9 h−1  are not naturally fermented by S. cerevisiae. It is crucial to convert both hexose and pentose sugars to bioethanol for the process to be economically feasible [The excessive use of nonrenewable fossil fuel as an energy source worldwide has resulted in an increased release of greenhouse gases into the atmosphere that is leading to global warming . Biofuelfeasible .Saccharomyces yeast from marula fruit and found Pichia kudriavzevii, a yeast known for its pentose-fermenting ability. This yeast was investigated by several authors for its ability to produce ethanol from D-xylose [Yeasts previously isolated from the gut of dung beetles, herbal concoctions, banana waste, and marula wine were screened for the ability to ferment both D-xylose and L-arabinose. Seven of the yeast isolates used in this study were obtained from marula wine. Yeasts associated with marula wine are typically good fermenters. Molelekoa et al.  isolatedD-xylose –27.C. shehatae, C. ergatensis, S. stipitis, and S. segobiensis) from passalid beetles. Makhuvele et al. [Candida tropicalis from the dung of dassie, kudu, rhino, and wildebeest. Santa-Maria et al. [Four yeast isolates  used here were purified from herbal concoctions. There is no information available on the screening of yeasts associated with herbal concoctions in terms of pentose fermentation. Only one yeast isolate from the gut of dung beetles and one from banana waste, respectively, were able to ferment both pentose sugars. These sources are known to be associated with pentose-fermenting organisms. Suh et al.  isolatede et al.  isolateda et al.  determina et al.  isolatedMeyerozyma guilliermondii, a close relative of M. caribbica. Martini et al. [M. guilliermondii. Species of Meyerozyma isolated from marula wine dominated, as 8 isolates were identified as M. caribbica and four as Cryptococcus terrestris, with only one strain of C. tropicalis isolated  on both pentose sugars. Araújo et al.  screenedi et al.  isolatedisolated . Martinidetected –35. Meye tequila .C. terrestris  on L-arabinose by M. caribbica D14YE6  grew at 40°C on agar plates using L-arabinose as carbon source, and these strains were included in the adaptation process  were subjected to evolutionary engineering. Only four yeasts, all belonging to M. caribbica , were capable of adapting up to 40°C in the presence of 3 g/l acetic acid. Strangely, no growth was observed when these yeasts were incubated in shake flasks at 40°C. It is known that oxygen solubility decreases with an increase in temperature in liquids (44). Therefore, it is possible that at 40°C in shake flasks, sufficient oxygen was not available to these yeasts to produce biomass. Similar results were reported by Abdel-Banat et al. [Kluyveromyces marxianus DMKU3-1042 grew on YPD plates at 48°C but failed to grow at the same temperature when inoculated in flasks containing liquid medium. This study is the first report on the adaptation of M. caribbica on acetic acid and elevated temperature with L-arabinose as a carbon source.Evolutionary engineering or adaptation is used to improve certain traits of microorganisms, such as inhibitor tolerance, temperature sensitivity, or the production of bioethanol from lignocellulosic substrates , 43. In  liquids . TherefoM. caribbica Mu 2.2f strain was the best ethanol producing strain. This yeast was capable of producing 4.3 g/L ethanol after 24 hours during growth on L-arabinose in the presence of 3 g/L acetic acid at 35°C  during growth on L-arabinose, with M. caribbica D28L3 producing the most (22.2 g/L) with a yield of 0.783 g/g  values  were used in a bioreactor (M. caribbica Mu 2.2f strain was observed at KLa 3.3 h−1 (5.7 g/L). Most studies investigating the effect of KLa on ethanol production were done on D-xylose as carbon source using yeasts such as S. stipitis, S. hagerdaliae, and S. shehatae [KLa of 3.3 h−1 for S. stipitis DSM 3651 after 72 hours in the absence of acetic acid. The adapted M. caribbica Mu 2.2f consumed all the L-arabinose at KLa 4.9 h−1 and produced the most biomass at this KLa  , 50. In oreactor . The maxshehatae , 50, 51.shehatae  obtainedthis KLa . Applicaoduction .M. caribbica Mu 2.2f strain produced more L-arabitol (0.900 g/g) from L-arabinose than ethanol (0.338 g/g). Sukpipat et al. [M. caribbica 5XY2 produced 30.3 g/L L-arabitol with a yield of 0.61 g/g from 50 g/L L-arabinose. Kumdam et al. [Debaryomyces nepalensis NCYC 3413 and reported significantly less ethanol (2.43 g/L) than L-arabitol (22.7 g/L) on L-arabinose with ethanol and L-arabitol yields of 0.03 and 0.26 g/g, respectively. Saha and Bothast [Candida entomaea NRRL Y-7785 and S. guilliermondii NRRL Y-2075 produced 33.0 and 31.5 g/L L-arabitol, respectively, from 50 g/L L-arabinose at 34°C with an L-arabitol yield of 0.66 and 0.63 g/g, respectively. Sundaramoorthy and Gummadi [P. manshurica from seawater produced 24.6 g/L L-arabitol, while the two soil yeast isolates produced 22.5 g/L with yields of 0.615 and 0.563 g/g, respectively. Watanabe et al. [Candida sp. NY7122 produced 10.69 g/L L-arabitol from 20 g/L L-arabinose when incubated at 37°C after 72 hours with a yield and productivity of 0.537 g/g and 0.148 g/L/h, respectively.L-arabitol is a five-carbon sugar alcohol often used as a natural sweetener in food and the pharmaceutical industry . In thist et al.  investigm et al.  examined Bothast  reported Gummadi  isolatede et al.  reportedM. caribbica Mu 2.2f were compared to similar yeasts in Tables M. caribbica Mu 2.2f produced an ethanol concentration of 5.7 g/L with a yield and productivity of 0.338 g/g and 0.158 g/L/h, respectively, in the presence of acetic acid after 36 hours. Ambrosiozyma monospora produced 4.1 g/L ethanol with a yield and productivity of 0.150 g/g and 0.014 g/L/h, respectively, in the absence of acetic acid after 14 days  as shown in M. caribbica Mu 2.2f has the ability to ferment L-arabinose in a shorter period to produce ethanol and L-arabitol when compared to other L-arabinose fermenting yeasts.The results obtained for ethanol and L-arabitol production for the adapted  14 days . McMillaThe ability of yeasts to ferment pentose sugars (D-xylose or L-arabinose) found in lignocellulosic biomass in the presence of inhibitors produced during pretreatment is important for second generation bioethanol production . L-arabiMeyerozyma caribbica Mu 2.2f could be considered for ethanol and L-arabitol production in the presence of acetic acid. Further studies are needed on this yeast to improve ethanol and L-arabitol production. Adaptation could be considered in shake flasks at higher temperatures to overcome the problems observed in this study.M. caribbica were able to ferment L-arabinose to ethanol and L-arabitol in the presence of 3 g/L acetic acid at 35°C. Adaptation improved the production of ethanol from L-arabinose by M. caribbica strains. The adapted M. caribbica Mu 2.2f strain produced 5.7 g/L ethanol with a yield of 0.338 g/g from L-arabinose at a KLa of 3.3 h−1. More L-arabitol than ethanol was produced at a KLa of 4.9 h−1 with a high yield of 0.900 g/g. The adapted M. caribbica Mu 2.2f strain could be a potential candidate in the fermentation of pentose rich lignocellulosic biomass, such as sugarcane bagasse, wheat straw, wheat bran, corn fibre, or brewery's spent grain. Therefore, the adapted M. caribbica Mu 2.2f strain could prove to be useful for both bioethanol and L-arabitol production under stressed conditions as compared to the documented pentose-fermenting yeasts under normal conditions.L-arabinose fermentation by yeasts is poorly documented compared to D-xylose. Four adapted strains of


