Chronic respiratory diseases and use of antiasthmatic medication during pregnancy may both play a role in the etiology of congenital anorectal malformations (ARM). However, it is unclear, whether the medication use or the underlying condition would be responsible. Therefore, our aim was to unravel the role of maternal chronic respiratory diseases from that of antiasthmatic medication in the etiology of ARM.We obtained 412 ARM patients and 2,137 population‐based controls from the Dutch AGORA data‐ and biobank. We used maternal questionnaires and follow‐up telephone interviews to obtain information on chronic respiratory diseases, antiasthmatic medication use, and potential confounders. Multivariable logistic regression analyses were performed to estimate odds ratios (ORs) with 95% confidence intervals (95% CI).We observed higher risk estimates among women with chronic respiratory diseases with and without medication use (1.4 [0.8–2.7] and 2.0 [0.8–5.0]), both in comparison to women without a chronic respiratory disease and without medication use. Furthermore, increased ORs of ARM were found for women using rescue medication (2.4 [0.8–7.3]) or a combination of maintenance and rescue medication (2.5 [0.9–6.7]). In addition, increased risk estimates were observed for women having nonallergic triggers (2.5 [1.0–6.3]) or experiencing exacerbations during the periconceptional period (3.5 [1.4–8.6]).Although the 95% CIs of most associations include the null value, the risk estimates all point towards an association between uncontrolled chronic respiratory disease, instead of antiasthmatic medication use, with ARM in offspring. Further in‐depth studies towards mechanisms of this newly identified risk factor are warranted. The prevalence ranges from 2 to 7 patients per 10,000 pregnancies worldwide,  data‐ and biobank of the Radboud university medical center (Radboudumc) in the Netherlands. AGORA includes clinical and questionnaire data, as well as DNA samples from children with birth defects or childhood cancer, population‐based control children, and their parents , type of trigger , and exacerbations in the periconceptional period . Within this study, the periconceptional period was defined as the 3 months before conception until the 10th week of pregnancy, as anorectal development is complete by that time , low birthweight , and preterm delivery (<37 weeks). Maternal characteristics were age at delivery, maternal education , twin pregnancy (vs. singleton pregnancy), parity (primiparity vs. multiparity), previous miscarriage, ART, recommended folic acid use (use of folic acid supplements or multivitamins containing folic acid from 4 weeks before until 10 weeks after conception), periconceptional smoking (any smoking), preexisting diabetes mellitus, and pre‐pregnancy body mass index (BMI). BMI was calculated using the reported weight (in kilograms) and height (in meters) before conception and was categorized in four groups .2.3We used univariable and multivariable logistic regression analyses to estimate crude and adjusted odds ratios (ORs) with 95% confidence intervals (95% CIs) for independent associations between ARM in offspring and maternal chronic respiratory diseases and antiasthmatic medication use. To unravel the roles of chronic respiratory diseases and antiasthmatic medication use, we subdivided the women into four categories: (a) having a chronic respiratory disease without antiasthmatic medication use, (b) antiasthmatic medication use without having a chronic respiratory disease, (c) having a chronic respiratory disease with antiasthmatic medication use, and (d) having no chronic respiratory disease nor antiasthmatic medication use (reference group). Antiasthmatic medication use was subdivided into maintenance use, rescue use, and a combination of maintenance and rescue use. The type of medication was not taken into account, only the self‐reported frequency of use. We also studied the associations of ARM with exacerbations in the periconceptional period and with the type of trigger. We considered year of birth, maternal age at delivery, maternal education, primiparity, previous miscarriage, ART, recommended use of folic acid supplements, periconceptional smoking, and pre‐pregnancy BMI as potential confounders in the analyses, as these factors were previously identified as risk factors for ARM or were found to be associated with ARM in this study.Potential confounders that changed the crude OR in bivariable analyses were included in the multivariable models, from which they were excluded when the OR did not change more than 10% from the fully adjusted OR upon removal. Only pre‐pregnancy BMI was found to be a true confounder in the analyses. Statistical analyses were performed using SPSS 22.0 for Windows .3In total, we included 412 ARM patients and 2,137 population‐based controls with available maternal questionnaire data. The majority of ARM patients had a perineal fistula (54%), followed by rectourethral fistulas (14%), vestibular fistulas (13%), ARM without fistula (4%), anal stenosis (3%), cloaca (2%), and rare/other types of ARM (9%). In total, 178 patients (43%) had additional major birth defects, mainly those that are also part of the VACTERL association.No substantial differences were observed between patients and controls regarding gender, descent, twin pregnancies, parity, recommended folic acid supplement use, and age of the child when completing the questionnaire by the parents Table . HoweverAltogether, 102 women were eligible for the telephone interview . We were able to reach 77 (75%) of these women and observed 88% agreement between the questionnaire and telephone interview for the presence of chronic respiratory diseases and 96% agreement for the use of antiasthmatic medication. As mentioned before, we combined the information from the questionnaire with the telephone interview.The risk of ARM seemed to be increased for offspring of women with chronic respiratory diseases (adjusted OR = 1.6 0.9–2.6]) (Table .6)  and adjusted OR = 2.0 [0.8–5.0]), both in comparison to the women without a chronic respiratory disease and without medication use (Table .7) and aFurthermore, we studied the chronic respiratory diseases and antiasthmatic medication use more in detail. Women using their medication on a rescue basis , or women who reported a combination of maintenance and rescue use showed increased risks of ARM in offspring (adjusted OR = 2.4 0.8–7.3] and adjusted OR = 2.5 [0.9–6.7]), compared to women without chronic respiratory diseases and no medication use . In contrast, no association with ARM in offspring was observed for women with allergic triggers (adjusted OR = 1.0 [0.3–2.8]). Women who experienced exacerbations during the periconceptional period were three times more likely to have a child with ARM in comparison with the reference group (adjusted OR = 3.2 [1.3–8.0]), whereas no increased risk was observed among women with chronic respiratory diseases without exacerbations (adjusted OR = 1.2 [0.5–2.8]). To study the effect of exacerbations independently from rescue medication use, we additionally corrected for the frequency of medication use. This led to ORs for women with and without exacerbations in the same order of magnitude (OR = 2.7 [0.8–9.5] and OR = 1.0 [0.3–3.5]).4In this study, we tried to disentangle the role of chronic respiratory diseases and antiasthmatic medication use during the periconceptional period in the etiology of ARM. Increased risk estimates of ARM in offspring were observed for women with chronic respiratory diseases, especially women without periconceptional antiasthmatic medication use, in comparison with women who neither have a chronic respiratory disease nor use antiasthmatic medication. The risk estimate of ARM in offspring was not increased in women with chronic respiratory diseases who used antiasthmatic medication, compared to women with chronic respiratory diseases without medication use. Furthermore, increased risk estimates of ARM in offspring were observed in particular among women using their medication on a rescue basis, women having nonallergic triggers, and women having exacerbations during the periconceptional period. Although all but one of the 95% CIs contained the null value, these associations all suggest an association between ARM in offspring and an uncontrolled chronic respiratory disease state.A strength of this study was the large number of representative and well‐characterized ARM patients and comparable population‐based controls. After applying several exclusion criteria, we observed additional birth defects in 43% of the patients, which corresponds with previous studies reporting 40–70% of ARM patients having additional birth defects  than in controls . These numbers are lower than the general rate of these conditions in the Netherlands in 2016, approximately 6% CBS, . HoweverBoth