Clostridium perfringens, Enterobacteriaceae, Enterococci and Mesophilic bacteria was found. Additionally, in litter, organic acids and essential oils dietary treatment drove to an overall decrease of Mesophilic bacteria and Enterococci counts. Overall, dietary strategy oriented to a supplementation of a mixture of organic acids and essential oils in broiler diets could offer some favorable perspectives in order to maintain adequate growth performance and gut healthiness either in term of morphology or of microbiology. Nevertheless, improving knowledge on the mechanisms of action of these natural additives together with a potential synergistic action is pivotal to clarify their potential as antibiotic replacers. Replacing antibiotics with natural alternative compounds in poultry feeding is being increased in the last few years to challenge the antibiotic resistance problem. Among natural compounds, organic acids and essential oils could be a favorable option. The goal of the trial was testing the dietary supplementation of a blend of organic acids and essential oils in broiler diets in order to evaluate growth performance and gut healthiness. The blend of organic acids and essential oils improved growth performances at the end of the growing period and favorably affected, to a certain extent, gut morphology at different gut districts. Moreover, a selective microbial control against p < 0.01) and feed conversion ratio at 37–47 days compared to BD treatment. OA&EO treatment improved gut morphology mostly at ileum and duodenum levels in terms of villi height, crypt depth, number of villi, mucosa thickness and villi area at 24 and 34 sampling days. A certain selective action against Clostridium perfringens in ileum of OA&EO group was shown at 33 (p = 0.053) and 46 days (p = 0.09) together with lower median values for Enterobacteriaceae, Enterococci, Mesophilic bacteria and Clostridium perfringens at ceca level. Overall, organic acids and essential oils supplementation improved growth performance in the final growth stage and some morphological gut traits and reduced to a certain extent Clostridium perfringens count in ileum.The goal of the trial was testing the effects of a blend of organic acids and essential oils dietary supplementation on growth performance and gut healthiness in broiler chickens. In total, 420 male Ross 308 chicks (1-day old) were randomly assigned to two dietary treatments: basal (BD) and organic acids and essential oils (OA&EO) diets . BD group received commercial diets whereas OA&EO group basal diets + 5 g/kg of microencapsulated organic acids and essential oils. OA&EO treatment improved the average daily gain ( The use of antibiotic as growth promoters (AGP) in the poultry industry was widespread all over the world. Initially they were used at low dosage in feedstuffs to improve growth performance and to prevent illness; in Europe, however, their use has been gradually restricted until their total ban starting from 2006 according to European Parliament and Council Regulation EC No. 1831/2003 and European directive 2004/28/EC of the of 31 March 2004 . Among aEssential oils (EO) are mixtures of volatile compounds produced by living organisms and isolated by physical means only (pressing and distillation) from a whole plant or plant part of known taxonomic origin that are proven to exert a natural antibiotic effect with less toxicity and without residues compared to AGP . The poultry industry has focused on these additives because they showed an increased nutrients digestion and absorption  and a reMicroencapsulated OA and EO, alone or mixed, as feed additive in broiler chickens improved performances and gut microflora , reducedA total of 420 male Ross 308 chicks (1-day-old) from the same hatching were placed in a large shed and were randomly allotted into two treatments with three replicates per treatment  and 70 chicks per replicate. The experimental trial was performed between May and July 2019 according to UE Regulation on using animals for scientific purpose . The dieA total of eight BD and eight OA&EO diet samples (two subsamples for each feed for each subperiod subsequently pooled) were collected during the trial, vacuum packed and stored at −30 °C until analyses. Dry matter, crude protein, lipid, crude fiber, ash, calcium, phosphorus, lysine and methionine were analyzed according to Commission Regulation (EC) No 152/2009  to samplSamples for histological analysis of the three segments  of the small intestine were immediately taken, after sacrifice, from three chickens for each replicate at every feed change (nine BD and nine OA&EO), for a total of 72 birds. 2] = π × Villus height [μm] × Villus width [μm] and expressed in Log10 basis. The criterion for villus selection was based on the presence of intact lamina propria. Villus height was measured from the tip of the villus to the villus-crypt junction, whereas crypt depth was defined as the depth of the invagination between adjacent villi.Three cm length-portions of each segment were collected during the trial at 11, 25, 34 and 46-day. A total of 216 samples were analyzed for morphometric analysis due to different treatments (BD and OA&EO) and age of chicks. Individual segments of intestine were rinsed with 0.9% of physiological saline and then fixed in 4% buffered paraformaldehyde. The fixed samples were dehydrated, cleared and infiltrated with paraffin in a tissue processor, Leica ASP300, and then embedded in paraffin blocks. The blocks were sectioned in slices of 2.5 µm of thickness, with two slices for every block. The sections were placed on glass slide and stained with hematoxylin and eosin (HE) with an automatic Stainer (Thermo Scientific Shandon varistain gemini) for histological analysis. Morphometric analysis was performed through a Leica DMLB microscope connected with a Nikon camera. Villus height, villus width, crypt depth and crypt width were individually measured on at least five intact villi per intestinal segment. In addition, intestinal mucosa thickness was measured, and villus surface area was calculated using the equation of Rubio et al. : Villus Mesophilic bacteria; Violet Red Bile Glucose Agar, aerobically incubated at 37 °C for 24 h, for the Enterobacteriaceae count; Tryptone Bile X-gluc agar, aerobically incubated at 40 °C for 24 h, for Escherichia coli determination; Bile Aesculine Azide agar, incubated at 37 °C for 24 h, for Enterococcus spp. determination; De Man Rogosa and Sharp agar, incubated at 37 °C for 72 h, for Lactic Acid Bacteria (LAB) determination; M17 agar, incubated at 37 °C for 72 h, for Lactococcus spp. determination; SC agar, incubated in anaerobiosis for 48 h, for determination of Clostridium perfringens. Results were expressed as Log10 Colony-Forming Unit CFU/g. The detection of Campylobacter spp. was assessed through the following protocol: 10 gram samples were enriched in 90 mL of Bolton’s broth and 5% (v/v) of lysed horse blood and subsequently homogenized through stomacher and incubated at 40 °C for 24 h in microaerobic condition. After incubation, 100 µl of the enriched broth was transferred to Modified Charcoal Cephoperazone Deoxycholate Agar (MCCDA) and incubated at 42 °C for 48 h in microaerobic condition. Presumptive Campylobacter colonies were observed under phase contrast microscopy  for spiral morphology and darting motility [Listeria Monocytogenes has been carried out according to the following protocol: 10 g samples were homogenized through stomacher in 90 ml of Half Fraser broth and aerobically incubated for 24 h at 30 °C. After the incubation, 1 ml of enriched broth eluted in 9 ml of Fraser Broth and incubated for 24 h a 30 °C. After the second incubation, 100 µl of enriched broth was transferred to Aloa Agar and incubated for 24 h at 30 °C [Salmonella spp. detection, 10 gram samples were homogenized through stomacher in 90 ml of Tryptone Soya Broth and aerobically incubated for 24 h at 37 °C. After incubation, 1 ml of enriched broth was transferred to Modified Semi-Solid Rappaport-Vassiliadis agar and incubated for 48 h at 40 °C. Presumptive Salmonella colonies were transferred on Xylose Lysine Desoxychloate agar and Brilliant Green Agar [As for the morphometric measurements, the microbiological analyses were assessed at every feed change . Ileal contents of the same 72 chickens used for histological measurements were collected approximately from 1 cm below Merckel diverticulum to 4 cm above caecum tonsils according to procedure of Gheisari et al. ; six samat 30 °C . Lastly,een Agar .p < 0.05, while trends were considered for 0.05 < p < 0.10. Statistical analyses were performed by the statistical software Minitab, version 16 .Data on broiler performance were analyzed using a one-way analysis of variance (ANOVA) to test the effect of the dietary treatment (OA&EO vs. BD). Data on gut intestinal morphometry were also analyzed using a one-way ANOVA to test the effect of dietary treatment within sampling day and intestinal section, while microbiological analyses in ileum and litter were processed with the same one-way ANOVA to test the effect of dietary treatment within sampling day. Microbial counts at caecum level were analyzed through a box-plot to show the distribution and the level of bacterial species. Differences between means were assessed using the Tukey’s adjustment for multiple comparisons. Significance was declared when The overall mortality was lower in OA&EO group compared to BD one . Animal performances are reported in p < 0.05) live weight compared to BD. On the other hand, the final weight at 47 d was significantly higher (p = 0.001) in the OA&EO group than in the BD one. As a consequence, in the 35–47 d growing phase average daily gain was significantly higher in the OA&EO group , whereas at 0–12 days, 12–25 days and 25–35 days, no significant differences were found in the growth rate between treatments. Feed intake tended to be lower (p < 0.10) at 0–12 days in the OA&EO group compared to its counterpart, while no differences were reported in the subsequent growing phases. Feed conversion ratio improved significantly (p < 0.01) in the last growing phase (35–47 days) in the OA&EO group compared to BD according to the differences in the average daily gain previously reported.No significant difference in live weight between treatments was found at day 12, while at 25 d and at 35 d, the OA&EO group showed a lower (p = 0.091) and crypt depth (p = 0.055) being higher in the OA&EO group compared to the BD one. Moreover, at the same sampling time, mucosa thickness of duodenum was significantly higher (p = 0.002) in OA&EO chicks compared to its counterpart. At day 34 in the ileal tract, OA&EO chicks showed an increased villi height (p = 0.001), an increased in tendency (p = 0.10) villi width, higher mucosa thickness (p = 0.057) and villi area, Log10 (p = 0.005), compared to BD chicks. At duodenum level, at the same sampling time, the number of villi was higher (p < 0.05) in the OA&EO treatment than in BD one. At day 46 in jejunum, villi height tended to be higher (p = 0.089) in OA&EO chicks compared to BD ones. Moreover, the epithelial structure of chicks’ gut fed with OA&EO and BD was different in terms of cells organization and inflammation as shown in Morphometric