Dryocoetes  are known vectors of fungi, such as the pathogenic species Grosmanniadryocoetidis involved in alpine fir (Abieslasiocarpa) mortality. Associations between hardwood-infesting Dryocoetes species and fungi in Europe have received very little research attention. Ectosymbiotic fungi residing in Ceratocystiopsis and Leptographium  were commonly detected in previous surveys of the Dryocoetesalni-associated mycobiome in Poland. The aim of this study was to accurately identify these isolates and to provide descriptions of the new species. The identification was conducted based on morphology and DNA sequence data for six loci . This revealed two new species, described here as Ceratocystiopsissynnematasp. nov. and Leptographiumalneumsp. nov. The host trees for the new species included Alnusincana and Populustremula. Ceratocystiopsissynnemata can be distinguished from its closely related species, C.pallidobrunnea, based on conidia morphology and conidiophores that aggregate in loosely arranged synnemata. Leptographiumalneum is closely related to Grosmanniacrassivaginata and differs from this species in having a larger ascomatal neck, and the presence of larger club-shaped cells.Bark beetles belonging to the genus  Dryocoetes (Coleoptera: Curculionidae: Scolytinae) are mainly secondary pests infesting dead, injured, and felled or windthrown conifer- and hardwood hosts. For this reason, most members of Dryocoetes have no or only minor economic importance, although Dryocoetesconfusus, the most destructive species in the genus, may cause extensive mortality of subalpine fir (Abieslasiocarpa) in North America , which has a wide geographical distribution, extending from France in the west to Siberia in the east, and from Fennoscandia in the north to Italy and Asia Minor in the south  that are well-recognized associates of bark- and wood-dwelling beetles  on conifers in western North America  and beta-tubulin sequence data carried out by Ophiostoma and Grosmannia, and redefined the latter genus to include all Leptographium with sexual morphs. Leptographium and Grosmannia, considering all available DNA sequence data for all species. The authors concluded that sequence data for additional gene regions would be necessary to fully resolve the delineation of Leptographium and Grosmannia. Leptographium and Grosmannia placed in Leptographiumsensu lato based on phylogenetic inference, should be treated in their current genera (Leptographium or Grosmannia). However, new species, excluding those residing in the G.penicillata species complex, should provisionally be treated in Leptographium, irrespective of their sexual or asexual morphs.d Halst. , and Oph P. Syd. . PhylogeLeptographiumsensu lato, members of Ceratocystiopsis are less widespread globally. The genus Ceratocystiopsis currently includes nearly 20 taxa, most of which are collected from plants infested by phloem and wood-breeding beetles. Ceratocystiopsis species have short-necked perithecia, elongated ascospores, and hyalorhinocladiella-like asexual morphs  during March–September 2018. The isolation procedures were the same as described by Alnusincana (L.) Moench and P.tremula infested by D.alni and from Malussylvestris (L.) Mill. infested by Scolytusmali (Bechstein) during studies conducted by Isolations were made from the bark beetle Luke), Finland. The ex-type isolates of the new species described in this study were deposited in the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands, and in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Herbarium specimens have been deposited in the Herbarium of the University of Turku (TUR), Finland. Three reference strains were obtained from collections. These included a living culture of Ceratocystiopsispallidobrunnea (WIN(M)51) from the culture collection of University of Manitoba (Canada), and cultures of Grosmanniacrassivaginata (CBS 119444) and Leptographiumpiriforme (CMW 52066) (Table www.MycoBank.org)  .ITS1-5.8S-ITS2), internal transcribed spacer 2 and large subunit (ITS2-28S), actin (ACT), beta-tubulin (TUB2), calmodulin (CAL) and the translation elongation factor 1-alpha (TEF1-α) using the primers listed in Table DNA extractions were done as described by DNA fragments were amplified in a 25 µL reaction mixture containing 0.25 µL of Phusion High-Fidelity DNA polymerase , 5 µL Phusion HF buffer (5x), 0.5 µL of dNTPs (10 mM), 0.75 µL DMSO (100%) and 0.5 µL of each primer (25 µM). Amplification reactions were performed in the LabCycler Gradient thermocycler . Amplification of the various loci was performed under the following conditions: a denaturation step at 98 °C for 30 s was followed by 35 cycles of 5 s at 98 °C, 10 s at 52–64 °C  and 30 s at 72 °C, and a final elongation step at 72 °C for 8 min. The PCR products were visualized under UV light on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetic Europe).ITS1-5.8S-ITS2-28S and ITS2-28S regions  and the products were resolved with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), at the DNA Research Centre  using the same primers that were used for the PCR. The sequences Table . Newly o regions . Alignmehttp://www.ncbi.nlm.nih.gov) and accession numbers for these sequences are presented in the corresponding phylogenetic trees  v6.06 program  were also used for 11 of our isolates, Grosmanniacrassivaginata, and L.piriforme . Aligned data sets of the protein-coding genes were compared to gene maps constructed by SNPs) for different gene regions between the new taxa and the phylogenetically closest related species were also identified by comparative sequence analysis.In the case of Taxon 2, the ITS2–28S dataset included most of the available sequences for reference species in ank Fig.  to show rme Fig. . Before MAFFT v7 . The ITS v.2.7.5 . The resML), Maximum Parsimony (MP) and Bayesian inference (BI). For ML and BI analyses, the best-fit substitution models for each aligned dataset were established using the corrected Akaike Information Criterion (AICc) in jModelTest 2.1.10 . The ML analysis included bootstrap analysis (1000 bootstrap pseudoreplicates) in order to assess node support values and the overall reliability of the tree topology.Phylogenetic trees were inferred for each of the datasets using three different methods: Maximum likelihood  and the ITS1-5.8S-ITS2–28S (Ceratocystiopsis) was GTR+I+G. The best evolutionary substitution model for TUB2 (Ceratocystiopsis) and for the combined ITS1-5.8S-ITS2–28S, ACT, TUB2, and TEF1-α, datasets for Leptographium was GTR+G.The best evolutionary substitution model for MP analyses were performed with PAUP* 4.0b10 (TBR) was selected as the branch swapping option. The tree length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI) and Rescaled Consistency Index (RC) were recorded for each analysed dataset after the trees were generated.* 4.0b10 . Gaps weBI analyses using Markov Chain Monte Carlo (MCMC) methods were carried out with MrBayes v3.1.2 , 20 g agar , 1 l deionized water] with or without host tree twigs to induce potential ascocarp formation. Autoclaved twigs with bark were positioned in the centre of the MEA agar plates. Fungal cultures were derived from single spores, and crossings were made following the technique described by i microscope  with an Invenio 5S digital camera  to capture photographic images. Microscopy was done as previously described by Morphological features were examined by mounting materials in 80% lactic acid on glass slides, and observing various fruiting structures using a Nikon Eclipse 50For each taxonomically relevant structure fifty measurements were made, whenever possible, with the Coolview 1.6.0 software . Averages, ranges and standard deviations were calculated for the measurements, and these are presented in the format ‘(min–)(mean-SD)–(mean+SD)(–max)’.Grosmanniacrassivaginata (isolate CBS 119144) were determined by analyzing the radial growth for five isolates in pure culture that represent each of the studied species  were determined 7 d (Taxon 1) and 4 d  after inoculation, and growth rates were calculated as mm/d.Growth characteristics for the two newly proposed species and synnemata that were arranged either singly or in groups topped with white mucilaginous spore drops. Taxon 2 produced short mononematous conidiophores with allantoid conidia, and stalked club-shaped cells. A sexual morph could be induced in all isolates of Taxon 2; the most distinct features observed in both the herbarium specimens and the studied isolates were the short ascomatal necks and falcate ascospores with gelatinous sheaths. Sexual morph was not observed for Taxon 1 in any of the crosses done between different isolates. Morphological differences among these new taxa and the most closely related species are listed in Tables The two new taxa showed differences with regards to growth rates in culture and color differences ranging from white (Taxon 1) to brownish gray (Taxon 2). Taxon 1 produced a hyalorhinocladiella-like asexual morph with simple and highly branched conidiophores, which often aggregate in loosely The optimal growth temperatures were 25 °C for Taxon 1 and 30 °C for Taxon 2. No growth was observed at 5 °C for Taxon 2.Ceratocystiopsis data set of ITS1-5.8S-ITS2-28S contained 1278 characters and for the TUB2 512 characters (including gaps). Alignments for the ITS2-28S