antiasthmatic medication use and chronic respiratory diseases show increased risk estimates of ARM in offspring in the current study. Our risk estimates for the effects of antiasthmatic medication use on ARM in offspring (adjusted OR = 1.4 [0.8–2.6]) were comparable to those found in the studies by Kallen & Otterblad Olausson  and KallIn our study, we observed elevated risk estimates for women who had chronic respiratory diseases and for women who used antiasthmatic medication, although the 95% CI did include the null value. In addition, women with a chronic respiratory disease without antiasthmatic medication use had the highest risk estimate of ARM in offspring. One could argue that these women stopped using their medication because they experienced fewer symptoms. However, the argument that we most often heard during the telephone interview was that women stopped using their medication because they were afraid to harm their unborn child. When we compared women with chronic respiratory diseases with and without medication use, no additional elevated risk was observed. Therefore, the results indicate that the chronic respiratory disease itself seems to be associated with ARM in offspring and not the associated medication use. However, were not able to draw any conclusions based on this information only.The telephone interviews helped us to obtain more detailed information to support our findings of the chronic respiratory disease being responsible. Especially women who used rescue medication, had nonallergic triggers, and experienced exacerbations during the periconceptional period had increased risk estimates of ARM in offspring. To study the independent effect of exacerbations, we corrected for the frequency of medication use. However, we found results in the same order of magnitude, indicating that the increased ORs among women experiencing exacerbations can be attributed to the exacerbations itself and not to the use of rescue medication. Both, the presence of exacerbations and the use of rescue medication point toward uncontrolled chronic respiratory diseases. In addition, nonallergic triggers, such as weather conditions, viral infections, and outdoor and indoor pollutants, are generally harder to avoid than allergic triggers; therefore, women may also experience more problems with controlling their chronic respiratory disease. Although the CIs for rescue medication, a combination of rescue and maintenance medication, and nonallergic triggers do contain the null value, possibly due to small numbers, their lower limits were very close to 1, and they all point in the same direction. Therefore, we conclude that these results suggest that uncontrolled chronic respiratory diseases may be associated with ARM in offspring. Blais et al. showed that women with exacerbations are two times more likely to have a child with a major birth defect, after exclusion of women using oral corticosteroids (Blais & Forget, Proposed mechanisms for adverse pregnancy outcomes in women with an uncontrolled chronic respiratory disease are, for example, maternal hypoxia or the release of inflammation mediators (Blais et al., In conclusion, even with the low frequency of women experiencing chronic respiratory diseases, our results suggest the involvement of uncontrolled maternal chronic respiratory diseases, with or without the use of rescue medication to alleviate exacerbations, in the development of ARM in offspring. This study suggested the involvement of uncontrolled maternal chronic diseases as a new mechanism in the etiology of birth defects that requires more in depth‐studies among larger groups of patients with all kinds of birth defects.The authors declare no potential conflict of interests.


Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by  Aspergillus flavus. Aflatoxin is well recognized for its potent toxicity, mutagenicity, teratogenicity, and carcinogenicity and is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxin contamination is a worldwide food safety concern and impacts both the marketability and safety of multiple food crops1. Most aflatoxin-related losses are primarily to the market rather than to human or animal health. Economic losses due to aflatoxin contamination are estimated at hundreds of millions of dollars globally, with maize and peanuts being the most seriously affected food crops2. Meanwhile, climate change have been predicted to significantly affect the infection of food crops by mycotoxigenic fungi and mycotoxin contamination, which will bring new challenges to the security of global staple food commodities3.Aflatoxin is a group of polyketide-derived secondary metabolites produced by the facultative plant parasite 4. Over the last decade, numerous research efforts have been undertaken to understand how environmental stress affects aflatoxigenic fungi and aflatoxin biosynthesis8. Although the function of aflatoxin production in fungal biology is still unclear, there is clear evidence that aflatoxin biosynthesis is regulated by oxidative stress. Oxidative stress can be induced by either temperature changes or reactive oxygen species (ROS), which are generated by host cells in oxidative bursts that occur during plant-pathogen interactions11. Even though both temperature change and oxidative stress are closely connected with ROS metabolism in fungal cell, the effects of temperature change-induced stress and extracellular ROS-induced oxidative stress on fungal cells have never been compared. Given that ROS play important roles in the regulation of both fungal pathogenesis and secondary metabolism13, a deep analysis of the responses of A. flavus to temperature stress, oxidative stress, and a combination of the two may further improve our understanding of fungal pathogenesis and the regulation of aflatoxin production in this fungus, thereby aiding efforts to prevent aflatoxin contamination under climate change.Climate change-induced environmental stresses, such as drought and temperature and oxidative stresses, can exacerbate aflatoxin contamination in food crops15. Meanwhile, fungal responses to environmental stimuli are highly dependent on the stage of growth16. Indeed, fungal cells have a complex “temporal switch” that regulates activation of genes involved in secondary metabolic pathways and controls the flow of primary metabolites through them. This results in maximal secondary metabolite production at specific time points in the fungal life cycle17. In laboratory conditions, aflatoxin production in A. flavus usually begins after 24–48 h of incubation in induction medium, at which time spore germination is complete, and tends to stop when fungal growth reaches stationary phase. Therefore, it is expected that A. flavus may exhibit different responses to environmental stress at different growth phases.In addition to environmental stress, fungal metabolism is also strongly affected by both the developmental stage and the cultivation conditions of the fungus. In solid substances, such as plant tissues and food products, fungi are usually immobilized and develop as colonies either on the surface or embedded within the solid material. However, studies on fungal growth and metabolism are typically conducted in broth medium, and data obtained from liquid cultures may not accurately describe the metabolic conditions occurring in immobilized growth formsA. flavus, we performed a global gene expression analysis on A. flavus at its aflatoxin production stage using a novel float culture method18. Genes that specifically responded to higher temperature-induced temperature stress, extracellular ROS (H2O2)-induced oxidative stress, or the dual effects of temperature and oxidative stresses were identified. Gene ontology (GO) enrichment revealed the close relationship between secondary metabolic processes and environmental stress responses. The response of mitochondrial function-related genes was also examined. These data can help us to better understand the response of A. flavus to environmental stress, provide insights into important mechanisms related to the regulation of its secondary metabolic systems, and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.In the present study, in order to improve our understanding of aflatoxin biosynthesis regulation in A. flavus to temperature stress and oxidative stress during aflatoxin production  and/or oxidative stress that was induced by H2O2 (5 mM) for an additional 24 h. Transcriptomes were sequenced on an Illumina Hi-seq 2500 system, and differences in the expression profiles among the different treatments were analysed. In total, 178.9 million quality-filtered reads, corresponding to 54,026,617,670 nucleotides, were generated from eight samples with an average of 22.4 million reads per sample. A total of 23,966 contigs were assembled through genome-guided transcriptome assembly using