parameters of villi  and depth and width of crypts in three segments of the small intestine  are reported in Enterobacteriaceae were found in samples from OA&EO treatment at days 34 and 46, from Enterococci at day 11, 25 and 34, from Escherichia coli at day 46 and from Mesophilic bacteria at day 25, 34 and 46, compared to the BD treatment. As above, in the OA&EO samples, the LAB count increased across all sampling days, although not at a statistical level but only as a numerical value. The bacterial count for Clostridum perfringens tended to be lower in intestinal content from OA&EO treatment at day 34 (p = 0.053) and at day 46 (p < 0.10) compared to the BD one.Bacterial counts of ileal digesta at different times and for each bacterial family and species are reported in Enterobacteriaceae, Enterococci, Mesophilic bacteria and Clostridium perfringens. The distribution of values showed a different trend, namely the presence of Escherichia coli stable in the caecum of OA&EO chickens, while floating in the caecum of chickens fed on basal diet. In litter sampled at different days within treatment, bacterial counts for each bacterial species are reported in Mesophilic bacteria and Enterococci counts were significantly lower (p < 0.05) in OA&EO litter compared to BD at day 41. Enterobacteriaceae count was lower (p < 0.10) in OA&EO at day 20, while an opposite trend was shown at day 41 when the latter bacterial count tended to be higher in the same group compared to BD one. With regard to Clostridum perfringens count, the litter of OA&EO group tended to have a lower (p < 0.10) total count compared to the BD one. At day 20 of sampling, Escherichia coli count was higher in the OA&EO litter compared to the BD one, whereas non-significant differences between treatments occurred at day 41. Within each period in the total of samples of gut tracts and in litter of chicks of both groups, the presence of Listeria monocytogenes, Salmonella spp. and Campylobacter spp. was not noticed.Bacterial counts of the caecum content at different days, treatment and for each bacterial species are reported in The goal of our experiment was testing the feasibility of dietary supplementation at 0.5% dosage of a microencapsulated blend of organic acids, such as citric and sorbic, and phytochemicals, such as thymol and vanillin, in terms of performance, gut integrity and microbial assessment at ileum and caecum level and in litter compared to an antibiotic-free basal diet. The main findings were a reduced mortality rate and an improved growth gain and feed conversion ratio in the last growing subperiod (35–47 days). The supplementation of organic acids and phytochemicals has been well-documented in swine either alone or in combination with or in partial replacement of allopathic growth promoters (antibiotics) due to their acidifying and antimicrobial properties ,16.In poultry, few studies focused on the use of a blend of OA and EO and often with controversial results if compared to medicated dietary treatment ,37,38. MGrowth performance were favorably affected by OA&EO treatment only at the final growing phase (35–47 days), showing a significant growth recover if compared to previous phases when growth rate decreased in chicks fed an OA&EO diet compared to those fed a basal diet. Probably, a delay in the adaptation to the supplement by OA&EO group that improved later growth performance could justify this finding. Gheisar et al. , using dThe gut ecosystem is composed by three crucial elements: microbial community, intestinal epithelial cells and immune system. These three elements could be affected, positively or negatively, by the diet, gender, background genotype, housing environment, litter and age of birds . PrebiotBogucka et al.  reportedThe combining increase of number of villi in duodenum segment together with the improved morphology of ileum tract could infer a more tonic intestine in OA&EO chickens compared to BD birds. Intestinal morphology of epithelial structure in the BD group showed villi fusion, loss of epithelium and presence of monocyte infiltrate; while in the OA&EO gut, structure and morphology appeared regular .Enterobacteriaceae count gradually increased in both groups, although any pathogenic strains were observed. The Enterococci count, although not different at a statistical level between treatments at each day of sampling, showed less fluctuation across sampling time in chickens supplemented with OA&EO compared to those fed on basal diet. The presence of LAB in the ileal digesta gradually increased in both group without statistical difference, although in chickens fed an OA&EO diet, it was numerically higher compared to BD chickens. Gheisar et al. [Lactobacillus counts in fecal microbiota of chicken fed with the same blend of OA and EO compared to control group. In contrast, the utilization of OA causing several reductions in ileal pH was shown to develop an unfavorable gut environment for the normal proliferation of Lactobacilli bacteria [The microbiological scenario in terms of the bacterial count of ileum digesta in chicken supplemented with OA&EO did not show significant higher values for most of the bacterial species analyzed compared to broilers fed a basal diet. The r et al.  reportedbacteria . Escherichia coli was not affected by dietary treatment, with an exception at day 25, when there was higher amount of E. coli in OA&EO chickens compared to BD ones. It is well known that the antimicrobial activity of essential oils such as thymol, eugenol and carvacrol, which showed high antimicrobial activity against pathogenic bacteria such as Escherichia coli and Salmonella typhimurium, is a potential risk factor of enteric infections [Lactobacillus but not always cause a reduction of E. coli in fecal matter [Mesophilic bacteria count showed the same increasing trend across the sampling time with no differences between treatments. The presence of fections . Neverthl matter . MesophiClostridium perfringens is claimed as the main causative agent of necrotic enteritis (NE) with serious damages in the poultry productive chain [Clostridium perfringens count after 25 days of growing period compared to BD chicks. We could hypothesize a gradual effect of OA&EO supplementation in controlling this pathogen, while in the BD group, a remarkable increase in Clostridium counts at 33 and 46 days of sampling was found. The lower presence of C. perfringens in ileum of OA&EO broilers is probably connected to the ultrastructural changes promoted by the prebiotic mix, which could increase the resistance capacity of broilers to intestinal infection caused by this pathogen [in vitro the growth of a number of bacteria, including several strains of Clostridia such as C. perfringens [in vitro an antibacterial effect of essential oils and favorable effects in stimulation of digestive enzymes, and in stabilizing gut microflora due to inactivation of the C. perfringens toxins thus reducing its colonization in the broiler gut.ve chain . OA&EO cpathogen . Furtherfringens ,49. Mitsfringens  reportedClostridia, is well known. Organic acids in the caeca as well as in the small intestine can affect the bacterial counts, as reported in literature [Enterobacteriaceae, Enterococci, Mesophilic bacteria and Clostridium perfringens in OA&EO caeca compared to BD ones. The range of values was extremely different between treatments if focused on Escherichia coli, which, even if it showed a median slightly higher in OA&EO treatment compared to BD, denoted a similar count value along sampling days. Clostridium perfringens had a similar range in both the treatment, but showed a lower value in OA&EO chickens.Organic acids are involved in antimicrobial activity through the reduction of digesta pH till in the upper parts of the intestinal tract , and it terature . In our Salmonella colonization in chicken caecum by enhancing the innate immune defense via increased synthesis of host defense peptides [Mesophilic bacteria and Enterococci counts recorded lower in OA&EO litter compared to BD one, at the last day of sampling. Nevertheless, Enterobacteriaceae did not follow the same trend, the concentration being lower at day 20 and higher at day 41 compared to the BD litter. Escherichia coli showed a similar trend found in ileum, being higher in OA&EO litter after the first half of the growing period, while comparable onwards with BD litter. Moreover, a certain reduction in C. perfringens on the litter, namely the ileum and caecum contents, was reported at the end of growing period.Organic acids, through lowering pH, protect chickens from various infections, especially at young ages. In addition, they have shown their ability in reducing peptides . Furtherpeptides . We coulClostridium perfringens count at ileum level in the late growing period and in litter and less Mesophilic bacteria and Enterococci counts in the litter of broilers fed on diet supplemented with organic acids and phytochemicals. Finally, there is a need to improve the understanding of the mechanism of action of these prebiotics, taking into account the dosage of supplement and potential interaction with other dietary ingredients, and also the great farm to farm variation in their main effects due to different cleanliness of the production environment and heterogeneity of gut microbiota. Moreover, a better comprehension of the synergistic effect of combinations of alternatives to antibiotics as growth promoters is still challenging. A 0.5% supplementation of a blend of microencapsulated organic acids (citric and sorbic acids) and essential oils (thymol and vanillin) in broiler chickens reduced the overall mortality rate and positively affected growth rate in the last period of growing cycle, thus also improving the feed conversion ratio. Moreover, a favorable effect on gut morphology was found in different intestinal segments in the last growing phases. In particular, an increased villi height, villi width, mucosa thickness and villus number after 25 days of supplementation was shown, together with an intact organization of epithelial structures till the end of the fattening cycle. With regard to microbiological aspects, the main finding was a certain reduction of