and the concatenated combined Leptographium data set of ITS1-5.8S-ITS2-28S+ACT+TUB2, TEF1-α, contained 637, and 13276 characters (including gaps), respectively. The exon/intron arrangement of the TUB2Ceratocystiopsis species complex data included exons 3, 4, 5 and 6, interrupted with introns 3, 4, and 5. The exon/intron arrangement of the TUB2Leptographium data included exons 3, 4, and 5/6, interrupted with introns 3 and 4, but lacking intron 5. The aligned TEF1-α gene region consisted of introns 3, 5 and exons 4/5, 6, while lacking intron 4. The alignment of the ACT dataset contained exons 5 and 6, interrupted with intron 5.Alignments for the ITS1-5.8S-ITS2-28S  provided trees with similar topologies (data not shown). In the TUB2 tree . Little intraspecific sequence variation was found within 6 isolates of Taxon 1. Intraspecific variability of the ITS1-5.8S-ITS2-28S, TUB2 and TEF1-α genes was detected for Taxon 1 in one position, i.e. 387, two positions, i.e. 212, 217, and one position i.e. 482, respectively . The intraspecific sequence variation was greater for 11 isolates of Taxon 2 than for Taxon 1. Intraspecific variability of the TUB2, TEF1-α, and ACT genes was detected for Taxon 2 in eight positions, i.e. 36, 82, 83, 87, 215, 230–232; nine positions, i.e. 14, 21, 31, 46, 101, 196, 272, 352, 549; and five positions, i.e. 402, 749, 754, 755, 766, respectively  refers to the synnematous conidiomata formed by this fungus.The epithet .Poland, Paprocice, from Sexual morph: not observed. Asexual morph: hyalorhinocladiella-like. Conidiophores micronematous or macronematous. The micronematous conidiophores, hyaline, consist of conidiogenous cells arising singly from the vegetative hyphae (6–)8.6–16.4(–23.2) × (0.6–)0.9–1.3(–1.6) µm. The macronematous conidiophores are much larger, (14.5–)17.3–39.8(–76.9) µm long than the preceding forms and from a basal cell, (3.1–)5.3–11.2(–17) × (0.9–)1.1–1.9(–2.6) µm. The basal cells branch lateral or penicillate and form 1–5 branches (mostly 1–2) producing conidiogenous cells at their apices. Conidiophores often aggregate in loosely synnemata, (43.2–)52.3–86.4(–114.7) µm long, (2.4–)3.6–8.2(–12.9) µm wide at the tip. Conidia hyaline, smooth, unicellular, oblong-elliptical, (2.4–)2.8–3.5(–4) × (1–)1.1–1.3(–1.4) µm. Cultural characteristics: Colonies with optimal growth at 25 °C on 2% MEA with radial growth rate 1.4 (± 0.1) mm/d, growth very well at 30 °C (1.3 mm/d) and 35 °C (1.0 mm/d). Colonies yellowish gray, margin smooth. Hyphae pale gray in color, smooth, submerged in the medium and aerial mycelium rare, not constricted at the septa, 0.4–2.6 (mean 1.1±0.6) µm diam., asexual morph moderately abundant, very abundant after adding twigs.Alnusincana, PopulustremulaDryocoetesalniPolandCeratocystiopsissynnemata can be distinguished from C.pallidobrunnea by the shape and size of the conidia. Ceratocystiopsissynnemata has shorter and oblong-elliptical conidia in contrast to the allantoid conidia of C.pallidobrunnea  refers to the species name of the bark beetle vector of this fungus, Dryocoetesalni.The epithet .Poland, Paprocice, from Sexual morph: Ascomata developing after 30 d on sterilized Populus twigs when two mating types were paired: superficially or partly embedded in the agar or wood, single. Bases light brown to dark brown, globose, unornamented, (59–)66–90(–108) μm in diameter, necks dark brown, cylindrical, straight or curved, (58–)68–88(–114) μm long (excluding ostiolar hyphae), (18.7–)20.7–27.9(–31) μm wide at base, (10.2–)11.8–15.3(–17.8) μm wide at the tip. Ostiolar hyphae present, pale brown, straight, non-septate or sporadically one-septate, numerous, divergent, pointed at the tip, (14.6–)15.9–19(–22.7) μm long, 5 to 12 in number. Asci not seen. Ascospores one-celled, hyaline, falcate in side view, (7.4–)8.1–11.1(–14.3) × (1.2–)1.5–2.1(–2.4) μm; fusiform in face view, (6.9–)7.4–8.8(–10.3) × (1.8–)2–2.8(–3.3) μm; end view not seen, excluding hyaline gelatinous sheath, (8.9–)10–11.5(–12.2) × (4.5–)5.5–6.7(–7) μm in face view including sheath, accumulated in orange yellow-colored mass at the tip of the neck. Gelatinous sheath 0.5–3 μm thick, oval in face view.Asexual morph: conidiophores macronematous, arising directly from hyphae, single solitary, without rhizoidal hyphae at the bases, often with barrel-shaped or globose cells, (48.1–)59.3–84.2(–102.9) μm in length. Stipes erect, light olivaceous, 1–4 septate (mostly 2), (7.6–)14.3–39.2(–48.5) μm long , (2–)2.4–5.4(–15.6) μm wide below primary branches, apical cell often strongly swollen, (3.2–)3.8–5.2(–6.1) μm wide at base, basal cell rarely swollen. Conidiophores often composed of barrel or globose cells. Conidiogenous apparatus (20–)26.5–38.6(–48.7) μm long  consisting of 2–3 series of branches-type B (more than two branches) 13.2–19(–23.7) × (0.8–)0.9–2(–3.5) μm. Conidia hyaline, mostly allantoid, sometimes oblong to obovoid (3.2–)3.7–5.9(–9.7) × (0.8–)1–1.8(–2.8) μm, accumulating around the conidiogenous apparatus as a creamy mucilaginous mass.ranches) . PrimaryCultural characteristics: Colonies with optimal growth at 30 °C on 2% MEA with radial growth rate 8.8 (± 0.9) mm/d, good growth observed at 35 °C (8.3 mm/d) and better than at 25 °C (7.9 mm/d). Colonies brownish gray with distinct silvery gloss, margin smooth. Hyphae olive yellow in color, smooth, submerged in the medium and aerial mycelium abundant, not constricted at the septa, 1.1–5.5 (mean 2.5±1) µm diam., asexual morph very abundant, which gives a shade of gray. Club-shaped cells terminal on septate hyphal branches present, (11.5–)14.8–25.6(–33.3) × (7.7–)11.3–15.1(–18.2) μm, born on a multicelled stalk, (7.2–)14.7–82.4(–124.2) μm long, (4.4–)5.1–7.7(–9.7) μm wide below primary septa, (2.9–)4–6(–7.4) μm wide at base. Perithecia and asexual morph co-occur in culture.Alnusincana, Malussylvestris, PopulustremulaDryocoetesalni, ScolytusmaliPolandLeptographiumalneum differs from Grosmanniacrassivaginata in having longer ascomatal necks , and the presence of larger club-shaped cells , , L.alneum has larger conidia, especially in regard to width 3.7–5.9(–9.7) × (0.8–)1–1.8(–2.8) µm: G.crassivaginata: (2.4–)3.2–5(–8.1) × (0.7–)0.9–1.3(–1.7) µm) . The optimal growth on MEA for L.alneum and G.crassivaginata (isolate CBS 119144) is 30 °C. However, L.alneum grows much faster than G.crassivaginata  and grows faster at 35 °C (8.3 mm/d) than at 25 °C (7.9 mm/d). In contrast, G.crassivaginata grows much faster at 25 °C (5.6 mm/d) than 35 °C (4.4 mm/d).Morphologically,  8–12 µm , 6.5–18.Dryocoetesalni on Alnusincana and Populustremula in hardwood ecosystems in Poland. DNA sequence comparisons and morphological features supported these as novel. The species were named Ceratocystiopsissynnemata and Leptographiumalneum. The results confirm earlier findings that many species of the Ophiostomatales are associated with hardwood-infesting bark beetles in Poland , especially with regard to the width of the conidia. Other differences are the presence of barrel-shaped or globose cells that make up the conidiophores, which in G.crassivaginata are occasionally only slightly swollen. There are also differences in characteristics of cultures. Leptographiumalneum has brownish gray cultures in contrast to olive brown culture of G.crassivaginata (isolate CBS 119144). Both species belong to fast-growing species on MEA, however L.alneum grows much faster than G.crassivaginata, especially at 35 °C.Based on DNA sequence comparisons, n Canada . MorpholLeptographium species are generally considered as saprotrophs or pathogens of conifer trees  is an associate of D.alni (L.alneum, was also associated with other ophiostomatoid species including C.synnemata and L.piriforme. Among them, only C.synnemata and L.alneum were commonly found in association with D.alni. The common occurrence of these species suggests their important role as fungal associates of D.alni in Poland. The results of the present study and other Polish findings (C.synnemata and L.alneum have been found only occasionally from other beetle species and therefore can be considered as regular and possible specific associates of D.alni. Leptographiumpiriforme is less specific and can be found with other beetle species (There was no information on  D.alni . Howeverfindings  indicate species  or hardw species  in low nOphiostomatales associated with D.alni in Europe. Two new species were described. Ophiostomatoid fungi on hardwoods have been relatively well investigated in Poland (Ophiostomatales associated with hardwoods-infesting bark beetles in Europe are still poorly understood.This work represents the most detailed survey of n Poland , but in