Trinity assembler19 with a size range of 301 to 23,487 bp  data, and the NCBI non-redundant (nr) protein database using an e-value threshold of 10–5. A total of 7123 (36.5%), 13,498 (69.1%), and 15,558 (76.7%) unigenes were annotated, respectively. Moreover, several previously uncharacterized genes were also found to exhibit high expression levels (FPKM > 200) in all tested samples, and many of them were differentially expressed under stress conditions  among the treatment groups , reductases (aflD and aflE), and dehydrogenases (aflH and aflM), which exhibited inhibition rates of 36.45% ~ 71.40% , the backbone enzyme of this cluster, also exhibited 37.04% and 48.77% inhibition under temperature stress and oxidative stress, respectively, and when temperature and oxidative stresses were both applied, its expression was reduced by 68.64%. The reductions of aflatoxin production in float cultures after treatment with temperature and oxidative stresses were also detected  analysis, and similar gene expression trends were observed  metabolism, including monooxygenases  were found to be downregulated in stress-treated samples in a manner similar to that observed for the aflatoxin biosynthetic genes. Expression of the genes encoding acyl-CoA desaturase and ceramide very long chain fatty acid hydroxylase, both of which are enzymes involved in lipid metabolism, was downregulated by oxidative stress. Among the genes involved in ergosterol biosynthesis, erg3, erg11, and erg25 were downregulated by both temperature and oxidative stress, whereas erg13 expression was upregulated by oxidative stress. Moreover, genes involved in the biosynthesis of imizoquins , a group of tripeptide-derived alkaloids that provide protection against oxidative stress, were upregulated by oxidative stress regardless of the presence or absence of temperature stress. Another secondary metabolite involved in fungal oxidative stress response is kojic acid. Genes encoding enzymes in the kojic acid biosynthetic pathway  were also found to be upregulated by the combined effects of temperature and oxidative stress. In particular, kojA was expressed at a much higher level (FPKM > 2500) than the other secondary metabolic genes.Furthermore, genes encoding glycolysis enzymes, such as glucokinase (EC 2.7. 1.2), glucose-6-phosphate isomerase EC 5.3.1.9), 6-phosphofructokinase (EC 2.7.1.11), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and enolase (EC 4.2.1.11), were found to be downregulated by both temperature stress and oxidative stress  was upregulated under stress conditions. A skn7-like (AFL2T_02624) transcription factor was also upregulated in response to both temperature stress and oxidative stress. Among the velvet protein family, the genes encoding LaeA and VeA were found to be upregulated by oxidative stress and temperature stress, respectively. The VelB-encoding gene exhibited decreased expression under the stress treatments, while the stress treatments increased expression of the VelC-encoding gene. The central regulatory genes for asexual sporulation, brlA, abaA, and wetA, were also examined. However, they showed no significant response to either temperature stress or oxidative stress. Several previously uncharacterized transcription factors were also found to be expressed under both the control and stress conditions , the ubiquinol-cytochrome C reductase , and the cytochrome C oxidase , were found to be affected by both temperature stress and oxidative stress Fig. . Genes e21. When compared with acute stress, mild stress is more commonly being induced in the natural environment. Meanwhile, it has been reported that mildly stressed fungal cells exhibit significantly promoted ability to survive when facing future insult23. In this study, secondary metabolic genes were found to be strongly affected in A. flavus under mild temperature stress and oxidative stress conditions. The co-regulation of different secondary metabolic pathways might contribute to maintaining cellular homeostasis and promoting cell survival under environmental stress.Environmental factors-induced abiotic stresses have complex influences on fungal secondary metabolism. Several studies have shown that mild abiotic stress and acute abiotic stress seem to affect mycotoxin biosynthesis differently24. ROS and their reactive products, such as peroxidised lipids, have been shown to affect aflatoxin production26. However, because the function of aflatoxin biosynthesis in A. flavus biology is still unclear, studies on the relationship between ROS and aflatoxin production have led to different hypotheses. Previous studies have shown that aflatoxin biosynthesis in A. flavus is accompanied by increased oxygen consumption and is followed by a subsequent boost in ROS production25. The aflatoxisomes have also been demonstrated as one of the major locations for ROS production inside A. flavus27. The multiple cytochrome p450 monooxygenases and monooxygenases in the aflatoxin biosynthesis pathway are likely responsible for this burst in ROS generation. These results have led to the hypothesis that the aflatoxin biosynthetic pathway is a source of endogenous ROS production. In our study, the expression of aflatoxin biosynthetic genes was downregulated under oxidative stress, possibly as a mechanism to reduce endogenous ROS production, thus maintaining cellular ROS homeostasis. It has been suggested that the burst of ROS production during aflatoxin production may contribute to enhancing conidial oxidative tolerance in aflatoxigenic fungi28.Among the in vitro compounds affecting aflatoxin biosynthesis, ROS have been of particular interest because they are produced in response to biotic and abiotic stress and can act as signals for the regulation of fungal growth, development, and metabolism29. Fountain et al. investigated the transcriptomic responses of different A. flavus isolates to H2O2-induced oxidative stress and found that aflatoxin biosynthetic genes were upregulated by oxidative stress in the moderate aflatoxin producer NRRL3357 but not in the high aflatoxin producer Tox430. The authors suggest that differences in fungal growth and development among the different isolates under oxidative stress may have led to this. Aflatoxin and most of other secondary metabolites are produced after the fungus completes its initial growth phase and is beginning the stage of development that is characterized by sporulation32. Aflatoxin biosynthetic genes are typically expressed at higher levels during the aflatoxin production period, which is approximately 2 to 6 d in conducive media. In this study, stress conditions were applied to cultures after 2 d incubation, which is when aflatoxin production started, and total RNA samples were isolated after a short exposure time (24 h) in order to capture early and direct responses rather than profiles from a later stage, when both fungal growth and aflatoxin production have usually ceased, and fungal cells have adapted to the stress condition. Thus, the results in our study that differ from those of previous studies most likely reflect differences in the treatment periods examined.However, several previous studies have hypothesized that the opposite is true. Since aflatoxin biosynthesis has also been shown to be induced by oxidative stress, it has been suggested that aflatoxin biosynthetic pathway may supplement the antioxidation system in aflatoxigenic fungi, functioning to protect against ROS A. flavus.In light of this, we hypothesize that aflatoxin production may serve different biological roles at different fungal growth stages. During early stages of fungal development, when sporulation has started, aflatoxin biosynthesis may function as a source of endogenous ROS production, which activates antioxidant enzyme expression and contributes to enhancing conidial oxidative tolerance; at the later stationary phase of growth, the aflatoxin biosynthetic pathway may instead function to fix excess oxygen into aflatoxin, which is then secreted from the fungal cells, thus providing a degree of oxidative tolerance in 17. These molecular switches may also react to environmental stress and, in response, adjust the expression patterns of the genes involved in primary and secondary metabolism to enhance the survival of fungal cells. Among the secondary metabolic genes detected in our transcriptomic analysis, biosynthetic genes for kojic acid and imizoquins were differentially expressed. Kojic acid is a potent antioxidant that is synthesized from glucose that does not undergo glycolysis; it is also an iron-chelating antioxidant compound capable of scavenging ROS33. Imizoquins are a family of tripeptide-derived alkaloids produced via a nonribosomal peptide synthetase-derived tripeptide; they also protect against oxidative stress and have been shown to be essential for normal germination in A. flavus34. Under