Concerns exist regarding the analgesia effect and safety of epidural steroid injection (ESI) after discectomy. There is an urgent need of studies that efficiently control for confounding, conduct comprehensive and consecutive observation of potential risks of ESI, and investigate its clinical applicability. We thus further designed a randomized controlled study to assess the efficacy of ESI on postoperative pain and complications in patients undergoing unilateral lumbar microdiscectomy.This prospective, blinded randomized controlled trial was conducted at our single hospital. This study was approved by the Committee at the No.2 People's Hospital of Yibin. All procedures were performed by a single surgeon and informed consent was obtained from each patient. Ninty eligible patients diagnosed at our institution with herniated lumbar disc during a period from June 2020 to July 2021 will be assessed. Group 1 was a mixture of 1 mL of 0.5% bupivacaine and 10 mg of triamcinolone acetonide in 1 mL. Group 2 was a mixture of 1 mL of 0.5% bupivacaine and 1 mL of normal saline. The primary outcome measure was the amount of morphine consumption from a patient-controlled analgesia pump at 12, 24, and 48 hours after surgery. The following secondary outcomes were also assessed: postoperative pain score, back pain score, functional disability, and adverse effect.We hypothesized that the ESI was associated with lower pain score, morphine consumption, and hospital stay, with no significant difference in complications for ESI application after lumbar discectomy in lumbar disc herniation when compared with placebo.This study protocol was registered in Research Registry (researchregistry5683). However, many patients with lumbar discectomy experience moderate to severe back pain and radicular leg pain. Lumbar discectomy will remove the mechanical cause of pain, but the painful inflammatory process might persist for a few more days. In addition, the inflammation together with the surgical intervention might negatively influence the long-term results by initiating fibrosis which is considered one of the reasons for late reappearance of pain.–4Lumbar disc herniation is considered to be one of the main causes of sciatica, and lumbar discectomy is the most popular surgical procedure performed in patients with sciatica in China.–8 Many surgeons routinely use ESI during lumbar discectomy to reduce traumatic nerve root inflammation and edema. However, glucocorticoids have multiple adverse effects. Administration of exogenous glucocorticoids dramatically reduces bone mineral density and increases the risk of fracture.–13Reduction of inflammation and edema of the affected nerve root should, theoretically, reduce the postoperative pain intensity. Epidural steroid injection (ESI) is widely used to treat pain originating from the spine, such as pain accompanying spinal stenosis, intervertebral disc herniation, and other degenerative spinal pathologies.–16 Other studies on perioperative steroid vary within administration, diagnostic criteria, surgical technique, and postoperative care. Furthermore, the number of patients in most studies has been low and 2 randomized controlled studies (RCTs) showed no significant effect. However, the other studies reported a positive effect of steroids on pain. Four previous meta-analyses were published–21; 2 meta-analyses reported that intraoperative ESI when compared to placebo was effective in reducing pain in the early stage and reducing consumption of analgesia with no statistically significant differences in terms of overall complications and infection in lumbar discectomy.,19 One meta-analysis reported that intraoperative ESI application offers some benefits in pain control with a significant reduction in the length of hospital stay. The most recent meta-analysis indicated a lower back and leg pain score, decreased morphine consumption, and shorter hospital stay without difference in complications for ESI application compared to placebo after lumbar discectomy in lumbar disc herniation that was not detected in previous meta-analyses. However, these meta-analyses still have many limitations.Previous studies on the effect of ESI following discectomy for herniated lumbar disc disease has been inconclusive and without long-term follow-up.Despite the evidence above, concerns exist regarding the analgesia effect and safety of ESI after discectomy. There is an urgent need of studies that efficiently control for confounding, conduct comprehensive and consecutive observation of potential risks of ESI, and investigate its clinical applicability. Considering different conclusion and limitation of previous studies, we thus further designed a RCT to assess the efficacy of ESI on postoperative pain and complications in patients undergoing unilateral lumbar microdiscectomy. We hypothesized that the ESI was associated with lower pain score, morphine consumption, and hospital stay, with no significant difference in complications for ESI application after lumbar discectomy in lumbar disc herniation when compared with placebo.22.1This prospective, blinded randomized controlled trial was conducted at our single hospital. This study was approved by the Committee at the No.2 People's Hospital of Yibin (YBH22470), and the protocol of the study was registered in Research Registry (researchregistry5683). All procedures were performed by a single surgeon and informed consent was obtained from each patient. Ninty eligible patients diagnosed at our institution with herniated lumbar disc during a period from June 2020 to July 2021 will be assessed.Eligible patients were those undergoing primary single-level unilateral lumbar discectomy. All patients involved in the study had been diagnosed with a prolapsed intervertebral lumbar disc with nerve compression demonstrated on magnetic resonance imaging, with clinically correlated clinical findings and had failed conservative treatment before enrolment. Excluded were patients with central or lateral spinal stenosis due to spondylosis or disc degeneration who needed bilateral decompression, laminectomy, or fusion, and patients with cauda equina syndrome who needed acute operative treatment.2.2The patients were randomly allocated by stratified block randomization using a computer-generated random number into the treatment group or the placebo group. The allocation was made by opening sequentially numbered opaque sealed envelopes. Concealment of allocation was maintained until the time of the study drugs or saline was injected. All other clinical personnel, participants, and outcome assessors were blinded to the intervention. After discharge, the participant's personal information was eliminated from the study number and was therefore not traceable back to the patients Fig. .2.3Group 1 was a mixture of 1 mL of 0.5% bupivacaine and 10 mg of triamcinolone acetonide in 1 mL. Group 2 was a mixture of 1 mL of 0.5% bupivacaine and 1 mL of normal saline. The trained assessor and the patient were blinded to treatment assignment for the duration of the study. Study and data monitor was not blind.In both groups, prophylactic cephalosporin 1500 mg intravenous was given 15 minutes before surgery. General anesthesia was standardized with remifentanil, propofol, rocuronium, glycopyron, and at the end of operation fentanyl 100 μg and toradol 30 mg. For treatment of postoperative nausea and vomiting dihydrobenzperidol 0.625 to 125 mg intravenous was given at induction of anesthesia and ondansetron 4 mg intravenous at the end of anesthesia. Both groups received postoperative pain treatment with acetaminophen 1 g 6 hourly and celecoxib 200 mg daily. When necessary oxycontin 20 mg twice daily, oxynorm 5 mg at demand, or morphine 50 to 100 μg/kg intravenous was given. Nutrition with protein supplements >1.3 g/kg, iron, and vitamins were given from the day before operation including the day of operation and until discharge.2.4The primary outcome measure was the amount of morphine consumption from a patient-controlled analgesia pump at 12, 24, and 48 hours after surgery. The following secondary outcomes were also assessed: postoperative pain score, back pain score, functional disability, and adverse effect.Postoperative pain intensity at rest and pain on cough were assessed at 12, 24, and 48 hours after surgery on a verbal numerical rating scale ranging from 0 (no pain) to 10 (worst pain possible). Back pain intensity was assessed before surgery and at 1 week, 1 month, and 3 months after surgery on a verbal numerical rating scale. Functional disability was assessed before surgery and at 1 and 3 months after surgery using the Oswestry Disability Index, a disease-specific questionnaire designed for use in patients with low back pain that evaluates the patient's overall level of functional disability in terms of percentage score, with higher values representing higher levels of disability. General health status was assessed before surgery and at 1 and 3 months after surgery using the Short Form SF-36 Health Survey Questionnaire, a validated general health questionnaire that document functional status, well-being, and patients’ overall evaluation of their health as a percentage score, with 100% representing the best possible function and 0% the worst possible function. Adverse effect such as sedation and nausea/vomiting was assessed during patients using a patient-controlled analgesia pump.2.5The sample size was calculated to detect a mean difference in 24-hour postoperative morphine consumption between ESI and placebo after unilateral lumbar microdiscectomy. For the previous meta-analysis, the mean and standard deviation of 24-hour postoperative morphine consumption in ESI group were 31.3 and 3.62, respectively. From this, it was determined that 50 subjects would be required to reach an α value of 0.05 and a power of 85%. It was estimated that the attrition rate due to canceled surgery or reasons of late patient ineligibility could be up to 20% and, therefore, to account for this, the final sample size selected was n = 140 (70 per group).2.6U variance analysis was used for discrete numerical variables that did not show normal distribution. Relationships between the categorical variables were determined by preparing crosstabs and using the χ2 test. P < .05 was accepted as statistically significant.The statistical analyses in this study were performed using the Statistical Package for the Social Sciences 20.0 software . Continuous variables were presented in the form of mean ± standard deviation or error. The Kolmogorov-Smirnov normality test was used to assess continuous variables. Group comparisons on the variables that showed normal distribution were performed using 1-way analysis of variance. Mann-Whitney 3,23 Recently, a shorter onset time for steroid effect has been found. The mechanism of this action is probably transmitted by cell membrane receptors decreasing release of glutamate and γ-amino butyric acid. Based on these action mechanisms, the effects of ESI administration after a lumbar discectomy have been studied extensively. However, the results have previously been inconclusive. Therefore, we further conducted a double-blinded RCT to assess the efficacy of ESI on postoperative pain and complications in patients undergoing unilateral lumbar microdiscectomy. The strength of this study was its prospective and randomized design.Steroids suppress the production of prostaglandins by inhibiting Cyclooxygenase-2 mRNA expression centrally and peripherally. Furthermore, steroids inhibit mediators of inflammatory hyperalgesia, such as tumor necrosis factor-α, interleukin-1β, and interleukin-6. These effects require protein synthesis and have a latency of onset. Another late effect of steroids is suppression of proliferation of intercellular proteoglycans and collagen, thereby reducing adhesion and formation of fibrosis.Conceptualization: Jianping Cai.Data curation: Jianping Cai.Formal analysis: Jianping Cai, Wei Jiang.Funding acquisition: Yuguang Song.Investigation: Jianping Cai, Wei Jiang.Methodology: Jianping Cai, Wei Jiang.Resources: Yuguang Song.Software: Jianping Cai, Wei Jiang.Supervision: Yuguang Song.Validation: Beiming Qiu.Visualization: Beiming Qiu.Writing – original draft: Jianping Cai.Writing – review & editing: Yuguang Song, Beiming Qiu.