O-acetate. Though all seasonal samples of CP induced an antiproliferative effect in M12.C3.F6 cells, CP from autumn showed the highest inhibitory activity (IC50: 5.9 ± 0.6 µg/mL). The DPPH assay pointed out that CP collected in autumn presented the highest antioxidant potential (IC50: 58.8 ± 6.7 µg/mL), followed by winter (65.7 ± 12.2 µg/mL) and spring (67.0 ± 7.5 µg/mL); meanwhile, the summer sample showed a lesser antioxidant capacity (IC50: 98.7 ± 2.5 µg/mL). The CAA assay demonstrated that CP induced a significant protective effect against ROS production elicited by H2O2 in M12.C3.F6 cells. Pretreatment of M12.C3.F6 cells with CP from spring and autumn (25 and 50 µg/mL for 1 h) showed the highest reduction in intracellular ROS induced by H2O2 (1 and 5 mM). These results indicate that the antiproliferative effect and cellular antioxidant activity of CP are modulated by quantitative fluctuations in its polyphenolic profile due to its collection time.The main chemical composition and pharmacological potential of propolis from arid and semi-arid regions of the Sonoran Desert have been previously reported. Caborca propolis (CP), from an arid zone of the Sonoran Desert, has shown a polyphenolic profile that suggests a mixed plant origin, presenting poplar-type markers, as well as a 6-methoxylated flavonoid, xanthomicrol, characteristic of Asteraceae plants. In addition, CP has shown significant antioxidant properties and antiproliferative activity on cancer cells. In this study, we analyzed the influence of collection time on the chemical constitution, antiproliferative activity and protective capacity of CP against reactive oxygen species (ROS), by using HPLC–UV–diode array detection (DAD) analysis, 3--2,5-Dimethyltetrazoliumbromide (MTT) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assays, as well as cellular antioxidant activity (CAA) assay on murine B-cell lymphoma M12.C3.F6 cells. HPLC–UV–DAD analyses of seasonally collected CP (one-year period) revealed quantitative differences among the most abundant CP constituents: pinocembrin, galangin, chrysin and pinobanksin-3- Under normal physiological conditions, slight and short-lived modulations of ROS are essential to regulate various signal pathways for different cellular processes, including development, proliferation and differentiation [2O2 could trigger the production of •OH that would damage proteins, lipids and DNA, as well as activate signal transduction pathways that mediate pathophysiological processes [Cellular redox homeostasis relies on the orchestrated generation and neutralization of reactive oxygen species (ROS), which are unstable and ephemeral molecules produced during aerobic metabolism. ROS encompass oxidizing species, such as superoxide anion  from the environmentally available plant chemistry, resulting in a chemically diverse resinous substance, wherein compounds other than polyphenols provide bioactivity into the pharmacological potential of several propolis types. The broad array of biological activities of propolis include antioxidant, antibacterial, antiparasitic, antifungic, immunomodulatory activity and antiproliferative effects, among others [To prevent the negative effects of an unbalanced load of ROS, as well as to modulate signaling pathways upregulated in cancer cells, a supportive and protective cellular neutralization is further provided by dietary bioactive phytochemical molecules with antioxidant potential ,9. Polypg others ,15,16,17g others ,23,24,25Propolis and polyphenols have demonstrated a great antioxidant activity, mostly by in vitro spectrophotometric assays, chemical methods based on the deactivation of free radicals, as well as either by hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms ,29,30,31In previous studies, we have reported the main chemical composition and pharmacological potential of propolis from the Sonoran Desert, collected in Caborca, Ures and Pueblo de Álamos regions. Caborca propolis (CP), an arid propolis sample from the Sonoran Desert, has been shown to be chemically constituted by polyphenolic compounds, including poplar chemical markers, such as pinocembrin, pinobanksin-3-O-acetate, chrysin and galangin , as well3 (≥ 99.5%), L-asparagine (98%), L-arginine monohydrochloride (≥ 98%), L-glutamine solution (200 mM), sodium pyruvate solution (100 mM), penicillin-streptomycin solution (1000 U/1 U per mL), ethanol, methanol , caffeic acid, phenethyl alcohol, p-toluene sulphonic acid, propidium iodide (PI), hexane, ethyl acetate, as well as HPLC grade methanol and formic acid (FA), were purchased from Sigma-Aldrich Co. . Hydrogen peroxide (30%) was purchased from J. T. Baker Chemicals. Fetal bovine serum (FBS) was purchased from Gibco . HPLC-grade water (18 mΩ) was prepared by a Milli-Q A10 purification system (Millipore Corp.). Pinocembrin (≥ 95%), chrysin (≥ 95%), galangin (≥ 95%) and pinobanksin-3-O-acetate (≥ 95%) were purified from propolis collected in Ures, Sonora, Mexico  by chromatographic isolation procedures over silica gel 60 , using progressive proportions of ethyl acetate in hexane as the mobile phase (Tedia Company). Purity was assessed by HPLC–UV–DAD, as well as by 1H NMR spectra analysis, using deuterated chloroform or acetone  in an Agilent 400/54 NMR spectrometer, operating at 400.02 MHz or 1H and 100.59 MHz for 13C  [Dimethyl sulfoxide (DMSO), 3--2,5-Dimethyltetrazoliumbromide (MTT), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2′,7′-dichlorofluorescin diacetate , Dulbecco’s Modified Eagle’s Medium (DMEM), NaHCOCA, USA) . CaffeicCA, USA) ,40. 2 and then covered with colony lid. Propolis traps were removed at the end of each season and were frozen (−80 °C) to allow propolis harvesting. The seasonal samples of CP (10 g) were cut into small pieces and extracted with methanol (50 mL) for three days, then filtered to concentrate the methanolic extracts under reduced pressure. Once the methanol was evaporated from the propolis methanolic extract, we removed the remaining waxes in CP by washing the obtained dry extracts with hexane (1:3 p/v), as previously published by our research group [Sonoran Desert propolis was collected from twelve hives of Tecolote farm , located in the arid region “El Arenoso”, between Caborca and Altar municipalities, Sonora, Mexico. Caborca propolis (CP) was collected over the four seasons of the year, from winter 2014 to fall 2015. Previous studies have shown the chemical stability of Sonoran propolis collected throughout the years ,20,36. Och group ,20,36. S2O (solvent A) and methanol (solvent B) at a flow rate of 1 mL/min. The gradient program adopted was as follows: time 0–2 min, A 70%, B 30%; 5 min, A 60%, B 40%; 10 min, A 55%, B 45%; 50 min, A 40%, B 60%; 80 min, A 20%, B 80%, followed by washing and re-equilibrating of the column. Samples were prepared at 1 mg/mL in MeOH and filtered through nylon membrane (0.22 µm). The UV spectra were recorded at a 200–600 nm range using a diode array detector (DAD). Detection of compounds was monitored and recorded at 280 nm [Seasonal samples of CP were analyzed on an Agilent 1290 Infinite series. The column (Zorbax Eclipse Plus C18 4.6 × 100 mm 2.5 Micron) was eluted using a binary gradient formed by 5% FA in Ht 280 nm . The cheFree radical scavenging activity was measured by DPPH assay as previously reported by Usia et al.  with som2O2 [4 cells, 50 μL) into each cell of a flat-bottom 96-well plate . After 24 h of incubation at standard culture conditions (37 °C under an atmosphere of 5% CO2), equal aliquots (50 μL) of DMEM containing different concentrations of each CP seasonal sample were added. Cells were incubated for 48 h. During the last 4 h of treatment, 10 μL of an MTT solution (5 mg/mL) were added to each well. Acidic isopropyl alcohol  was used to dissolve the formazan crystals. The absorbance was measured by an ELISA plate reader , using a test wavelength of 570 nm with a reference wavelength of 650 nm. The antiproliferative activity of seasonal samples of CP is reported as the IC50 value, obtained by linear regression analyses of each seasonal CP treatment.The M12.C3.F6 cell line (murine B-cell lymphoma) was provided by Emil R. Unanue, Department of Pathology and Immunology, Washington University in St. Louis, MO, USA. The B-cell lymphoma M12.C3.F6 cell line was used