the oxidative stress condition, A. flavus may regulate the flow of primary metabolites, so that it can accumulate kojic acid and imizoquins to relieve the oxidative stress in its cells, thus resulting in fewer primary metabolites produced via the aflatoxin biosynthetic pathway. In fact, a negative correlation between aflatoxin and kojic acid biosynthesis has been reported33.Since fungal secondary metabolites are synthesized at their highest levels at specific time points in the fungal life cycle, it has been suggested that fungal cells possess complex molecular switches that regulate the activation of secondary metabolic pathway genes and the flow of primary metabolites through these pathways2 to H2O, thus introducing a branch in the cytochrome-based electron transfer chain; this results in fewer protons migrating across the inner mitochondrial membrane, leading to lower ATP production through oxidative phosphorylation36. According to our results, the gene encoding AOX is upregulated under oxidative stress. The activity of AOX has been found to be associated with stress response, ROS control, cellular redox state, cellular energy demand, and metabolic homeostasis in different fungal cells38. As AOX is absent in mammals, it has also been investigated as a potential drug target for pathogenic fungi40.In this study, we found that the genes encoding mitochondrial complexes I and III were downregulated by temperature and oxidative stress. Under physiological conditions, endogenous ROS are generated mainly by the mitochondrial electron transport chain through these two respiratory chain components. Under stress conditions, besides depending on the antioxidation system, fungal cells may also downregulate cellular processes that generate ROS in order to avoid cellular oxidative damage. By contrast, the gene encoding mitochondrial complex IV was found to be upregulated. These different responses could be due to the fact that mitochondrial complex IV is not directly involved in ROS generation. Furthermore, when complex I and complex III were inhibited, the fungal cells may have promoted complex IV in order to maintain the cellular ATP supply. AOX is localized on the matrix side of the inner mitochondrial membrane, and it couples ubiquinol oxidation directly to the reduction of O41. In order to maintain cellular homeostasis during the aflatoxin production stage, fungal cells may rely on alternative respiratory pathways for reoxidation of NADH when oxidative phosphorylation is inhibited. Previous studies have found that aflatoxin biosynthesis implies a boost in oxygen uptake, which is followed by an increase of endogenous ROS production, and this occurs during the transition between trophophase and idiophase, when secondary metabolites begin to be abundantly produced25. AOX was also found to affect oxygen uptake in Aspergillus spp.43. Additionally, AOX plays a role in the production of sterigmatocystin, which is the penultimate intermediate in the biosynthesis of aflatoxin B144. These observations clearly indicate that AOX plays important roles in the regulation of aflatoxin biosynthesis. Taken together, these facts suggest that AOX might be an effective target for the control of aflatoxin contamination. Further study will be required to elucidate the role of AOX in the regulation of secondary metabolism and the significance of this enzyme in antiaflatoxigenic practice.AOX maintains mitochondrial respiration and is essential for metabolic functions in fungal cells when the classical electron transport chain is inhibited, maintaining fungal growth and viability. Meanwhile, secondary metabolism, such as aflatoxin biosynthesis, requires considerable amounts of ATP and NADPHA. flavus to environmental stress, which suggests that there is post-transcriptional regulation of protein accumulation in response to environmental stress in A. flavus46. The detection of DEGs involved in protein metabolism and modification and ribosome function may also be indicative of post-transcriptional regulation of temperature and oxidative stress response  used in this study was collected from the Korea Culture Center of Microorganisms . Conidial suspensions for inoculation were prepared using fresh conidia harvested from potato dextrose agar (PDA) after 5 d incubation in the dark at 28 °C using 0.05% sterilized Tween 20 solution and diluted to the concentration of 106 conidia/mL with autoclaved potato dextrose broth . Fungal incubation was conducted using a float culture method18, in which a polyvinylidene difluoride membrane  was placed on the surface of autoclaved PDB in a 90-mm Petri dish. Fifteen microliters of fresh conidial suspension were inoculated onto the centre of the membrane. To test biomass and aflatoxin production, float culture plates were supplied with 25 mL of PDB and incubated for 1 to 7 d at a constant temperature of 28 °C. To test the transcriptomic responses of A. flavus to temperature stress and oxidative stress, each float culture plate was first supplied with 10 mL PDB. After 48 h of incubation at 28 °C, another 15 mL PDB with or without H2O2  was added into each plate, which was then further incubated at 28 °C or 33 °C for 24 h. Two biological replicate cultures were performed for each treatment combination. All plates were incubated in the dark under stationary conditions.The 18. Each test was conducted with three biological replicates for each sample and three technical replicates of each biological replicate.To analyse biomass and aflatoxin production in float cultures, membranes  and culture broths were collected. Fungal biomass production was determined based on the dry weight of fungal colony on each membrane. Aflatoxin production was determined by a high-performance liquid chromatography system  using the previously described methodA. flavus was removed from the membrane of each float culture and then immediately flash frozen in liquid nitrogen. Total RNA was then extracted from the mycelia using a QIAzol Lysis reagent kit  according to the manufacturer’s instructions. The total RNA was then purified using a Riboclear plus kit . Quality and quantity of RNA samples were analysed using the Caliper LabChip GX system . RNA samples with quality scores more than 7 were used for transcriptome library construction and RNA sequencing.After incubation, the whole fungal colony of Transcriptome libraries were constructed using the NEXTflex Directional RNA-seq kit according to the manufacturer’s instructions  with two biological replicates for each sample. Libraries were quantified using the KAPA qPCR kit . The libraries were sequenced on an Illumina HiSeq 2500 system to generate 151 bp × 2 paired-end reads for each sample at the National Instrumentation Center of Environmental Management, College of Agriculture and Life Science, Seoul National University .http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). Quality reads (> Q20) were trimmed to remove adaptor sequences and then used to perform genome-guided transcriptome assembly aligned to the A. flavus genome using Trinity assembler version 2.8.6. To remove redundancy, assembled contigs were clustered at 95% identity using CD-hit. Relative transcript abundances among the different samples were analysed in the cuffdiff program based on FPKM. DEGs were defined as genes with a log2 fold change > 0.5 and FDR < 0.05. Annotation of the assembled transcripts was performed by aligning their sequences against the uniprot database, the A. flavus coding sequences (CDS), and the NCBI non-redundant (nr) protein database with and e-value threshold of 10–5 using the BLASTx algorithm. All DEGs were annotated according to Gene Ontology  databases to establish functional categories. P-values < 0.05 were considered significant.Raw sequencing reads obtained from Illumina sequencing for each sample were first subjected to quality checking using FastQC . A Bio-Rad CFX96 system  was used to perform qRT-PCR. Gene expressions were tested using the Rotor-Gene SYBR green PCR kit (Qiagen) with specific primers , according to the manufacturer's instruction. The eluate was dried using N2 gas and then resuspended using 1 mL of 0.1% acetic acid–acetonitrile–methyl alcohol . Suspensions were filtered through a syringe filter  and stored at − 20 °C until analysis. Aflatoxin detection was performed using a high-performance liquid chromatograph (HPLC) system  with a reversed-phase C18 column . The mobile phase was pumped for 30 min at a constant flow rate of 0.8 mL/min. Aflatoxin was detected in the 50 μL samples using a fluorescence detector  and quantified based on a calibration curve of standard solutions of aflatoxin B1. The recovery rate of the chromatographic procedure for aflatoxin B1 was 85%, and the limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.036 and 0.109 ppb, respectivelySupplementary Information.Supplementary Data.