Dictyostelium as a model microbe in biomedicine.This MS opens a window to the general audience on the potential of  Dictyostelium discoideum is one of eight non‐mammalian model organisms recognized by the National Institute of Health for the study of human pathology. The use of this slime mould is possible owing to similarities in cell structure, behaviour and intracellular signalling with mammalian cells. Its haploid set of chromosomes completely sequenced amenable to genetic manipulation, its unique and short life cycle with unicellular and multicellular stages, and phenotypic richness encoding many human orthologues, make Dictyostelium a representative and simple model organism to unveil cellular processes in human disease. Dictyostelium studies within the biomedical field have provided fundamental knowledge in the areas of bacterial infection, immune cell chemotaxis, autophagy/phagocytosis and mitochondrial and neurological disorders. Consequently, Dictyostelium has been used to the development of related pharmacological treatments. Herein, we review the utilization of Dictyostelium as a model organism in biomedicine. Dictyostelium discoideum is a protist that has long been regarded as a valuable and attractive tool for the study of eukaryotic cell biology because a high number of conserved functions and host‐pathogen interactions comparable to human cells  and similar to other model organisms such as D. melanogaster or C. elegans, while higher than in S. cerevisiae or S. pombe   in the United States for its utility in the study of fundamental molecular processes of human medical importance , the transcriptional changes after infection, the protein composition of LCV and also to study some of the Legionella virulence factors and cellular targets , phospholipase C (plc), calnexin (cnxA), calreticulin (crtA) and cytoskeleton‐associated proteins  strains, that are more susceptible of Legionella infection  and WAVE (scrA), small G proteins of the Rho family  and actin‐binding proteins such as coronin (corA), a well‐conserved protein in Dictyostelium  , PI3K (pikA) (inhibition of PI3K would let a higher Legionella replication) and the PI‐5‐phosphatase (Dd5P4), helping the fusion of vesicle and lysosome , myosin I (myoA and myoB), profilin (proA and proB) and Nramp1 (nramp1)  overexpression, a central cellular energy sensor, helps for a higher proliferation of L. pneumophila , myosin  and atg1 . These and other host key proteins in D. Discoideum have been unravelled  signal changes cytoskeletal dynamics  contributes to front‐rear cell polarity by controlling localization and cellular levels of activated Rac   spatial gradient    and PI3K (pikA)  and PAKa (pakA)  or amyloid beta peptide, in D. discoideum they are not and therefore, they can be mutated   and is essential in Dictyostelium differentiation , a key enzyme in myo‐inositol biosynthesis pathway, is responsible for metabolic changes resulting in elevated protein degradation, glucose breakdown and high levels of amino acids   , observed in human cells, not only related to heart arrhythmias, but also to epilepsy and delayed neurodevelopment , a group of inherited progressive degenerative brain diseases characterized clinically by a decline of mental and other capacities, epilepsy and vision loss through retinal degeneration     which encode lysosomal enzymes, Ddj1 (CLN4 orthologue) and Kctd9 (CLN14 orthologue) which are membrane proteins, Cln5 (CLN5 orthologue) a soluble lysosomal protein, Grn (CLN11 orthologue) a granulin domain‐containing protein and Cln3 (CLN3 orthologue)  and Kil2 (CLN12 orthologue) coding for transmembrane proteins that localize in different organelles  have been annotated in D. discoideum , ATG9 (atg9) and ATG16 function on phagocytosis  and Sqstm1 (sqstm1) , Sqtm1, ATG5 (atg5) or ATG1, which are involved in protein degradation, or ATG8, ATG5 and ATG1 involved in Hirano bodies‐like aggregates degradation in Alzheimer  involved in Parkinson’s disease and Chorea‐acanthocytosis , orthologue of VCP/p97 in humans, is related with cell death by autophagy in the presence of aberrant mitochondria and has been linked to IBMPFD , HSP (hereditary spastic paraplegia), and a form of ALS  , and it is related with tumour suppression and neurological disorders as a consequence of psychological stress , TAK1 (DDB_G0267514) and CaMKK2 (DDB_G0279405)  in D. discoideum, is not only related with neurological disorder but also causes developmental diseases, respiratory enzyme deficiencies and early infancy death  could be a good treatment for this pathology , Alzheimer, Huntington and Parkinson . VPA is the most highly prescribed epilepsy treatment worldwide, also used to prevent bipolar disorder and migraine, since it has been demonstrated to have neuroprotective effects in neurodegenerative conditions  and PIP₃ (protecting against its reduction)  and presenilin‐1 (PsenB). Both proteins are conserved in human, PP2A and PS1 respectively. The orthologue for the phosphatase 2A regulatory subunit is the subunit B56 of the PP2A protein, involved in many functions such as cell proliferation, signal transduction, apoptosis and related with some cancers . Other approaches include the study of naringenin and aminobisphosphonates  as the target of aminobisphosphonate bone resorption inhibitors in mammalian osteoclasts  and this information provided insight into the proteins involved in the process in order to eventually understand better how to fight these human pathogens. D. discoideum also has de ability to chemotax, like human leucocytes or tumour cells, and that is why it has been chosen as the key model organism for the study of eukaryotic chemotaxis. D. discoideum is an exceptional model organism to study a wide range of neurological disorders, many of them characterized by altered mitochondrial dynamics, structure and/or function, proven D. discoideum as a mitochondrial disease system. Thanks to D. discoideum it was possible to identify and study proteins involved in those neurological disorders, in part because D. discoideum cells do not exhibit variation in symptoms, thus simplifying the study on mitochondrial diseases. Studied proteins related to the aforementioned diseases are involved in actin cytoskeleton, endocytosis, transport, metabolism and signalling pathways including but not limited to RAS and Notch , to reduce the reliance on the use of animal tissue and whole‐animal experiments (Otto et al., tch Fig. . The anaism Fig. . A totalThe authors declare no conflict of interest.Table S1.Dictyostelium proteins involved in Legionella pneumophila infection.Click here for additional data file.Table S2.Dictyostelium proteins involved in directed cell migration.Click here for additional data file.Table S3.Dictyostelium proteins involved in neurological disorders.Click here for additional data file.Table S4.Dictyostelium proteins involved in autophagy/phagocytosis.Click here for additional data file.Table S5.Dictyostelium proteins involved in mitochondrial syndromes.Click here for additional data file.Table S6. GO Term analysis for biological processes associated to the proteins studied in Dictyostelium.Click here for additional data file.


BTk in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect. Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6  These steps can be experimentally detected as two subsequent mixing events: lipid mixing following stalk formation and content mixing following the fusion pore expansion.6 The free energy accumulated in the intermediate states defines the fusion energy barriers and determines the rate of the two mixing steps.Membrane fusion is fundamental in numerous developmental, physiological, and pathological processes, including fertilization, muscle formation, enveloped virus infection, and neural activity.7 but accelerate content mixing.8 Another conflicting factor is membrane tension (“tension” for brevity); however, unlike the well-documented role of lipid composition,13 the effect of tension is much less understood.The mechanical and physical properties of the fusing membranes and their surroundings affect the energy of the fusion intermediates. In some cases, the same property may have opposite effects at different stages, increasing the energy of one and decreasing the energy of the other. A prominent example of such a conflict is the membrane lipid composition, namely, the lipid’s intrinsic curvature. Lipids with positive intrinsic curvature, such as lysophosphatidylcholine (LPC), inhibit lipid mixing15 Tension in cellular membranes can originate from osmotic pressure difference, membrane—cytoskeleton interaction, and substrate adhesion.16 Tension varies across orders of magnitude within an individual cell, ranging from 0.005–0.015 mN/m in the inner membrane compartments, such as the endoplasmic reticulum and Golgi,17 to 0.1 mN/m in the plasma membrane.18 It also varies between cell types; for example, membrane tension can be as high as 0.45 mN/m in migrating cells20 due to membrane flow and friction with the substrate and cytoskeleton anchors. The maximal tension a typical lipid membrane can sustain before rupture is approximately 3–4 mN/m.21Tension is the free energy per unit area needed to stretch a flat membrane element, either by smoothing membrane undulations, pulling an area from a reservoir, or lipid stretching/compressing in the lateral direction.16 This effect was computationally studied24 and observed experimentally.27 In contrast, the initial steps leading to the merger of the proximal monolayers and stalk formation are inhibited by tension because stalk formation involves pulling additional lipids from the surrounding membranes, which is energetically unfavorable.16 In line with this is the observation of high tension blocking the lipid mixing step in hemagglutinin-mediated membrane fusion.28 However, despite the large variability of membrane tension in cellular membranes and the importance of membrane fusion, the effect of tension on the lipid mixing step has not been theoretically or experimentally systematically addressed before.The fusion of tense membranes is energetically favorable overall, as it relaxes membrane stretching stress. However, similarly to lipid intrinsic curvature, tension also has a dual effect on the fusion energy barriers. Diaphragm expansion and fusion pore formation are favored at high tension since both involve release of lipids from the fusion site to the surrounding reservoir, relaxing the stress.Here we combine optical tweezers, micropipette aspiration, and confocal fluorescence microscopy to manipulate and monitor the hemifusion processes of membranes under tension. We experimentally demonstrate that the lipid mixing time delay increases with tension and that the lipid mixing energy barrier agrees with the theoretically predicted change in the stalk formation energy. We further predict that the mean distance between the membranes in the pre-fusion configuration determines the relative contribution of tension to the stalk energy, with larger separation resulting in a larger contribution.