as a cellular model due to its culture characteristics, such as non-adherent features, as well as its sensitivity to cytotoxic drugs and susceptibility to oxidative stress induction with H2O2 ,36,43. M2O2  with som2 atmosphere), an aliquot (1 mL) containing two different concentrations (25 and 50 µg/mL) of seasonal samples of CP was added. CAPE (5 µM) was used as a control, according to its protective effect against ROS, as previously demonstrated in M12.C3.F6 cells [The cellular redox state was measured in M12.C3.F6 cells treated with seasonal samples of CP, according to the intracellular ROS level detected by using the cellular antioxidant activity assay (CAA) adapted to flow cytometry ,33 with F6 cells . g, 7 min, 4 °C) with cold PBS (pH 7.2). Then, cells were incubated with DCFH-DA  protected from light for 30 min at 37 °C in a 5% CO2. The DCFH-DA probe precursor is intracellularly de-esterified to DCFH2, a molecule that is oxidized to the fluorescent probe DCF by cellular ROS [2O2 (1 and 5 mM) for 10 min at room temperature. H2O2 at the highest concentration tested (5 mM) did not affect cell viability. After 10 min with H2O2, the reaction was diluted out with cold PBS, and cells were washed once with cold PBS, and incubated with Propidium Iodide (PI) (1 µg/mL) for 10 min at room temperature in the dark. After 1-h treatment, cells were harvested and washed twice  [Finally, cells were washed and resuspended in cold PBS and immediately analyzed by flow cytometry . Only metabolically active cells with uncompromised cell membranes were analyzed for each condition, according to DCF and PI fluorescence. ROS production in M12.C3.F6 was estimated by a displacement in the mean fluorescence intensity (MFI) of DCF in comparison to DMSO under the same oxidative conditions  . CAA expp < 0.05) between the CAA of each CP treatment condition group to the DMSO control were analyzed by means of a non-parametric Mann–Whitney U test. In addition, a Kruskal–Wallis test was used to compare differences in population distribution amongst CP treatments (p < 0.05), according to each concentration (25 and 50 µg/mL). Antiproliferative data were analyzed by one-way ANOVA with Tukey’s comparison post-test. DPPH data were analyzed by two-way ANOVA with a Bonferroni post-test. Differences with a value of p less than 0.05 were considered significant . The results shown were obtained by at least three independent experiments carried out in triplicate. CAA analysis was performed at least by two independent experiments at each tested condition. Data were graphed (mean ± standard deviation) using Prism 5 software v5.01 (2007). Differences in medians (O-acetate) [O-acetate, presented their highest concentration in summer samples , followed by winter ; meanwhile, their presence was significantly lower in autumn , and even lower in the spring seasonal samples . These results indicated that the concentration of poplar-type flavonoids is quantitatively affected in CP by seasonal shifts, even though there is a qualitative resemblance found in the chemical profile of CP among seasonal samples  , we obse samples . p < 0.05), in comparison to the dissolvent control (DMSO). CP produced during the autumn season induced the highest significant inhibitory effect (p < 0.05) in M12.C3.F6 cell proliferation (IC50: 5.9 ± 0.6 µg/mL), in comparison to spring (9.2 ± 1.3 µg/mL), summer (9.5 ± 0.4 µg/mL) and winter (12.1 ± 0.5 µg/mL) propolis (50: 1.4 ± 0.1 µM), according to previous studies [Once we found quantitative variations in the polyphenolic profile of seasonal samples of CP, we determined whether those changes in the relative abundance of CP constituents would inflict modifications on the pharmacological potential of CP. By using MTT assays, we observed that the samples collected over the four seasons of the year induced a significant antiproliferative effect in M12.C3.F6 cells  after 48propolis . These d studies ,36,40. 50: 58.8 ± 6.7, 65.7 ± 12.2 and 67.0 ± 7.5 µg/mL, respectively). CP collected in summer showed a lesser antioxidant capacity among the seasonal samples (IC50: 98.7 ± 2.5 µg/mL). Statistical differences were found among the antioxidant activity of CP seasonal samples at 100 µg/mL; CP from autumn, winter and spring were able to reduce DPPH radical up to 80% at 100 µg/mL, while CP from summer reduced 54% of the DPPH radical at the same concentration ( <p 0.05). Ascorbic acid (AA) was used as antioxidant control . Given the methodological characteristics of the assay, we analyzed only the metabolically active cell population with membrane integrity. By considering DCF and PI fluorescence, none of the CP seasonal treatments, together with H2O2 treatment conditions, significantly affected cell viability or membrane integrity during the 1 h period , wherein lower quadrants (Q3 and Q4) comprise viable cells (negative to PI). At least 90 to 95% of M12.C3.F6 cells (Q3 and Q4) were viable and morphologically similar to those cells pretreated with dissolvent control (DMSO). Cellular morphology distribution of treated M12.C3.F6 cells with CP seasonal samples and H2O2 (1 and 5 mM) did not appear to be affected, in comparison to the forward scatter (FSC) and side scatter (SSC) parameters of cells treated with DMSO and H2O2 (data not shown). CAPE was included as a CAA control based on previous studies [2O2, similar to CAPE, showing a remarkable fluorescence displacement from Q4 to Q3  of intracellular ROS after H2O2-stress induction, but also morphological protection against oxidative stress.In order to evaluate the antioxidant potential of seasonal samples of CP against biological ROS in the cellular environment, a cellular antioxidant activity (CAA) assay was performed in M12.C3.F6 cells under different oxidative stress conditions . Particularly, CP from autumn and spring  induced a 2.2- to 2.7-fold reduction in ROS at basal conditions , in comparison with DMSO treatment  and 5 mM H2O2  of DCF, as a measure of ROS intracellular burden in M12.C3.F6 cells, pretreatment with methanolic extracts of seasonal samples of CP  induced a significant reduction in ROS burden at basal conditions, in comparison to the DMSO control, according to a Mann–Whitney .05, 2O2 . Meanwhi.05, 2O2 . CP coll2O2) in M12.C3.F6 cells , indicating a high protective effect at this concentration (50 µg/mL) under the same conditions, in comparison with DMSO treatment  was as high as 88.3% and 77.2%, respectively; meanwhile, at 5 mM H2O2 their CAA was 72.3% and 63.0%, respectively, an antioxidant effect as high as that induced by CAPE  induced a significant CAA ( <p 0.05). CP from spring and autumn induced the highest protective effect in M12.C3.F6 cells against different conditions of oxidative stress (1 and 5 mM H2O2), at both 25 and 50 µg/mL treatments. CP collected in summer and winter seasons induced a significant CAA; however, these CP seasonal samples were significantly less active than those from spring and autumn, according to the Kruskal–Wallis test  in the Sonoran Desert, particularly collected from autumn to spring, showed significant antiproliferative activity in human and murine cancer cell lines, as well as a significant free radical scavenging activity, measured by a chemical assay ,39. In tO-acetate amongst the seasonal samples. In previous studies, propolis from the semi-arid region of Ures (UP), a propolis type mainly derived from poplar tree exudates (P. fremontii), presented greater amounts of pinocembrin (218.4 ± 1.5 mg/g) than the sample collected from Caborca (60.6 ± 1.2 mg/g). In addition, the sample from UP (at 100 µg/mL) showed a lower antioxidant capacity (23.4 ± 1.5%) than CP at the same concentration (86.0 ± 0.3%). Furthermore, pinocembrin was reported as a flavonoid with no significant free radical scavenging potential (IC50: > 100 µM) [The polyphenolic profile of CP evidenced quantitative fluctuations, mostly between poplar-type constituents among the seasonal samples. CP collected during the summer season showed the highest concentration of pinocembrin and pinobanksin-3- 100 µM) ,39. ThisThe pharmacological potential of polyphenolic compounds is broadly described, including their antiproliferative effect and ROS scavenging capacity ,11. In a50) from autumn, in comparison to a previously studied sample from November to March, showed corresponding bioactivity (IC50: 4.1 µg/mL), suggesting that the inhibitory effect of CP on cancer cell proliferation is mostly due to the phenology of persistently selected plants