Most studies about dry eye disease (DED) chose unilateral eye for investigation and drew conclusions based on monocular results, whereas most studies involving tear proteomics were based on the results of pooling tears from a group of DED patients. Patients with DED were consecutively enrolled for binocular clinical tests, tear biochemical markers of DED, and tear proteome. We found that bilateral eyes of DED patients may have similar but different ocular surface performance and tear proteome. Most ocular surface homeostatic markers and tear biomarkers were not significantly different in the bilateral eyes of DED subjects, and most clinical parameters and tear biomarkers were correlated significantly between bilateral eyes. However, discrepant binocular presentation in the markers of ocular surface homeostasis and the associations with tear proteins suggested that one eye’s performance cannot represent that of the other eye or both eyes. Therefore, in studies for elucidating tear film homeostasis of DED, we may lose some important messages hidden in the fellow eye if we collected clinical and proteomic data only from a unilateral eye. For mechanistic studies, it is recommended that researchers collect tear samples from the eye with more severe DED under sensitive criteria for identifying the more severe eye and evaluating the tear biochemical and proteomic markers with binocular concordance drawn in prior binocular studies. Dry eye disease (DED) is a multifactorial ocular surface disease characterized by a homeostatic loss of the tear film along with ocular distressing symptoms, of which tear film hyperosmolarity and instability, ocular surface inflammation, and neurosensory deviations play etiological roles . It is aTo meet the need for diagnosis and to clarify the loss of ocular surface homeostasis in DED, a set of qualified clinical evaluation is required . Ocular However, most of the studies about DED chose unilateral eye for investigation and drew conclusions based on monocular results ,17, wherIn order to clarify the binocular issue in the research of DED, this study aimed to identify the binocular concordant and discrepant inferences about the association between the loss of ocular surface homeostasis and changes in tear biomarkers in patients with dry eye.A total of 23 female participants who met the inclusion and exclusion criteria of DED was enrolled in this study . The meaThere was no significant difference in tear biochemical markers between two eyes of the DED subject, including the concentration of matrix metallopeptidase 9 (MMP-9), the concentration of lactoferrin, and the ratio of MMP-9/lactoferrin. a–c. MeanUtilizing the liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we found no significant difference in the number of identified peptides or proteins in the tears of bilateral eyes a. For co−4, p = 0.9172). In addition, binocular difference and correlation of polymeric immunoglobulin receptor were shown in the Six always presented peptides were adopted for mass spectral intensity analysis. There were no significant differences in these peptides’ standardized signal intensities between these DED subjects’ right and left eyes . Howeverp = 0.0210) but was not significant in the right eye . Moreover, the correlation between nasal and temporal bulbar redness reached statistical significance in the left eye  but did not reached statistical difference in the right eye . Furthermore, the correlation between meiboscale and Oxford staining score was significantly negative in the right eye , while this correlation was positive in the left eye but did not reach significance . Although there were significant positive correlations among NIKBUT_f, NIKBUT_av, and assessable time during the test, the correlation between NIKBUT_f and assessable time was weaker than that between NIKBUT_f and NIKBUT_av, and that between NIKBUT_av and assessable time.Comparing the Spearman correlation matrices of bilateral eyes, we found the color code patterns were similar but not the same . The corp-value in the rectangle of Comparing the Spearman correlation matrices of bilateral eyes, we also found the color code patterns were very similar but not the same . In addiConcordant binocular associations were shown between MMP-9 and two clinical parameters, central TMH and temporal bulbar redness. MMP-9 concentration had a significant negative correlation with central TMH a, but a We hypothesized that bilateral eyes of DED patients might have similar but different tear performance. Thus, we proposed a novel tear proteomics approach to compare binocular ocular surface homeostatic indexes and tear proteomic biomarkers in patients with DED. Moreover, we compared the drawn connections between these clinical parameters and tear biomarkers from bilateral eyes. In this study, we found most clinical parameters and tear biomarkers were not significantly different in DED subjects’ bilateral eyes. In the exception, in terms of clinical performance, NIKBUT_av and assessable time during the NIKBUT test of the left eye were significantly lower than that of the right eye . Most ocIn our previous study , we founKoh et al. pointed out forced eye-opening required for the NIKBUT assessment influences the measurement of TMH, possibly due to reflex tear secretion, even in patients with aqueous-deficient dry eye . In our In clinical practice, tear film instability (shorter NIKBUT_f), less tear secretion , inflammatory ocular surface , and ocular surface staining (Oxford staining score) are commonly used as indicators of DED severity. Although the four severity indices of DED did not show statistical significance between right and left eyes , a signiThe tear proteomics approach was limited as a laboratory tool and varied by MS-based proteomic strategies for small volume of tear fluid sample. Patients with DED have a small volume of tears, and many researchers adopted the pooling and eluted tear sample for discovering the subtle changes of tear proteomes. We adopted a standardized procedure via normal saline flush to collect tear samples for obtaining the individual tear mass spectrum of each subject. Moreover, we directly explored the association of lactoferrin-corrected standardized spectral intensities of tear peptides with ocular surface homeostatic markers. For quantifying the mass spectral intensity of a tear protein, some researchers used isotopic labeling protein quantification ,23, whilThe flush method is a verified tear fluid sampling procedure , but it In a clinical trial of DED treatment, it is wise to treat and collect the data of the eye with more severity since it has a greater room for improvement in dry eye parameters or clinical scores. The researcher may observe more significant progress or faster response to clinical interventions in the eye with more severe DED. However, because DED is a bilateral eye disease, various DED severity indices may indicate a different eye with severe DED. In this study, we found that the clinical performance was associated with biochemical and proteomic markers, in which the associations are similar in bilateral eyes. For mechanistic studies that establish strict criteria for identifying the more severe DED eye, we recommend researchers to collect tear samples from the eye with more severe DED instead of a unilateral eye and evaluate the tear biochemical and proteomic markers with binocular concordance as suggested in this study.This prospective case serial study enrolled female DED patients at the corneal department of Kaohsiung Chang Gung Memorial Hospital (CGMH) from 1 November 2019 to 30 June 2020. Informed consent was obtained from all subjects, and all procedures adhered to the Declaration of Helsinki. Institutional Review Board/Ethics Committee approval (code no. 201900954B0) was obtained from the Committee of Medical Ethics and Human Experiments of CGMH, Taiwan. Subjects were included if they met OSDI > 13, and at least one positive for NIKBUT < 10 s or Oxford staining score > 1. Subjects were excluded if they aged less than 20 years, were in pregnancy, had diabetes mellitus, had acute ocular inflammation or glaucoma, or underwent ocular or eyelid surgery within six months. Both eyes of each subject were evaluated for ocular surface homeostasis and tear biomarkers, including tear biochemical markers and proteome.Each subject completed a dry eye questionnaire, OSDI . Each suAfter the above examinations for at least 30 min, tears were always collected from the right eye and then the left eye. Each subject was instructed to lie down on an operation table in a supine position for tear collection. Tear fluid samples were collected according to a standardized eye-flush technique without topical anesthesia . A physig in a microcentrifuge tube for 10 min at 4 °C and the supernatant fluid was frozen at −20 °C. Finally, the cornea was stained with fluorescence and observed by blue-light illuminating slit-lamp to assess corneal staining via Oxford scheme [The collected tears were immediately centrifugated at 6,000× d scheme . The abo® 5M, Oculus, GmbH, Wetzlar, Germany).Three primary ocular surface homeostatic markers were adopted in this study to determine DED subjects’ binocular clinical performance, including tear secretion, ocular surface inflammation, tear film stability, and meibography. Each patient was noninvasively examined, and all the above homeostatic markers were objectively obtained via a tear film analyzer  kit . Following the manufacturer’s instructions, one μL tear fluid sample was diluted with the ELISA buffer. The analysis was performed with a Microplate Reader  at a wavelength of 450 nm. The assay was performed twice for each tear sample at room temperature. The concentration of tear components was corrected by a 2.5 × dilution factor estimated by a priori test . LactofeThe concentrations of MMP-9 of all tear samples were measured by using a human MMP-9 ELISA kit . Under the