τ, which is the time from the membrane contact to the initiation of lipid mixing. This time is measured from the contact time to the time fluorescence intensity increases on the bead.We introduced a new setup that combines micropipette aspiration, optically trapped membrane-coated beads, and confocal fluorescence imaging. An aspirated giant unilamellar vesicle (GUV) and an optically trapped membrane-coated bead are brought into contact to initiate the hemifusion process, as illustrated in 30To validate our method, we performed control experiments with lipids of different intrinsic curvatures, which have a well-documented effect on the lipid mixing time delay. The early fusion stages are characterized by a strong negative splaying of the lipid’s tails. Therefore, membranes with a higher fraction of lipids with positive intrinsic curvature, such as LPC, inhibit stalk formation, while lipids with negative intrinsic curvature, such as oleic acid and cholesterol, promote it.μM increased the lipid mixing time delay from 69 ± 11 s to 108 ± 17 s for membranes containing 30% cholesterol (μM resulted in no lipid mixing during the experiment (10 min). To conclude, our control experiments agree well with numerous previous studies, demonstrating the validity of our new experimental approach.lesterol . Measure2+ concentration, resulting in a very short lipid mixing time delay (see 2+). Following the validation of our method, we measured the effect of tension on the lipid mixing time delay. We compared the lipid mixing time delay under different tensions, ranging from 0.003 ± 0.002 to 0.125 ± 0.002 mN/m. Under a high tension of 0.3 mN/m, no lipid mixing events were observed during the experiment (10 min). The measurements were also performed on loose vesicles and at high Ca31 Therefore, we speculated that the prolonged lipid mixing time delay is due to the increased stalk energy. To test this hypothesis, we calculated the dependence of the stalk energy on tension using continuum elastic theory.Lipid mixing must follow the merger of the proximal monolayers of the fusing membranes and the formation of a hemifusion stalk , which is the initial lipidic connection between the membranes. Previous studies found stalk formation to be the major barrier to lipid mixing.0F represents all terms independent of the tension increase, such as the elastic energy associated with lipid monolayer deformations, dehydration energy, and the contribution of residual tension originating from membrane thermal undulations, which is typically in the range of μN/m.32 The second contribution, FT, is due to the product of membrane area change and tension and is given by A is the sum of the area withdrawn from both monolayers to form the stalk  lipid composition used here.36 As a result, the water gap equilibrium distance between the membranes is 15 nm without tension and is reduced by 6 nm at 0.12 mN/m , 1,2-dioleoyl-sn-glycero-3-phospho-L-serine , and cholesterol (Sigma) were mixed at a final lipid concentration of 0.25 mM and labeled with 0.1% Rhodamine-PE . GUVs were grown on ITO slides (Nanion Technologies) by gently spreading 30 μL of the lipid solution and evaporating the solvent by an argon stream, followed by desiccation under a mild vacuum for at least 2 h. GUVs were then grown by electroformation in 275 mL of 300 mM sucrose solution using a Vesicle Prep Pro instrument (Nanion Technologies). First, the electroformation voltage was increased stepwise to 3 V, 15 Hz) and applied at 55 °C for 2 h, followed by a slow decrease of voltage and frequency . The solvent was evaporated under an argon stream, followed by desiccation under a mild vacuum overnight. Next, the lipids were rehydrated with 1 mL of HEPES buffer . Liposomes were produced by extrusion through a 100 nm polycarbonate filter at 25 °C using a mini extruder (Avanti Polar Lipids). Polystyrene nonporous particles  of 3.15 μm diameter were suspended in Milli-Q water and washed through 3 cycles of vortexing followed by centrifugation for 5 min at 1000g. Washed beads were introduced into a 600 μL liposome dispersion and continuously mixed on a rotator overnight to form a continuous lipid bilayer of the beads.51 Next, beads were washed three times with a HEPES buffer to remove any free liposomes.DOPC, DOPS, and cholesterol were mixed at a final lipid concentration of 0.25 mM in chloroform and labeled with 0.1% Oregon Green 488 1,2-dihexadecanoyl-The experiments were performed using a C-trap confocal fluorescence optical tweezers setup (Lumicks) made of an inverted microscope based on a water-immersion objective (NA 1.2) with a condenser top lens. The optical trap is generated by a 10 W 1064 nm laser. The displacement of the optically trapped beads from the center was measured and converted into a force signal by back-focal-plane interferometry of the condenser lens with a position-sensitive detector. The samples were illuminated by a bright field 850 nm LED and imaged in transmission onto a metal-oxide semi-conductor camera (CMOS).The C-trap includes three fiber-coupled excitation lasers with 488, 561, and 638 nm wave-lengths. Scanning is performed using a fast tip/tilt piezo mirror. For confocal detection, the emitted fluorescence was descanned, separated from the excitation by a dichroic mirror, and filtered using emission filters . Photons were counted by using a fiber-coupled single photon counting module. The multimode fibers serve as pinholes, providing background rejection.PDMS walls were placed on a glass slide  and mounted onto an automated XY-stage. GUVs and coated beads were added into the chamber containing glucose buffer  and allowed to settle for 15 min before the experiment. The 488 and 561 nm lasers were used for confocal imaging to excite Oregon Green and RH-PE (respectively), with emission detected in three channels .μm diameter  connected to a pump , was integrated into our optical tweezers instrument. By controlling the aspiration pressure, membrane tension on the GUV was modified according to52γasp is the aspiration tension, ΔP is the micropipette suction pressure, Rve is the vesicle radius, and Rpip is the micropipette radius. Before each experiment, the zero-suction pressure was found by aspirating a bead into the pipette and reducing the suction pressure until the bead stopped moving. For most experiments, multiple measurements under different tensions were performed on the same GUV (2–4 measurements).A micropipette aspiration setup, including a micromanipulator (Sensapex) holding a capillary of 5 2+ was added with a concentration of 8.3 mM . To initiate the hemifusion, CaTo ensure sealing between the GUV and the micropipette, the GUV was aspirated under a high tension of 0.2 mN/m before each measurement. Then measurements were performed at the desired suction pressure/tension values.55 However, micropipette aspiration is a well-established method that allows direct control over tension.52 We validated our method by conducting bending rigidity measurements for pure DOPC membranes  and cholesterol (negative intrinsic curvature). All the hemifusion measurements were performed under a mild tension of 0.04 ± 0.01 mN/m.μM) and allowed to settle for 15 min before the measurement. LPC concentration was below the CMC; therefore it easily inserted into the proximal monolayer.56 GUVs and membrane-coated beads were prepared for cholesterol measurements with a lipid composition containing different DOPC:cholesterol:DOPS ratios .GUVs and membrane-coated beads were prepared for LPC measurements with a lipid composition of 50:30:20 (DOPC:cholesterol:DOPS). An LPC-supplemented buffer was prepared by rehydrating an LPC film (previously desiccated under a mild vacuum for 2 h) with a glucose buffer and further sonication for 1 min. The vesicles and beads were added to the experimental chamber containing an LPC-supplemented buffer . More details can be found in a previous publication39 and the code itself.The second contribution comes from the mechanical work of pulling lipids from the surrounding membranes to form the stalk against membrane tension. We consider the surrounding membranes to be much larger than the stalk and allow the lipids to freely exchange between the stalk in its vicinity and the surrounding membranes, which act as a reservoir. Such exchange is accompanied by thermodynamic work and, consequently, is related to the system’s free energy changes. This thermodynamic work per monolayer area is the monolayer tension and is defined as  elastic  and tens elastic  energiesAppendix AAppendix BMovie AMovie B


Saccharomyces bayanus is a yeast species which is not commonly used for the production of alcoholic beverages, but it is able to produce volatiles that add desirable aroma. Since there is little information regarding the application of that microorganism for the production of apple brandies and how it affects volatile profile of finished products, we decided to address that issue. The aim of the study was to determine the impact of S. bayanus on the profile of volatile compounds and sensory properties of apple spirits obtained from three apple cultivars  in comparison to spirits obtained from fermentation carried out spontaneously or with Saccharomyces cerevisiae. Obtained brandies were analysed using gas chromatography–flame ionization detector (GC–FID), solid phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) and sensorially. In our study, brandies produced from musts fermented by S. bayanus demonstrated the highest concentration of ethyl esters and increased concentrations of isoamyl acetate, 2-phenylethyl acetate, ethyl palmitate and hexanol. Moreover, our results support the hypothesis that non-Saccharomyces yeasts which are present during spontaneous fermentation and demonstrate higher β-glucosidase activities enhance aroma of alcoholic beverages through releasing aroma compounds from glycosidic forms, e.g., α-phellandrene, (E)-β-fanesene, -α-farnesene, α-farnesene, and farnesol. Considering results obtained in sensory analysis, we proved that S. bayanus is suitable for the production of apple brandies, improving their flavour. Brandies obtained from musts fermented by S. bayanus obtained the highest average range for “overall note” parameter in sensory analysis.Qualitative and quantitative profiles of volatiles in alcoholic beverages depend mainly on the quality of raw materials, yeasts used for fermentation, and processing technique.  