by honeybees [2O2 conditions, indicating the high antioxidant potential of CP. The CAA results are in agreement with those variations observed in the polyphenolic profile of seasonal samples, wherein CP from autumn and spring has the lowest amount of poplar flavonoids amongst the seasonal samples, as well as the highest CAA, indicating that CP has a differential antioxidant potential clearly influenced by seasonal effect.CP has been previously reported as an antiproliferative polyphenolic matrix from the Sonoran Desert . Herein,oneybees . In prevoneybees ,20. Thesoneybees . Other soneybees . The CAAPopulus nigra) extract, as well as that of its main phenolic compounds, has been determined on human fibroblasts by CAA assay. P. nigra exudates showed moderate antioxidant properties, reducing DCF fluorescence by up to 53% at a 200 ug/mL treatment, whereas pinocembrin and pinobanksin reduced DCF fluorescence by 39 and 27% at 200 uM, respectively [P. nigra extract is in agreement with our results, since P. nigra exudates are described as the main botanical source of European poplar-type propolis [P. fremontii buds are not necessarily correlated to those antioxidant compounds present in CP. In this study, we found that the DPPH assay was not able to discern between the antioxidant potential of CP collected during spring, autumn and winter , although this difference was not significant. Via the DPPH assay, we determined that the free radical scavenging potential of CP for autumn, winter and spring (at 50 µg/mL) was 46.8 ± 4.7, 43.7 ± 7.8 and 41.1 ± 4.2 %, respectively. By using the CAA assay, we found that CP from winter exerted a significantly lower protective effect against biological ROS in M12.C3.F6 cells, in comparison to the antioxidant potential induced by CP from autumn and spring. At 50 µg/mL and at 1.0 mM H2O2, the CAA of CP from autumn, winter and spring resulted in 81.9 ± 5.5, 28.4 ± 12.4 and 86.7 ± 3.5 %, respectively; meanwhile, CAA was 76.0 ± 6.2, 12.0 ± 17.0 and 85.8 ± 5.0 %, respectively, at the same concentration and 5.0 mM H2O2. Herein, CAA provided a sensitive strategy that allowed us to deeply analyze the seasonal effect on ROS scavenging potential of CP into biological context.The antioxidant activity of aqueous poplar bud , which affords a more stringent method. Therefore, in order to pharmacologically characterize a bioactive substance, it is important to analyze its antioxidant potential in assays that consider the cellular environment, such as CAA.Ascorbic acid is one of the most used antioxidant control compounds in the DPPH assay; however, previous studies have demonstrated that its efficiency in a chemical assay could not be properly related to that obtained by CAA . CAA hasP. fremontii exudates are the main botanical source. However, the biological properties of those propolis samples, specifically their antiproliferative effect on cancer cells, suggested the slight contribution of other bioactive resinous materials from different sources other than poplar trees, which enhanced their pharmacological properties [P. fremontii, as well as the previously identified xanthomicrol, a lipophilic flavonoid not biosynthesized by poplars, suggesting its mixed plant origin [Ambrosia deltoidea and Encelia farinosa, which were previously reported as contributing plants to propolis production throughout the Sonoran Desert [Recently, we have determined the plant origin of propolis collected from the semi-arid region of Ures (UP), wherein operties . Here, Ct origin . Therefot origin , pointinn Desert ,38.In conclusion, these results indicate that CP is a source of bioactive compounds that induce a remarkable antiproliferative effect, as well as antioxidant activity and pharmacological potential, which are is clearly influenced by quantitative variations in the polyphenolic profile of CP throughout the year, mostly in poplar-type constituents. According to our results, the most bioactive collection time for CP is during the autumn and spring seasons. Further studies are needed to characterize the molecular basis of the bioactivity of CP.


Human samples: Autopsies on 205 subjects  were performed within 3 days of death. Cause of death was classified as either hypothermia  or non-cold exposure . Cortisol levels were determined in blood samples obtained from the left and right cardiac chambers and common iliac veins using a chemiluminescent enzyme immunoassay. Adrenal gland tissues samples were stained for cortisol using a rabbit anti-human polyclonal antibody. Cell culture: AtT20, a mouse ACTH secretory cell line, and Y-1, a corticosterone secretory cell line derived from a mouse adrenal tumor, were analyzed in mono-and co-culture, and times courses of ACTH (in AtT20) and corticosterone (in Y-1) secretion were assessed after low temperature exposure mimicking hypothermia and compared with data for samples collected postmortem for other cases of death. However, no correlation between ACTH concentration and cortisol levels was observed in hypothermia cases. Immunohistologic analyses of samples from hypothermia cases showed that cortisol staining was localized primarily to the nucleus rather than the cytoplasm of cells in the zona fasciculata of the adrenal gland. During both mono-culture and co-culture, AtT20 cells secreted high levels of ACTH after 10–15 minutes of cold exposure, whereas corticosterone secretion by Y-1 cells increased slowly during the first 15–20 minutes of cold exposure. Similar to autopsy results, no correlation was detected between ACTH levels and corticosterone secretion, either in mono-culture or co-culture experiments. These results suggested that ACTH-independent cortisol secretion may function as a stress response during cold exposure.We previously showed that postmortem serum levels of adrenocorticotropic hormone (ACTH) were significantly higher in cases of hypothermia (cold exposure) than other causes of death. This study examined how the human hypothalamic-pituitary-adrenal axis, and specifically cortisol, responds to hypothermia.  Many reports have documented the pathologic changes observed in human affected by hypothermia due to cold exposure, and “classic” morphologic findings supporting a diagnosis of hypothermia have been established –7. Howevbody) drops below 35°C relative to exhaustion of heat production and failure of heat loss prevention over time [body drops, the hypothalamus (thermoregulation center) triggers a series of reactions that function to produce heat and prevent heat loss. Heat production involves the secretion of stress-response hormones , which triggers an overall increase in lipid metabolism, particularly ketogenesis [Generalized hypothermia occurs when the body temperature , and the median age was 60 years (range 15–98 years). Cortisol levels were determined in blood samples collected aseptically from the left and right cardiac chambers and the common iliac vein using syringes.n = 14) or control. Cause of death in the latter group included blunt injury , sharp-instrument injury (n = 8), fire fatality (n = 43), asphyxia (n = 28), intoxication , drowning (n = 12), hyperthermia , acute ischemic heart disease (n = 20), and natural causes (n = 22). This study involved autopsy cases from 2010 to 2019. Case profiles are shown in Cause of death was determined based on findings from a complete autopsy as well as macromorphological, micropathologic, and toxicologic examinations. Cases were classified as either hypothermia  for histopathologic and immunohistochemical analyses.