manufacturer’s instructions, four μL tear sample was diluted with the ELISA buffer to a final volume of 100 μL and carried out at 37 °C. The analysis was performed with the same Microplate Reader  at a wavelength of 450 nm. The assay was repeated twice for each tear sample. MMP-9 production increases in response to hyperosmolar situations of the ocular surface, contributing to the disruption of the corneal barrier and increasing the severity of DED .The analysis of tear proteome was based on our previous work . In brie2O were mixed with the sample. At 37 °C, the mixed sample was incubated for six h. Subsequently, 0.5 μL of 0.5 M dithiothreitol solution was added to the sample and incubated for 30 min at 56 °C. By adding 1.5 μL of 0.5 M iodoacetamide, the sample was then incubated in the dark environment for 30 min at room temperature for alkylating reduced cysteine residues.Five μL of 0.2 g/L trypsin, ten μL of 100 mM ammonium bicarbonate, and 27.5 μL ddH2O. After incubation overnight at 37 °C, the sample was heated for 5 min at 100 °C. Each sample was lyophilized at −80 °C. The sample was restored with 25 μL of 0.1% trifluoroacetic acid. Lastly, three μL of the sample was analyzed via liquid chromatography coupled tandem mass spectrometry.After that, the sample was added with 0.5 μL of 0.5 M dithiothreitol solution and incubated for 30 min at 37 °C. The sample was then added with 5.0 μL of 0.2 g/L trypsin in a 1:30 mass ratio (trypsin/protein) and reached the last volume of 100 μL with dilution 42.5 μL ddHAccording to the Jian study , an HCT The mass spectra for the eluted fractions were acquired as successive sets of scan modes. The MS scan gained the intensity of ions in the range of 200 to 2000 m/z, and a specific ion was selected for a tandem MS/MS scan. The centroid MS/MS data of enzyme-digested fragments were collected using HyStar 3.2, Bio-Tools, and WarpLC software . Then, the data were submitted to a search program (MASCOT) for searching Swiss-Prot databases of Homo Sapiens with the following settings: A mass tolerance of 0.3 Da for precursor and fragment ions, carbamidomethyl cysteine residues as fixed modifications, one missed cleavage acceptable for trypsin digestion, and oxidized methionine residues for an optional improvement.https://sample-size.net/sample-size-study-paired-t-test/). According to the preliminary results in binocular NIKBUT_av differences of the first ten patients in this study, we estimated the sample size by adopted the significance level (α) as 0.05, the desired power (1-β) as 0.8, the standard deviation of the change of 4.3, and the estimated effect size of 3. Accordingly, the estimated sample size was at least 16 subjects for identifying the binocular difference, and 23 subjects were determined to be the sample size of this study.A free online calculator developed and maintained by the Clinical and Translational Sciences Institute (CTSI) of UCSF was used to calculate the sample size , and the equivalent spectral intensity of lactoferrin was calculated for each subject. The standardized signal intensity of a specific peptide was then calculated by the absolute spectral amplitude of the specific peptide divided by the absolute spectral amplitude of lactoferrin from the same mass spectrum and multiplied by the equivalent lactoferrin spectral intensity of the same subject.p-value of < 0.05 was considered statistically significant.The statistical analysis was performed by GraphPad Prism version 8.4.3 for Windows . The Wilcoxon matched-pairs signed rank test was used to compare the indexes of ocular surface homeostasis, tear biochemical markers of DED, and tear proteome between two DED patients’ eyes. A general linear regression model and the Spearman rank correlation coefficient was used to explore the association among the selected parameters for the same eye and different eyes. A Bilateral eyes of DED patients may have similar but different ocular surface performance and tear composition. Discrepant binocular presentation in markers of ocular surface homeostasis and tear proteins suggested that the performance of one eye cannot represent that of the other eye or both eyes. Therefore, in the study for elucidating tear film homeostasis, we may lose some important messages hidden in the fellow eye if we collected clinical and proteomic data only from a unilateral eye.


Intracortical microelectrodes are valuable tools used to study and treat neurological diseases. Due in large part to the oxidative stress and inflammatory response occurring after electrode implantation, the signal quality of these electrodes decreases over time. To alleviate this response, resveratrol, a natural antioxidant which elicits neuroprotective effects through reduction of oxidative stress, was utilized. This work compares traditional systemic delivery of resveratrol to the novel cyclodextrin polymer (pCD) local delivery approach presented herein, both in vitro and in vivo. The pCD displayed an extended resveratrol release for 100 days, as well as 60 days of free radical scavenging activity in vitro. In vivo results indicated that our pCD delivery system successfully delivered resveratrol to the brain with a sustained release for the entire short-duration study (up to 7 days). Interestingly, significantly greater concentrations of resveratrol metabolites were found at the intracortical probe implantation site compared to the systemic administration of resveratrol. Together, our pilot results provide support for the possibility of improving the delivery of resveratrol in an attempt to stabilize long-term neural interfacing applications.  Intracortical microelectrodes (IME) are a neuroscience tool that can be utilized for studying the functions of the nervous system . IMEs caFortunately, the use of antioxidants can reduce oxidative stress and the incidence rate of neuronal damage . NaturalThe diluent and frequency of intraperitoneal injections was optimized to 30 mg/kg of resveratrol in either 5% ethanol in corn oil or 10:90 propylene glycol and corn oil diluent at a frequency of either biweekly IP administration or every other day, respectively. Histological findings demonstrated significantly more astrocyte reactivity at two weeks post implantation from animals receiving IP injections of resveratrol every other day compared to control animals not receiving any injections  is a widely used constituent in pharmaceutical and FDA-approved applications, including foods, cosmetics, and therapeutics ,34. CD-bTo establish an expected affinity release profile from CD polymers, modelling software was used to characterize the energy associated with CD/resveratrol (host/guest) inclusion complexes . Using PExperimental studies confirmed that the CD/resveratrol host/guest complex allows for resveratrol loading in the polymerized form of CD as well. The maximum drug, in weight percent, that can be loaded into pCD, as well as released from pCD disks, is reported for each CD monomer type  in Three forms of pCD , were evaluated in vitro through drug release studies to determine the optimal form of pCD for long-term, consistent resveratrol delivery. Under physiological conditions in vitro, resveratrol was released from all pCD disk types at low but consistent dosages for 100 days after a small initial burst within the first 24 h . In the The antioxidative activity of resveratrol loaded into pCD disks was assessed using a DPPH scavenging assay. Resveratrol released from loaded pCD under physiological conditions in vitro, displayed sustained free radical scavenging activity for 50 days. A decrease in the scavenging activity began to occur after 50 days in vitro, correlating to the decrease of resveratrol concentration over time observed in There was an average of 10–20 μg/day of resveratrol released from the pCD during the first 30 days. The average antioxidative activity during days 1–30 was 30%, which matched the standard curve for 10 μg of free resveratrol also having 30% antioxidative activity (depicted by the shaded area in p = 0.05) of IP administered resveratrol concentration from 18 h to 7 days. There were no significant decreases of resveratrol concentration from pCD disks, indicating a sustained and controlled release of resveratrol from the pCD loaded disks. Mass Spectrometry analysis of tissue extracted adjacent to the neural probes indicated low bioavailability of resveratrol from the IP injection and the pCD disk one hour post implantation . There wp = 0.05) and mono-glucuronide (p = 0.05) around the implant site from animals receiving resveratrol through the pCD disk compared to the IP injection. There were no significant differences of dihydro monosulfate, dihyro, and disulfate found between the treatment groups. Mass Spectrometry analysis was performed to quantitatively assess the resveratrol metabolites around the neural probes at 18 h post implantation . There wOxidative stress has been linked as a contributor to the failure of intracortical microelectrodes ,7,9. AntCyclodextrins are cyclic oligosaccharides comprised of six or more glucose residues which come together to create a truncated cone shape. This cone- or ring-shaped structure allows the molecule to act as a ‘pocket’ for holding small molecule drugs. The inner space of the pocket is hydrophobic, allowing poorly soluble drug molecules, such as resveratrol, to have a high affinity for localizing in this area, thus creating a reversible host/guest inclusion complex. The insolubilization of CD can be achieved by forming a high molecular weight polymer, in which the inclusion complexes can be exploited to alter the rate of release beyond the rate of diffusion alone. The von Recum lab has shown the affinity within the inclusion complex can reduce the initial drug burst release, allowing the release of the drug to be delivered over a long period of time ,50,51,52Previous work demonstrated various formulations of cyclodextrin ability to improve resveratrol biodistribution, utilizing the CD/resveratrol inclusion complex to overcome resveratrol’s low aqueous solubility and bioavailability ,54,55. HResveratrol has been used as an antioxidant among countless applications such as cancer, ischemia, and neurological diseases and injuries ,65,66,67Mass spectrometry analysis demonstrated that resveratrol was at the electrode interface following IP delivery and pCD delivery at 18 h post neural probe implantation. A significant decrease in IP delivered resveratrol concentration surrounding the electrode by 7 days in vivo was observed, which supported the known low bioavailability of systemically delivered resveratrol ,43,76,77A unique property of CD polymers is the possibility for refilling the polymer with additional drugs once the initial stock of loaded drug is depleted. Once the drug is introduced to the local area in which the pCD is implanted, the drug will be taken up by the CD polymer based on the same affinity and diffusion characteristics, allowing for refilling of the CD pockets and a second round of drug delivery to occur. It has also been shown that this process is not affected by competition from local biomolecules . This abLightly epichlorohydrin-crosslinked cyclodextrin prepolymers  were purchased from CycloLab Ltd., . 