Fruit distillates are valued worldwide due to their unique flavour. Fruit distillates  are produced by alcoholic fermentation and distillation of fleshy fruit or must of those fruits, berry or vegetable, with or without stones, and ethanol content obtained after distillation is less than 86% . SubstanThe origin of volatile compounds in apple alcoholic beverages is very diverse. Parts of the volatiles are already present in fruits. Some of the components, which contribute to the flavour of alcoholic beverages, originate from the raw material but are released/transformed throughout fermentation by microorganisms . NumerouSaccharomyces species . In the SPME–GC–MS method, 2 mL of saturated saline with an internal standard solution  and 0.05 mL of spirit were added into a 10 mL vial. The SPME device  coated with polydimethylsiloxane (100 μm) fiber was first conditioned by inserting it into the GC injector port at 250 °C for 1 h. For sampling, the fiber was inserted into the headspace under stirring (300 rpm) for 30 min at 60 °C. Subsequently, the SPME device was introduced in the injector port of the Agilent Technologies 7890B chromatograph system (Agilent Technologies) equipped with LECO Pegasus HT, High Throughput TOFMS (time-of-flight mass spectrometry), and was kept in the inlet for 3 min. The SPME process was automated using the GERSTEL MultiPurpose Sampler (MPS).m/z = 40 to m/z = 400.Separation was conducted with a Rtx-1ms capillary column . The detector temperature was 250 °C, and the column was heated using the following program: 40 °C for three minutes at an increment of 8 °C/min to 230 °C, then maintaining a constant temperature for 9 min. The carrier gas was helium at a 1.0 mL/min constant flow. EIMS electron energy 70 eV; ion source temperature and connection parts: 250 °C. Analyte transfer was performed in splitless mode; the MSD (mass spectrometer detector) was set to scan mode from Volatiles were identified using mass spectral libraries and linear retention indices, calculated from a series of n-alkanes from C6 to C30. The amount of components was determined semi-quantitatively by measuring the relative peak area of each identified compound, according to the NIST database, in relation to that of the internal standard . This semi-quantification approach was already performed in many previous scientific studies ,50,51.Sensory analysis of apple brandies was based on eight aroma descriptors  rated on a five-point hedonistic scale. For overall note, 0 meant least acceptable and 5 most acceptable. For aroma descriptors, 0 was used for the least intensity of aroma and 5 was used for the highest intensity of a particular aroma. We applied quantitative descriptive analysis (QDA). The overall note in our paper defines the general acceptance of tested samples. Panellists were selected among scientific staff working in the Faculty of Food Technology and Human Nutrition who previously graduated from that faculty and obtained extensive course of sensory analysis as a part of their curriculum. Sensory assessment was determined using a set of standards provided to panellists prior to analysis ,52. FirsAll experiments were conducted in at least five physical replicates and each analysis was carried out for each replicate. Results were presented as arithmetic means ± standard deviation. Statistical analysis was performed in the R 3.6.1  program. We applied the Shapiro–Wilk test to assess the normality of data distribution. Then, we carried out analysis of multivariate analysis of variance (MANOVA) with the post hoc Tukey test.S. bayanus is an appropriate strain of yeast for brandies production. In musts fermented spontaneously, the adaptation period of microorganisms to fermentation conditions was the longest and fermentation was slower in comparison to other tested variants. Samples fermented by S. cerevisiae demonstrated the highest fermentation efficiency and ethanol content. In our study, the best acetate ester producer was S. bayanus. The exception was ethyl acetate, which was present in the highest concentration in samples fermented spontaneously. Highest concentrations of acetaldehyde, methanol, and a majority of fusel alcohols were in samples obtained by spontaneous fermentation. Terpenes were found in the highest concentrations in spirits obtained from musts fermented spontaneously. These results support other reports that linked non-Saccharomyces yeasts with higher β-glucosidase activities, enhancing the aromas of alcoholic beverages through releasing aroma compounds from glycosidic forms. Apple brandies obtained from the Topaz cultivar fermented by S. cerevisiae demonstrated the most diverse profile of volatile compounds and the best aroma, described as floral, mildly sweet, grassy, fruity, alcoholic, and intensively citrus. Moreover, their oenological parameters that are the most important in the production of alcoholic beverages were the most favourable. Sensory results justify application of S. bayanus for the production of apple brandies because they improve the quality of alcoholic beverages. Brandies obtained from musts fermented by S. bayanus received the highest average score for the “overall note” parameter. In overall, apple brandies obtained after spontaneous fermentation obtained lower results in sensory analysis. It also must be considered that in many cases, products, including beverages, obtained in this type of processes might pose potential risks to human health due to the presence of some toxic substances  or some pathogens  [Our research confirmed the hypothesis that various types of fermentation significantly influence chemical composition of fermented musts, as well as the volatile profile and sensory characteristics of obtained spirits, and that O157:H7) ,54.However, further studies should be carried out to evaluate if the obtained profiles of volatile compounds of apple brandies might be associated with the presence of particular microorganisms rather than the applied apple cultivar.


B. vulgaris L.) is a moderately salt tolerant glycophyte, but little is known about the different mechanisms of sugar beet response to salt and alkaline stresses. The aim of this study was to investigate the influence of neutral salt  and alkaline salt (Na2CO3) treatment on physiological and transcriptome changes in sugar beet. We found that a low level of neutral salt  or alkaline salt  significantly enhanced total biomass, leaf area and photosynthesis indictors in sugar beet. Under a high concentration of alkaline salt , the growth of plants was not significantly affected compared with the control. But a high level of neutral salt  significantly inhibited plant growth and photosynthesis. Furthermore, sugar beet tends to synthesize higher levels of soluble sugar and reducing sugar to cope with high neutral salt stress, and more drastic changes in indole acetic acid (IAA) and abscisic acid (ABA) contents were detected. We used next-generation RNA-Seq technique to analyze transcriptional changes under neutral salt and alkaline salt treatment in sugar beet. Overall, 4,773 and 2,251 differentially expressed genes (DEGs) were identified in leaves and roots, respectively. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that genes involving cutin, suberine and wax biosynthesis, sesquiterpenoid and triterpenoid biosynthesis and flavonoid biosynthesis had simultaneously changed expression under low neutral salt or alkaline salt, so these genes may be related to stimulating sugar beet growth in both low salt treatments. Genes enriched in monoterpenoid biosynthesis, amino acids metabolism and starch and sucrose metabolism were specifically regulated to respond to the high alkaline salt. Meanwhile, compared with high alkaline salt, high neutral salt induced the expression change of genes involved in DNA replication, and decreased the expression of genes participating in cutin, suberine and wax biosynthesis, and linoleic acid metabolism. These results indicate the presence of different mechanisms responsible for sugar beet responses to neutral salt and alkaline salt stresses.The salinization and alkalization of soil are widespread environmental problems. Sugar beet ( Soil salinization and alkalization frequently occur at the same time in nature  and associated with high pH  scavenging system and maintaining ion homeostasis . Many geBeta vulgaris L.) is an important crop, which is primarily cultivated for extracting sugars from its tap root , and the detail of the soil was listed in 2SO4, at 1:1 Na+ molar ratio) and alkaline salt treatments (AS) (Na2CO3). The neutral salt or alkaline salt treatments contained two concentrations of each respective salt , resulting in a total of 4 kinds of treatment  with varying salinity and pH . Total root length was determined by a root measuring system based on an optical scanner (WinRHIZO). Using a LI-6400 portable photosynthesis system , net photosynthetic rate, stomatal conductance and intercellular carbon dioxide concentration were measured on harvest day . Chlorophyll content was measured using the method described by –1 for 8 min at 4°C. The resulting supernatant was treated with C-18 solid phase extraction column, and the analyses were eluted from the column by 2.5 mL 100% (v/v) methanol from the column. The extract was vacuum evaporated to remove methanol, and the evaporated residue was dissolved with 2ml PBS buffer containing Tween 20 (0.1% [v/v]) and 1 g L–1 gelatin. ELISA measurement was performed on a 96-well microtitration plate provided by the manufacturer. Each well had 50 μL of extracts or standards added, and 50 μL of 5 μg mL–1 antibodies against GA, IAA, and ABA, respectively. The plate was incubated for 1 h at 37°C, and then washed four times with washing buffer (PBS buffer containing 0.1% Tween 20). 100 μL of 1.25 μg mL–1 IgG-horseradish peroxidase substrate was added to each well and the reaction lasted 30 min at 30°C. The microtitration plate was washed four times as above, and then 100 μL color-appearing solution containing 1 mg mL–1O-phenylenediamine and 0.008% (v/v) H2O2 was added to each well. The reaction was stopped by adding 50 μL of 2M H2SO4. The OD values at 490 nm of each sample were determined by ELISA Reader.The IAA, ABA, and GA contents were measured by enzyme-linked immunosorbent (ELISA) kits provided by China Agricultural University . Fresh lLeaf tissue  was used to extract soluble sugar in 7 mL of 80% ethanol and repeated three times at 80°C for 1.5 h. Ethanol was evaporated in the boiled water bath. Then 5 mL of 0.15% anthrone solution was added. The reaction mixture was incubated at 95°C for 20 min and left at room temperature for 20 min. The absorbance was measured at 620 nm. The content of soluble sugar was estimated using a standard curve. To assess the reducing sugars, the DNSA reagent  was mixed with ethanol extract (1 mL). The reaction mixture was heated for 12 min, and then 2 mL of distilled water was added .