®analyzer  [Blood samples were immediately centrifuged to prepare serum, and ACTH and cortisol levels were measured using an AIA-360Germany) ,40. ThisSerum samples (150 μL each) were placed in the test cups, and both hormones were measured using the above-mentioned immunoassays. The lower (and upper) reported values for the ACTH and cortisol assays were 2.0 (2000.0) pg/mL and 28.0 (1656.0) nmol/L, respectively.Blood oxyhemoglobin was determined using a CO-oximeter system  in hypothermia patients , 42. Blo2O2-methanol for 10 min to inactivate endogenous peroxidases. After washing in PBS for 5 min, slides were incubated overnight with anti-cortisol-binding globulin antibody . Immunoreactivity was visualized by the polymer method using Dako Envision+ Dual Link System-HRP  and the Dako liquid DAB+ Substrate Chromogen System , according to the manufacturer’s instructions and with hematoxylin counterstaining [Harvested adrenal glands were fixed in 4% paraformaldehyde in PBS (pH 7.2) for 12 h, embedded in paraffin, and sectioned at a thickness of 4μm. Deparaffinization  of each section was followed by heat-mediated antigen retrieval in citrate buffer (pH 7.0) for 10 min, after which each section was immersed in 0.3% Hstaining ,31. The staining ,44.6 tumor cells on the back of the neck. Adrenalectomy was performed by making a mid-incision on the back of young animals 2–3 days before tumor grafting. Adrenalectomized animals were provided 0.5% saline as drinking water and injected with 0.5 mg of Percorten  every 3 weeks. Establishment of clonal cell line of AtT-20  was complete. AtT-201 K cells used in this study were independently established in our laboratory from a transplanted tumor; thus, AtT-20 animals were designated ‘Ms’ mice. Mouse Y-1 adrenocortical tumor cells (ATCC CCL-79), were established in a male mouse [2 for AtT20 and 57,803 cells/cm2 for Y-1, and the cells were allowed to proliferate until they covered the surface of the culture dished. The culture medium for Y-1 cells was replaced once every 2 days. Once the AtT20 and Y-1 cells reached confluence, they were transferred to 4°C and maintained. The amount of ACTH and corticosterone in the culture medium was measured at 5,10,1,20,30,40,60,180, and 360 min; at 12 and 24 h; and at 3 and 5 days. ACTH was measured using a mouse ACTH assay kit  [Mono-culture models of ACTH-secreting AtT20 pituitary cells –49 and cle mouse –61. For c., USA) ,63, and c., USA) –47. At tWe developed a co-culture system for AtT20 ACTH-secreting cells (ECACC no.87021902) ,49 and Y6 pores/cm2. Initially, corticosterone-secreting Y-1 cells were cultured on the bottom of the filter with the filter placed upside down so that the cells formed a mono layer. Subsequently, the filter was placed upright in the culture medium. Schroten H, (2016) established this method in a choroid plexus model [Initially, both AtT20 and Y-1 cells were cultured separately at 37°C, with AtT20 and Y-1 cells on the top and bottom of the filter, respectively. The cells were then co-cultured at 4°C. The insert for 6-well plate  used to separate the AtT20 and Y-1 cells had a diameter of 23.1 mm, pore size of 3.0 μm, and pore density of 2×10us model –72, and us model .2. As the Y-1 cells formed tights junctions, movement of ACTH between the cells was prevented. Thereafter, the culture medium was replaced once every 2 days. Once culturing of the Y-1 cells was complete, AtT20 (ACTH secreting) cells were similarly grown on the other side of the filter . The filter was immersed in the culture medium by placing the ACTH-secreting (AtT20) cells side facing up and corticosterone-secreting (Y-1) cells side facing down. Levels of ACTH and corticosterone in the culture medium were measured at 5, 10, 15, 20, 30, 40, and 60 min, as indicated above. After measurement of both hormones, the adherent cells were dissociated from the filter using trypsin and counted. Accurate hormone concentrations were calculated using a correction formula and the measured values.Excessive growth on the filter was controlled by trypsinization to maintain a single layer of cells; the number of Y-1 cells on the filter was limited to 57,803/cmU test. The Games-Howell test was used for analyses involving multiple comparisons. All analyses were performed using Microsoft Excel and IBM SPSS statistic viewer 24. Lines in each box represent the median, whereas lines outside each box represent the 90% confidence interval. The sensitivity and specificity for distinguishing between two groups using cut-off cortisol values based on blood collection site  were estimated using receiver operating characteristic (ROC) curve analysis. Areas under the curve were calculated and analyzed using a 1-tailed test. The optimal compromise between sensitivity and specificity was determined graphically.For comparisons between groups, we used the nonparametric Mann-Whitney This study was evaluated by the Independent Ethics Committee of the Osaka City University Graduate School of Medicine. According to the Independent Ethics Committee of the Osaka City University Graduate School of Medicine, informed consent from opt-out for the autopsy data analysis was approved. (authorization no.4153). The Ethics Committee approval for the study was valid for the period 2010–2019.R = 0.249; p>0.05, right cardiac blood: y = 0.279x+11.002; R = 0.163; p>0.05, iliac vein: y = 0.299x+11.814; R = 0.152; p>0.05), survival period , sex and related differences, or age.Serum cortisol levels were not associated with postmortem period . Cortisol levels by sex were as follows. Males: left cardiac blood 1.5–154.6 μg/dL; median 20.8 μg/dL, right cardiac blood 1.7–136.7 μg/dL; median 19.1 μg/dL, iliac vein blood 1.1–150.1 μg/dL; median 20.3 μg/dL. Females: left cardiac blood 0.7–284.5 μg/dL; median 30.5 μg/dL, right cardiac blood 1.0–272.9 μg/dL; median 25.2 μg/dL, iliac vein blood 1.3–297.4 μg/dL; median 26.3 μg/dL.Males and females did not exhibit significant differences in left cardiac blood, right cardiac blood, or iliac vein blood : left cardiac blood 1.9–22.4 μg/dL; median 13.0 μg/dL, right cardiac blood 1.4–21.7 μg/dL; median 12.6 μg/dL, iliac vein blood 2–24.8 μg/dL; median 14.8 μg/dL. 20s (n = 7): left cardiac blood 4.6–42.7 μg/dL; median 13.0 μg/dL, right cardiac blood 4.7–13.0 μg/dL, median 13.0 μg/dL, iliac vein blood 6.4–44.7 μg/dL; median 18.3 μg/dL. 30s (n = 7): left cardiac blood 2.7–43.4 μg/dL; median 15.5 μg/dL, right cardiac blood 1.7–33.8 μg/dL; median 11.6 μg/dL, iliac vein blood 0–40.0 μg/dL; median 10.8 μg/dL. 40s (n = 19): left cardiac blood 1.5–55.2 μg/dL, median 15.7 μg/dL, right cardiac blood 1.7–53.2 μg/dL; median 14.1 μg/dL, iliac vein blood 1.1–57.2 μg/dL; median 15.4 μg/dL. 50s (n = 14): left cardiac blood 6.1–154.6 μg/dL; median 40.3 μg/dL, right cardiac blood 8.3–136.7 μg/dL; median 36.9 μg/dL, iliac vein blood 7.8–150.4 μg/dL. 60s (n = 37): left cardiac blood 2.9–60.0 μg/dL; median 17.7 μg/dL, right cardiac blood 3.4–60.0 μg/dL; median 15.5 μg/dL, iliac vein blood 2.9–72.8 μg/dL; median 16.8 μg/dL. 70s (n = 30): left cardiac blood 3.8–86.8 μg/dL; median 24.9 μg/dL, right cardiac blood 3.3–81.5 μg/dL; median 23.7 μg/dL, iliac vein blood 4.5–109.6 μg/dL; median 2.5 μg/dL. 80s (n = 25): left cardiac blood 0–60 μg/dL; median 15.5 μg/dL, right cardiac blood 2.6–60.0 μg/dL; median 16.4 μg/dL, iliac vein blood 0–60 μg/dL; median 14.9 μg/dL. 90s (n = 5): left cardiac blood 11.4–28.7 μg/dL; median 20.9 μg/dL, right cardiac blood 9.4–17.9 μg/dL; median 18.9 μg/dL; iliac vein blood 9.4–17.0 μg/dL; median 17.0 μg/dL. Females: 10s (n = 1): left cardiac blood 2 μg/dL, right cardiac blood 1.9 μg/dL, iliac vein blood 2.4 μg/dL. 20s (n = 3): left cardiac blood 0.7–3.0 μg/dL; median 1.9 μg/dL, right cardiac blood 0.0–1.7 μg/dL; median 0.9 μg/dL, iliac vein blood 1.3–2.4 μg/dL; median 1.7 μg/dL. 30s (n = 2): left cardiac blood 3.0 μg/dL and 3.3 μg/dL, right cardiac blood 1.9 μg/dL and 2.4 μg/dL; iliac vein blood 2.9 μg/dL and 3.8 μg/dL. 40s (n = 6): left cardiac blood 3.3–7.1 μg/dL; median 5.4 μg/dL, right cardiac blood 3.1–6.3 μg/dL; median 4.6 μg/dL, iliac vein blood 3.8–6.9 μg/dL; median 5.0 μg/dL. 50s (n = 7): left cardiac blood 7.6–11.1 μg/dL; median 8.8 μg/dL, right cardiac blood 6.5–8.7 μg/dL; median 8.7 μg/dL, iliac vein blood 3.8–6.9 μg/dL; median 5.0 μg/dL. 60s (n = 3): left cardiac blood 13.1–13.8 μg/dL; median 13.3 μg/dL, right cardiac blood 9.0–11.0 μg/dL; median 10.2 μg/dL, iliac vein blood 10.9–11.4; 11.1 μg/dL. 70s (n = 12): left cardiac blood 13.9–19.4 μg/dL; median 15.9 μg/dL, right cardiac blood 11.5–15.3 μg/dL; median 13.4 μg/dL, iliac vein blood 12.8–16.3 μg/dL; median 14.0 μg/dL. 80s (n = 19): left cardiac blood 19.8–58.2 μg/dL; median 33.0 μg/dL, right cardiac blood 16.1–45.3 μg/dL; median 28.1 μg/dL, iliac vein blood 16.8–60.0 μg/dL; median 29.7 μg/dL. 90s (n = 5) left cardiac blood 86.5–284.5 μg/dL; median 154.9 μg/dL, right cardiac blood 50.8–272.9 μg/dL; median 119.4 μg/dL, iliac vein blood 68.6–297.4 μg/dL; median 131.7 μg/dL.Cortisol levels in each age groups of males and females were as follows. Males: 0–24 h: left cardiac blood 1.5–75.3 μg/dL; median 17.0 μg/dL, right cardiac blood 1.7–76.2 μg/dL; median 1.7–76.2 μg/dL; median 15.6 μg/dL, iliac vein blood 1.1–76.9 μg/dL; median 16.8 μg/dL. 24–48 h: left cardiac blood 2.9–154.6 μg/dL; median 24.5 μg/dL, right cardiac blood 2.4–136.7 μg/dL; median 22.2 μg/dL; iliac vein blood 3.0–150.4 μg/dL; median 24.6 μg/dL. 48–72 h: left cardiac blood 0.0–55.2 μg/dL, median 18.8 μg/dL, right cardiac blood 3.3–53.2 μg/dL; median 18.7 μg/dL, iliac vein blood 0.0–57.2 μg/dL; median 16.8 μg/dL. Females: 0–24 h: left cardiac blood 0.0–66.9 μg/dL, median 17.7 μg/dL; right cardiac blood 0.0–50.8 μg/dL; median 16.6 μg/dL, iliac vein blood 1.6–38.7 μg/dL, median 17.7 μg/dL. 24–48 h: left cardiac blood 0.7–284.5 μg/dL; median 29.6 μg/dL, right cardiac blood 1.1.