1,6-diisocyanatohexane (HDI) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) from Sigma Aldrich , N,N-dimethylformamide  from Applied Biosystems , and trans-resveratrol (RSV) from PureBulk  were used as received. All other reagents, solvents, and chemicals were purchased from Thermo Fisher Scientific  in the highest grade available.Molecular structure data files for resveratrol and all CD types were downloaded from the PubChem database. These structures were converted to PDBQT format and loaded into PyRx  with resveratrol serving as a ligand and CD as a host. The Autodock Vina algorithm was used to predict host/guest affinity interaction energies. Finally, PyMOL  was used to visualize host/guest models in 3D space . Polymerized CD hydrogel disks were synthesized from lightly crosslinked CD prepolymer . BrieflyResveratrol was dissolved in DMF at 5 wt % and pCD disks were exposed to this loading solution for 3 days at room temperature to allow for drug loading. Optimization of resveratrol concentrations for drug loading into pCD indicated that an increase in resveratrol concentration during drug loading can increase the weight percent loading achieved in pCD polymer disks, therefore determining the loading concentration utilized in the study herein . Loaded Resveratrol-loaded pCD disks (8 mm) were incubated with 1 mL of phosphate buffer saline  at 37 °C with mild shaking. Every 24 ± 4 h, PBS release media was removed for collection, and replaced with fresh PBS to create an infinite sink condition. The collected PBS was analyzed to quantify the concentration of resveratrol at each time point, by spectrophotometric analysis at 321 nm. A standard curve was generated using a known amount of resveratrol in PBS. To estimate the in vitro antioxidative efficacy of resveratrol delivered from pCD, a 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay was used. DPPH is a stable free radical which is reduced when scavenged. Briefly, a DPPH (100 μM) in 200 proof ethanol solution was made, following previously reported methods ,93,94. FA) is the absorbance value of the neat DPPH solution and (B) is the absorbance value of the DPPH solution in the presence of the pCD released resveratrol. In order to ensure consistency and repeatability, all samples were tested in triplicate. Here,  were used. These animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Louis Stokes Cleveland Department of Veterans Affairs Medical Center. Animals were implanted with neural probes in the motor cortex for either 1 h, 18 h or 7 days. Animals receiving resveratrol through an intraperitoneal injection were compared to animals receiving resveratrol through local gamma-CD polymer delivery and to control animals that did not receive any resveratrol. There were three animals used per group and at each time point.2) for about four minutes. Once the surgical plane of anesthesia was reached, anesthesia was maintained with isoflurane at 2.5% by a nose cone. The animal’s head around the surgical site was shaved, and Marcaine (0.25%) was applied subcutaneously around the surgical site. Carprofen (5 mg/kg) and Cefazolin (25 mg/kg) were applied subcutaneously as analgesics and antibiotics, respectively. The animal was placed onto a stereotaxic frame with 1%–2.5% isoflurane flowing through the nosecone to maintain anesthesia. The surgical site was sterilized with alternating cotton tipped applicators of chlorhexidine gluconate (CHG) and isopropanol scrubs. Animal body temperature was maintained on a circulating water pad and vitals  were monitored using a MouseSTAT® Pulse Oximeter & Heart Rate Monitor, . For the surgical procedures, rats were anesthetized in an isoflurane chamber . Using a 2 mm drill bit, the skull was drilled to gain access to the dura. A fine 45° angle dura pick was used to reflect the dura and uncover the brain. A resveratrol loaded cyclodextrin disk was placed into the craniotomy for the animals receiving local delivery of resveratrol. Implants were placed into the cortex , approximately 2 mm deep, manually, while avoiding vasculature. Kwik-Cast  was applied over the implant site to keep the brain insulated. Teets cold cure dental cement was used to construct a stable headcap covering the implant hole and tab. The skin was pulled back together and sutured closed with 5-0 monofilament polypropylene suture , and coated with triple antibiotic ointment. Analgesia and antibiotics were administered for three days postoperatively. Resveratrol  was weighed in 50 mg aliquots in centrifuge tubes and autoclave sterilized at 121 °C for 20 min. Assays measuring resveratrol quantification and antioxidant activity confirmed no differences between the autoclaved sterile resveratrol and the non-sterile resveratrol . A stock2, followed by decapitation and brain harvesting. A 3 mm biopsy punch was utilized to collect tissue samples around the implant site for bioavailability analysis. Biopsied tissue samples were homogenized in 1 mL of ethanol solution using a Tissue-Tearer at 10,000 rpm speed . Following which, homogenates were centrifuged at 2500 rpm for five minutes, and the aqueous supernatant was transferred to clean Eppendorf tubes further analysis.Animals were euthanized using COAs a means for quantifying resveratrol in brain tissue, samples were sent out for LC-MS. Supernatant aliquots (200 µL) were shipped on ice to PhenoSwitch Bioscience . Supernatants were dried under nitrogen and then reconstituted with 200 µL of 50% acetonitrile and 50% water containing 10 mM ammonium acetate and 10 ng/mL of internal standard—Resveratrol-13C6. Standard curves were generated by spiking a known amount of resveratrol standard into control tissue samples  and 10 ng/mL of internal standard and were utilized to quantify resveratrol. Known metabolites of resveratrol  were evaluated utilizing LC-MS/MS using reported precursor and product ion. Their identity was validated at the MS/MS level. The absolute amount of metabolite was estimated based on the ratio over resveratrol internal standards signal. Acquisition was performed with an ABSciex TripleTOF 5600  equipped with an electrospray interface with a 50 μm iD capillary and coupled to an Eksigent μUHPLC . Analyst TF 1.7 software was used to control the instrument and for data processing and acquisition. The source voltage was set to −4.5 kV and maintained at 350 °C, curtain gas was set at 30 psi, gas one at 23 psi and gas two at 30 psi. Acquisition was performed in negative product ion mode. The following precursor ions were used for quantification: 227.07 – 185.05 (resveratrol), 233.08 – 191.07 . Separation was performed on a reversed phase Luna Omega Polar C18 column 1 mm i.d., 1.6 μm particles, 50 mm long  which was maintained at 50 °C. Samples were injected by loop overfilling into a 5 μL loop. For the 2 min LC gradient, the mobile phase consisted of the following solvent A (10 mM NH4 acetate in water) and solvent B (10 mM NH4 acetate in acetonitrile) at a flow rate of 50 μL/min. Quantification was done using the area under the curve with the Sciex MultiQuant software .n = 3). Data displayed are the mean of each condition, and error bars represent the standard deviation of the triplicate set. ANOVA was done using Prism to determine reported statistical significance. Significance is defined to be a p-value < 0.05. If not otherwise stated, experiments were carried out in triplicate for statistical analysis (The objective of this pilot study was to present a proof of concept approach to locally deliver resveratrol to the neural probe implantation site, utilizing a novel polymerized cyclodextrin disk loaded with resveratrol. In vitro analysis of the pCD model demonstrated consistent, controlled release of resveratrol from the pCD disks for 100 days, with a slight decrease of release rate around day 60. Evaluation of the antioxidant activity of the released resveratrol discovered stable, free radical scavenging activity for 60 days, which was related to the drop off in released resveratrol around day 60. In vivo analysis of the pCD localized resveratrol delivery system verified a continuous delivery of resveratrol to the microelectrode implantation site for up to 7 days. Thus, the delivery system presented here collectively provides support for the promise of a potential localized, sustained drug delivery system to the brain. Further investigation will include the optimization of reloading pCD disks systemically, circumventing the need for a secondary invasive operation, as well as investigation of several therapeutic drugs delivered in a temporal pattern, deemed necessary to treat the dynamic tissue response to implanted neural microelectrodes.