–1 for 15 min. 2 mL of supernatant was then mixed with 2 mL acid ninhydrin and 2 mL glacial acetic acid, and the mixture was incubated for 30 min at 95°C. 4 mL of toluene was next added to the reaction mixture with full shaking, and the proline content was determined by measuring absorbance of the toluene at 520 nm. The free amino acid contents were estimated by detecting the absorbance at 570 nm. The amount of free amino acid was determined using the amino acid standard curve . A total of thirty RNA samples were used to construct the transcriptome sequence library and each library was prepared using 5 μg of total RNA. After quantifying by TBS380, the thirty paired-end libraries were sequenced using Illumina HiSeq PE 2 bp × 151 bp high-throughput sequencing platform.Total RNA was extracted from root and leaves using TRIzol Reagent  according to the manufacturer’s instructions, and RNA was digested with DNaseI  at 37°C for 30 min. The quantity and quality of total RNA were determined by NanoDrop ND2000 spectrophotometer  and 2100 Bioanalyser (Agilent), and the poly (A) mRNA was enriched through the oligo (dT) magnetic bead method. The cDNA library for transcriptome sequencing was constructed following TruSeq1. The expression of each gene was calculated by FRKM (Fragments Per Kilobase of exon model per Million mapped reads). The Cuffdiff2 software was used to identify genes that were differentially expressed between two samples. The false discovery rate (FDR) was used to determine the threshold of the p-value. Genes with FDR ≤ 0.05, | log2(fold change)| ≥ 1 and FRKM ≥ 1 were considered as significant differentially expressed genes (DEGs).Raw data generated by the sequencing platform was subjected to quality control (QC) to remove low-quality sequences, which include reads with adapter contamination, reads <70 bp after removing adapter sequences, reads containing more than 8% unknown bases, and reads in which >50% of the bases have a quality score <5. The clean reads were then mapped to the reference sequence (RefBeet-1.2.2) using Hisat23 and KOBAS4  functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted by Goatoolsd KOBAS4 . DEGs we18S rRNA gene as an internal control gene  to validate the transcriptome analysis using qRT-PCR. Total RNA from the roots and leaves of sugar beet was extracted with TRIzol reagent  and cDNA was synthesized using a reverse transcription kit from TOYOBO (Japan) according to the manufacturer’s instructions. The qRT-PCR analysis was performed using Bio-Rad Quantitative PCR System  and the SYBR Green Real-time PCR Master Mix Kit . PCR amplification was performed under the following conditions: 20 s at 95°C, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and then 72°C for 20 s. Three biological replicates were carried out in each reaction, and normalization was done using rol gene . All priThe data obtained were expressed as means and standard errors, and all of the experiments were repeated three times. Analysis of variance (ANOVA) between the physiological parameters of plants in the control and salt treatments were performed using SPSS 13.0 . The qRT-PCR data of the 6 genes were subjected to ANOVA analyses.Sugar beet exposed to four kinds of salt treatment for 25 days showed different phenotypes and growth status . A low l2 concentration and stomatal conductance increased significantly in both NS25 and AS25 . Thus, neutral salt causes more drastic changes in ABA level.As the response of plants to salt stress is closely influenced by plant hormone signaling, the influence of neutral and salt stress on the levels of GA, ABA, and IAA were examined in sugar beet . Both loBeta Vulgaris referenced genome, and between 87.96 and 92.63% of the clean reads were mapped to the reference database in each of the thirty libraries  ≥ 1 or log2 FC ≤ -1 and FPKM ≥ 1) between the three salt-treated and control samples. This analysis revealed variations between different salt treatments  during each salt treatment, we compared the expression level of each gene between salt treatment and control samples. The expression of each gene was calculated by fragments per kilobase of exon model per million fragments mapped (FPKM) based on the gene length and its mapped fragments count. All annotated genes that were used to further identify DEGs in sugar beet response to neutral and alkaline salt treatment are listed in eatments . Compareeatments . Moreoveeatments . DEGs ateatments . Thus, ieatments . These DTo investigate possible biological functions of DEGs that determine the response of sugar beet under different salt treatments, GO enrichment analysis were conducted and DEGs were assigned to GO terms in three ontologies involved in cellular components, biological processes and molecular function , 9. BaseS in different KEGG pathways  was down-regulated in the roots of NS25 and AS25  can enhance the growth of sugar beet seedlings, the enrichment pathways of DEGreatment . Severaland AS25 . Furtherand AS25 . For exaand AS25 . TherefoS in AS100 may be involved in the alkaline salt tolerance phenotype of sugar beet. In this study, the pathway of monoterpenoid biosynthesis was specifically enriched in leaves at AS100 treatment, meanwhile, amino acids metabolism and starch and sucrose metabolism were specifically found in the roots at AS100 treatment compared with NS100  obviously inhibited the growth status of sugar beet. Therefore, several genes may be only differentially expressed in response to a high level of neutral salt environment. It is noteworthy that pathways involved in DNA replication were only enriched significantly in leaves under NS100 condition compared with AS100. Moreover, pathways related to cutin, suberine and wax biosynthesis, and linoleic acid metabolism were only identified in roots at NS100 . We specS were selected for qRT-PCR assays. The six genes were involved in the metabolic pathways mentioned obviously , which grows naturally along the Atlantic coasts of Western Europe  in leaves of sugar beet, respectively. Thus we speculated that the accumulation of primary alcohol in cuticular wax was involved in growth stimulation under a low level of salt treatment.Several genes involved in cutin, suberine and wax biosynthesis, sesquiterpenoid and triterpenoid biosynthesis were simultaneously up-regulated in leaves treated with low level neutral and alkaline salt. The cuticle is a continuous hydrophobic layer that coats most aerial surfaces of terrestrial plants . The maiin wheat . Moreovesqualene monooxygenase (LOC104908747) and b-amyrin synthase (LOC104901642) were induced to express in the leaves of sugar beet under low concentration of neutral salt and alkaline salt. Thus, the biosynthesis of triterpenoids and oleanolic acid may be related to promoting growth.Triterpenoids, containing carbon skeleton based on six isoprene units, constitute one of the largest groups of secondary metabolites in plants . Triterpchalcone synthase (LOC104902217) and flavonol synthase (LOC104883093) were down-regulated in root under a low level of alkaline salt treatment, and chalcone synthase  and cytochrome P450 (LOC104886776) decreased the expression in root under a low level of neutral salt treatment  plants over-expressing a linalool synthase gene led to the accumulation of linalool. Moreover, transgenic plants exhibited strong resistance to citrus canker and up-regulation of defense-related genes  under high level of alkaline salt stress in sugar beet leaf. The induced expression of linalool synthase may cause the high level of linalool and lead sugar beet to adapt to alkaline salt stress. In addition, neomenthol dehydrogenase (LOC104888569), an important enzyme in the metabolism of monoterpenes, is also specifically induced to express under alkaline salt stress in leaf. Neomenthol dehydrogenase participates in the conversion L-menthone to neomenthol  and salt stress tolerance. Salt stress significantly increased PME31 expression, and knock-down mutants in PME31 adapted hypersensitive phenotypes to salt stress   genes were dramatically increased in NS100. MCM protein functions as DNA replication licensing factor which ensures that DNA in the genome is replicated only once per cell division cycle. Recently, the transcript of pea MCM6 was reported to be up-regulated in response to salt and cold stress, and over-expression of MCM6 enhanced salinity stress tolerance without affecting yield , only had specially increasing expression in the leaves of sugar beet in NS100. This report suggested that the DNA replication pathway was specifically involved in sugar beet in response to high neutral salt stress, and provided a new pathway for manipulating neutral salt stress tolerance.DNA replication is the biological process by which can exact copy of a DNA molecule is created and genetic information is faithfully transmitted in all living organisms. It is evident that salinity stress affects the cellular molecules involved in DNA replication including helicases, DNA polymerase and DNA replication licensing factor . In our ng yield . In addiolerance . Similarlinoleate 9S-lipoxygenase 5  was only down regulated in the roots of NS100, which may lead to decreasing the level of oxylipins and tolerance of sugar beet to high neutral salt stress. Furthermore, different from low concentration of neutral salt and alkaline salt, high concentration of neutral salt inhibited the expression of genes related to cutin, suberine and wax biosynthesis, such as alcohol-forming fatty acyl-CoA reductase  and omega-hydroxypalmitate O-feruloyl transferase (LOC104893839). These results indicate that the effect of neutral salt stress on epidermal wax synthesis may lead to growth inhibition in sugar beet.Linoleic acid is known as an important polyunsaturated fatty acid, required for normal plant growth. Lipoxygenase catalyzes the oxygenation of linoleic acid (18:2), which is a key reaction in biosynthesis of plant oxylipins . It is kIn this study, we found a low level of neutral or alkaline salts can enhance photosynthesis and stimulate the growth of sugar beet. Moreover, it indicated that high neutral salt and alkaline salt stresses are different, and the inhibitory effects of high level of neutral salt on sugar beet growth and photosynthesis were greater than those of alkaline salt. On the other hand, the high level of neutral salt induced the hormone content as well as some drastic change in osmotic adjustment substance content. Besides, the pathways of cutin, suberine and wax biosynthesis, sesquiterpenoid, and triterpenoid biosynthesis and flavonoid biosynthesis were involved in low salt treatment enhancing sugar beet growth. Our findings suggest the presence of different mechanisms involved in plant responses to neutral salt and alkaline salt stresses. The pathways of monoterpenoid biosynthesis, amino acids metabolism and starch and sucrose metabolism were specifically enriched in sugar beet at high alkaline salt and related to alkaline salt tolerance of sugar beet. However, high level of neutral salt only significantly affected the expression of genes participating in DNA replication, cutin, suberine and wax biosynthesis, and linoleic acid metabolism compared with high alkaline salt. These findings will enhance our understanding of sugar beet response mechanisms under neutral salt and alkaline salt stresses.PRJNA634158.The datasets analyzed for this study can be found in the National Center for Biotechnology Information (NCBI) and can be accessed in the Short Read Archive (SRA) under the accession number GG, YW, RL, ZL, LY, and CL conducted most of the physiological and biochemical analyses. YW, GG, and RL carried out the transcriptome analyses. YW, GG, and PS revised and contributed to writing the manuscript. All authors read and approved the final manuscript.The 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.