-272.9 μg/dL; median 27.4 μg/dL, iliac vein blood 1.3–297.4 μg/dL; median 31.0 μg/dL. 48–72 h: left cardiac blood 10.2–121.8 μg/dL; median 54.4 μg/dL, right cardiac blood 0.0–99.6 μg/dL; median 30.0 μg/dL, iliac vein blood 0.0–90.4 μg/dL; median 29.7 μg/dL.Cortisol levels were also classified by postmortem period in each sex as follows . R = 0.97–0.98) with blood collection site, particularly the left and right cardiac chambers and external iliac vein. A strong correlation was found between left cardiac vein blood, right cardiac vein blood, and iliac vein blood, respectively .Cortisol levels exhibited a correlation ; right cardiac blood, 20–100 μg/dL , females: 36.5–99.6 μg/dL (median 62.5 μg/dL), p>0.05); iliac vein, 20–130 μg/dL , females: 36.5–99.6 μg/dL (median 60.5 μg/dL), p>0.05) versus left cardiac blood, 0–50 μg/dL , females: 0.7–284.5 μg/dL (median 27.0 μg/dL), p>0.05); right cardiac blood, 0–40 μg/dL , females: 1.0–272.9 μg/dL (median 21.5 μg/dL), p>0.05); iliac vein, 0–20 μg/dL , females: 1.3–297.4 μg/dL (median 23.6 μg/dL), p>0.05). There was no significant difference in cortisol levels between hypothermia and other causes of death for either males or females. Furthermore, most cases exhibited lower cortisol levels, except in hyperthermia cases . There was no correlation between ACTH concentration and cortisol in hypothermia cases at any of the collection sites tested .At all blood collection sites, cortisol levels were approximately three times higher in hypothermia cases than in cases involving other causes of death  and lower (other cause of deaths) cortisol levels were determined using ROC curve analysis and estimated as 30 μg/mL (0.917 and 0.852) for the left cardiac chamber, 25 μg/mL (0.917 and 0.836) for the right cardiac chamber, and 30 μg/mL (0.917 and 0.872) for the common iliac veins.Cortisol immunostaining analysis indicated that in hypothermia cases, cortisol was primarily localized in the nucleus, whereas cortisol staining was predominant in the cytoplasm in cases involving other causes of death . the GraIn the mono-culture models, ACTH- and corticosterone-secreting cells were cultured separately at 4°C to ensure the absence of ACTH in the culture of corticosterone-secreting Y-1 cells . AtT20 cIn the co-culture model , ACTH seThe correlation between cortisol levels and blood collection site in the present study suggests there were differences in cortisol levels at the various collection sites tested. Therefore, we assessed the relationship between cortisol levels in blood collected from each site and cause of death and found that cortisol levels in cases of hypothermia were three times higher than those in other causes of death. As stress hormones, ACTH and cortisol may exhibit regional differences in terms of increases with an acute change in survival period. However, ACTH and cortisol levels were highly correlated in the blood. It was thought that this result was associated with death characterized by a long survival period rather than sudden death due to causes such as acute cardiac death, based on the respective half-lives of ACTH and cortisol. No significant correlations were observed between cortisol levels and causes of death other than hypothermia. There was no correlation between ACTH concentration and cortisol levels in hypothermia, suggesting that cortisol can be produced by the adrenal gland during cold stress without stimulation by ACTH.O-methylated metabolites, urinary free cortisol, blood cortisol, as well as blood, vitreous humor, and pericardial fluid for ketone bodies and free fatty acids. These biomarkers are increased in response to either cold-associated stress or bioenergetic ketogenesis crisis and significantly contribute to the diagnosis by excluding death by hypothermia, perimortem elevation of adrenal cortex hormones might reflect more of a decrease in metabolism and hepatic clearance than an increase in ACTH [We cultured an adrenal cortex at low temperature using culture fluid that did not contain ACTH. An increase in ACTH-independent corticosterone was observed. We did not use cells cultured from human samples for this experiment because the cells that normally secrete ACTH and cortisol in humans have not been established. We therefore used a mouse cell culture mode. Such ACTH-independent production of cortisol might be protective during prolonged (but not acute) periods of cold stress, as cold exposure promotes glucose production . Recentl in ACTH –84. One  in ACTH ,85–88. ICortisol is produced primarily in the zona fasciculata of the adrenal gland. Cell counts and nuclear and cytoplasm staining by technicians blinded to cause of death showed that during hypothermia, cortisol staining was primarily localized in the nucleus rather than the cytoplasm. Furthermore, nuclear staining of cortisol was significantly greater in cases of hypothermia than cases involving other causes of death, whereas no significant difference between groups was noted in terms of cytoplasmic staining. These findings support studies showing that glucocorticoid receptors are inactive in the cytoplasm, as they are complexed with other proteins . When glIn this study, we used a novel co-culture system to assess ACTH and corticosterone secretion secondary to cold stimulation. Experiments involving cultured cells are generally initiated at the point each hormone is at a stable concentration. Thus, increases after initiation of the experiment are significant. The increased levels of ACTH and cortisol in the blood following cold exposure did not differ by blood collection site. This suggested that the survival period of the case involving death due to cold exposure was long and that cortisol levels were stable at each blood sampling site. In contrast, the cell culture experiment used medium that did not contain ACTH. This experiment was started after the number of cells in the culture flask was stable. Thus, the point at which measurements began was not the start time of ACTH secretion from ACTH-producing cells after cold exposure. In other words, in the cell culture model experiment, the increases in ACTH and corticosterone production after the start of measurement were significant.We demonstrated that ACTH and corticosterone secretion levels and patterns differed and were not correlated. Mono-culture of ACTH- and corticosterone-secreting cells under ACTH-free conditions at 4°C resulted in a sudden peak in ACTH at 10 min that decreased after 30 min. This can be explained by the half-life of mouse ACTH . HoweverThere are some limitations to this study. The correlation between ACTH and corticosterone levels in mouse cell culture may differ from that observed in human autopsy examples. The half-life of hormones may also differ in the cell culture models and in humans. Furthermore, it is necessary to examine differences between human cortisol and mouse corticosterone and address problems associated with temperature setting in the cell culture model .In conclusion, the present study showed that serum cortisol level can be used as a biomarker for cold exposure and that cortisol production in response to cold stress does not depend on ACTH-based activation. As immunostaining for cortisol revealed high expression levels in the nucleus after cold exposure, it is possible that cortisol production following cold exposure is independent of ACTH stimulation.S1 FigRelationship between the cortisol level in blood collected at different sites and sex (a), age (b), survival period (c), and postmortem period (d) in all cases. The difference in males and females was associated with the difference in number. However, in terms of age, survival period, and postmortem period, there was a correlation, so the number did not affect the cortisol level. There was no difference between males and females in terms of collection site.(PPTX)Click here for additional data file.S2 FigLeft and right cardiac blood (a), left cardiac blood-iliac vein blood (b), and right cardiac blood-iliac vein blood (c).(PPTX)Click here for additional data file.