Cuon alpinus), restricted today largely to South and Southeast Asia, was widespread throughout Eurasia and even reached North America during the Pleistocene. Like many other species, it suffered from a huge range loss towards the end of the Pleistocene and went extinct in most of its former distribution. The fossil record of the dhole is scattered and the identification of fossils can be complicated by an overlap in size and a high morphological similarity between dholes and other canid species. We generated almost complete mitochondrial genomes for six putative dhole fossils from Europe. By using three lines of evidence, i.e., the number of reads mapping to various canid mitochondrial genomes, the evaluation and quantification of the mapping evenness along the reference genomes and phylogenetic analysis, we were able to identify two out of six samples as dhole, whereas four samples represent wolf fossils. This highlights the contribution genetic data can make when trying to identify the species affiliation of fossil specimens. The ancient dhole sequences are highly divergent when compared to modern dhole sequences, but the scarcity of dhole data for comparison impedes a more extensive analysis. The Asiatic wild dog ( Many species’ common names are derived from their modern distribution, although this may not reflect the full range of a species’ former distribution, as in the case of the Tasmanian devil . In factPhylogenetic analyses of mitochondrial genomes, as well as of extensive nuclear data, place the dhole as a sister lineage to a clade containing wolves, dogs, coyotes and jackals e.g., ,16,17), ,17, 16,1The identification of ancient dhole remains is complicated, as fossil remains from dholes are both rare and difficult to distinguish from those of other canids. Although dholes are generally smaller and more slender than co-distributed canids , there is some overlap in dimensions ,10,23. FIn this study, we analyse ancient DNA sequences from several (putative) dhole specimens, which have been identified based on morphological traits. We are able to identify the remains at species level with high confidence. To our knowledge, these data represent the first genetic data from Pleistocene dholes, allowing for an initial insight into the genetic structure and diversity that was lost when the dhole disappeared from much of its range at the end of the Pleistocene.Cuon alpinus or Cuon alpinus europaeus) based on morphological characteristics of the bones analysed or bones that had been found in close proximity and were assumed to originate from the same animal [Our dataset encompasses six putative ancient dhole samples, and one modern sample. The ancient samples originate from the Czech Republic, Bulgaria, and Romania (one site each). Ancient samples have been identified as dhole . DetailDNA for the modern sample was extracted using a QIAamp DNA blood mini kit following the manufacturer’s instructions. First, this high-molecular-weight DNA extract  was used as input for long range polymerase chain reactions (LR PCRs) to amplify the complete mitochondrial genome. For this purpose, three primer pairs  were desDNA of six ancient samples was extracted using 15–54 mg of bone powder following a protocol specifically adapted for the recovery of short DNA fragments from ancient samples . Negativ2 (25 mM)) and finally with TET buffer. The final amplification reaction was carried out in a total reaction volume of 60 µL using 30 µL of the purified capture product as template. The following PCR conditions were applied: initial denaturation at 95 °C for 2 min followed by the predetermined number of cycles (via qPCR) at 95 °C for 30 s, 60 °C for 45 s, 72 °C for 45 s and, after cycling, a final extension at 72 °C for 3 min. Amplified products were purified using the Qiagen MinElute kit following the manufacturer’s instructions using two times 10 µL of EB buffer for the final elution.SsDNA libraries of ancient samples were enriched for mitochondrial reads by performing two consecutive rounds of in-solution capture, following , with slAll libraries were sequenced on an Illumina Nextseq 500 machine, using custom primers to sequence read 1 and the index of read 2 ,35. AnciIllumina-specific adapter sequences were trimmed from all raw reads using cutadapt  with dePre-processed reads were mapped, parsed, filtered for reads with low mapping quality (MAPQ < 30) and duplicates removed using BWA aln v.0.7.8 and samtools v0.1.19 with default settings ,40. Ten https://github.com/lh3/seqtk). Data were treated as described above, except for the duplicate removal, for which Picard’s MarkDuplicates tool  was used.Published data for a modern gray wolf were downloaded from the Sequence Read Archive . AdapteAs a means to quantify how uniform reads were distributed across the reference genome, the mapping evenness was calculated . https://www.geneious.com/). Consensus sequences were created based on the read alignment to the canid reference to which the highest number of reads could be mapped. A minimum read depth of three and a 50% consensus threshold were applied.Read alignments were visualised, curated and consensus sequences were created using Geneious v.10.2.3 (Canis simensis) due to its high amount of missing data; [https://www.geneious.com/). The MSA was modified as follows: removal of the control region based on the annotation of published genomes (Geneious) and removal of all columns containing gaps or uncalled positions (Ns) using MEGA . The ML tree was reconstructed using a TIM2 + I + G model , as it was identified as the best model using the Bayesian information criterion (BIC) by jModeltest  with default settings, applying a support threshold of 50%. Additionally, a Bayesian phylogeny was calculated using the MrBayes v3.2.6 [https://www.geneious.com/) with the following settings: HKY model with four γ categories  sequence served as outgroup.A multiple sequence alignment (MSA) was created by aligning the consensus sequences of the modern and ancient dhole samples to 41 canid sequences . Thu. ThuCani  pluv2.1.7, ,59), usi, usiCanis v3.2.6  plugin oodeltest ,59 usingTo evaluate genetic diversity within species, pairwise differences were calculated for species for which four or more sequences were available (similar to ). CalculSequences from samples Y-38 and Y-39 were compared to a 246 bp long dhole mitochondrial sequence fragment  by creatSequencing after in-solution capture yielded between 1 and 5.5 million raw reads per sample. After pre-processing, between 997,439 and 4,199,293 reads were available for mapping. For the modern sample, about six million raw read pairs were generated, of which 5,242,575 could be merged and were used for mapping .BLAST results of pre-processed reads (prior to mapping) revealed a pattern typical for ancient samples . All ancFor the ancient samples, between 741 and 26,169 reads (median: 3894) could be mapped to various canid mitochondrial genomes . For allTo further describe the overall quality of the read alignments . Fou. FouCaniTo further investigate the relationship between samples Y-38 and Y-39 and modern dholes, we reconstructed a median-joining network using publicly available sequences of a 246 bp mitochondrial fragment . After tTo estimate the genetic diversity within and compare it between species, pairwise distances were calculated . Based oHomotherium fossil [Equus ovidovi specimens outside the formerly known spatial and temporal range [The correct species identification of a fossil specimen can be of exceptional importance. Several studies have shown that one single sample can make a huge difference, e.g., the molecular confirmation of the first Late Pleistocene Eurasian m fossil , the idem fossil  or the ial range . These fFossil specimens can be identified by morphological characters and measurements, by using molecular data or by a combination of both approaches (summarised and compared in ). IdentiIn all ancient samples, DNA from exogenous and contaminant sources was present , which iCanis species  the amount of reads that could be mapped to different canid reference mitogenomes , (2) theThe evenness of the read alignment is a second possibility to evaluate species identification. If sequence reads are mapped to an intraspecific mitogenome or very closely related species, even mapping of reads across the entire reference genome is expected, resulting in little variability in the level of coverage. Highly variable regions  or repetitive elements would lead to lower or higher coverage, but these represent only a small portion of the mitochondrial genome. If the reference used for mapping is more distantly related to the sample, fewer reads will be mapped and only to conserved sites at which sequence similarity is high, leading to uneven coverage compare . TherefoA potential drawback of the evenness-of-coverage metric exists for cases when cross-species hybridisation enrichment was applied in the laboratory to enrich for a particular genetic region . It has been shown that cross-species capture—i.e., when baits from a related species are used for enrichment—can introduce strong biases for regions with the highest sequence similarity between bait and target ,75. We uCanis . Conseqs .,8,10.7,8Cranial elements are considered easier to identify, as the cranium, mandible and dental composition often contain characteristic features unique to a particular species . Due . Due CanTo our knowledge, the data gained from the two samples identified as dholes represent the first ancient DNA sequences published for this species. However, due to a lack of modern data available for comparison, only limited population genetic insights or phylogeographic comparisons are possible at present. To date, genetic data from dholes have been limited to a small number of modern and historical samples (e.g., ,19,20,22Phylogenetic analysis of complete mitochondrial genomes from the two ancient, one newly sequenced, and three published modern dholes reveals considerable differences between ancient and modern dhole sequences  and placThe approximate age of the two verified dhole specimen (35 to 45 ka, In this study, we reconstructed mitochondrial genomes of six fossil specimens initially identified as dhole and found that more than half had to be reassigned as gray wolves. This underlines the importance of genetic analysis for the identification of fossil specimens, especially if findings originate from postcranial elements or are highly fragmented and species affiliation is difficult to determine. Phylogenetic analyses point towards a considerable divergence between ancient and modern dhole sequences and a loss of genetic diversity in modern dholes. However, the database of modern dhole sequences available for comparative analysis is scarce. The generation of further genetic data, both mitochondrial and nuclear, on the population level, is mandatory to evaluate the findings of the current study and extend our knowledge of the phylogenetic history and genetic diversity of the dhole.