To evaluate the specifications and technique properties of the new Femto LDV Z8 in creating intrastromal refractive lenticules during small incision lenticule extraction (SMILE).  Six enucleated porcine eyeballs were equally divided into two groups (Femto LDV Z8 or VisuMax) and were randomly assigned to three experienced refractive surgeons who performed SMILE on each group. Five intraoperative time parameters and surgeons' satisfaction on the surgical procedure were compared between two groups. Postoperatively, the roughness of the lenticule surfaces and the irregularity of edges were observed by scanning electron microscopy (SEM) and were also compared between the two groups. p=0.039; anterior, ×250, p=0.337; posterior, ×180, p=0.006; and posterior, ×250, p=0.007).  Longer time on suction peak pressure, total laser application, and total surgery were spent in the Femto LDV Z8 group as compared with the VisuMax group. The Femto LDV Z8 group applied OCT scanning and offsetting before performing the laser procedure, which expended more time for these crucial steps. The widest range of surgeons' satisfaction scores was found in the step of lenticule interface identification of the Femto LDV Z8 group. The roughness scores of the anterior and posterior lenticule surfaces were statistically less in the Femto LDV Z8 group than in the VisuMax group (anterior, ×180,  Femto LDV Z8 showed promising performances as a novel SMILE equipment for the correction of myopia. It has special and unique features for SMILE procedures, which need more learning and researching processes. With its low-energy high-frequency nJ-level laser system, the Femto LDV Z8 provided smoother lenticule surface than VisuMax. Small incision lenticule extraction (SMILE) is a femtosecond laser procedure which was first performed using the VisuMax Laser System . , 2 This Since Femto LDV Z8 has not yet acquired approval from the China Food and Drug Administration for commercial and patient use, enucleated porcine eyeballs were utilized in lieu of human eyes for this study. Six enucleated porcine eyeballs were retrieved from local abattoirs within five hours postmortem. These eyeballs, which were numbered from Eye 1 to Eye 6, then were divided into 2 groups (Femto LDV Z8 group and VisuMax group) before delegating to three experienced refractive surgeons. Eyes 1, 2, and 3 were assigned to the Femto LDV Z8 group, while Eyes 4, 5, and 6 were assigned to the VisuMax group. Small incision lenticule extraction was performed accordingly in the following order: Dr. W for Eye 1 and Eye 4, Dr. L for Eye 2 and Eye 5, and Dr. Z for Eye 3 and Eye 6. A Femto LDV Z8 model machine from a medical exhibition in China and a VisuMax Laser commercial machine from Hebei Eye Hospital, China, were used for this study. The same treatment parameters were set on the two laser platforms preoperatively. These included cap thickness, refractive correction, lenticule diameter , and lenticule sidecut angle. All the surgeons in this study had more than 10 years of refractive surgical experience, each of them having more than 1000 cases of FS-Lasik operations using Femto LDV Z6 and more than 500 cases of SMILE operations using VisuMax.The machine specifications of the two femtosecond laser platforms are shown in In the current study, the differences in suction peak pressure time, OCT scanning time, offsetting time, total laser application time, and total surgery time were compared between the two laser platforms during the surgical procedures of the 6 eyes. After the completion of each procedure, each surgeon's satisfaction was scored using a self-10-point scale based on an evaluating method for a general surgical study.  The paraAll lenticules were removed and divided into halves by microscopic scissors and were prepared for scanning electron microscopy (SEM). The anterior surface, posterior surface, and lenticule edge of each of the lenticules were examined at ×180 and ×250 magnification using a Hitachi S–3500 N Scanning Electron Microscope , and images were taken. The SEM images were handed over to three observers in a randomized order to evaluate the roughness of anterior and posterior lenticule surfaces and the irregularity of lenticule edge. The subjective scoring is carried on as a self-10-point scale from 1 for “smoothest/most regular” to 10 for “roughest/most irregular” according to methods used by previous studies. .p < 0.05 was considered statistically significant.The statistical package IBM SPSS Statistics 20  was used in the current study. Data were expressed as mean–standard deviation. The descriptive statistical analysis was performed for comparing the differences of the two laser platforms on suction peak pressure time, OCT scanning time, offsetting time, and total laser application time, as well as the surgeon's satisfaction during the surgical processes. Score data of lenticule surface roughness and edge irregularity were compared between the two laser platform groups using a Mann–Whitney nonparametric test. Each laser platform was successfully used for three complete SMILE procedures without complications.p=0.006 and p=0.007, respectively). SEM images of the anterior lenticule surfaces were statistically smoother in the Femto LDV Z8 group than in the VisuMax group at ×180 magnification (p=0.039), but no statistical difference at ×250 magnification (p=0.102). Meanwhile, there was no statistical difference in the irregularity score of the lenticule edge between the two groups at ×180 magnification (p=0.337). To the best of our knowledge, this is the first published report of utilizing Femto LDV Z8 for SMILE procedures. In contrast with the VisuMax, the Femto LDV Z8 has unique features as follows.One notable feature of Femto LDV models is the low-energy yet high-frequency nJ-level laser system see . With thThis speculation could be proven by the roughness performance of the lenticule surface in our study. Although the target incision settings were similar in both laser platforms, smoother surfaces were found on the lenticules created by the Femto LDV Z8 than by the VisuMax see . BecauseAnother special characteristic of the Femto LDV Z8 is its intraoperative OCT and adjustable offsetting. The high-resolution OCT images provided automatic detections of pupil edges, entire structures of cornea layers, and the front surface of lens and iris. In addition, the quantifiable and adjustable offsetting function made it possible for individualized designing the lenticule position without loading and unloading suctions back and forth, which would increase the precision of SMILE procedures and decrease the possibility of surgical complications. On the contrary, because there was no offsetting function in VisuMax, surgeons had to release suction and repeat the docking procedure when the lenticule centration was not satisfied.As a new SMILE platform, Femto LDV Z8 meant a new learning curve even for the experienced surgeons, as those in our study. Because of the wide range of ease score, the biggest challenge seemed to be in the step of lenticule interface identification see . GeneralLike its older generations, the Femto LDV Z8 uses a flat contact glass on its docking system which directly gets in contact with the cornea to minimize lenticule distortion and avoid suction loss during laser applications. Previous studies have proven that the flat suction cone in Femto LDV models induced a significantly higher IOP than the curved suction cone of VisuMax. , 19 In WThere are several limitations in this study because this is the first experience in SMILE using Femto LDV Z8. First, SMILE was performed on porcine eyes rather than human cadaver eyes. Because of the lack of human cadaver eyes, porcine cornea was thought as a close model for human cornea and was widely used in previous studies. Second is the number of SMILE cases performed in the two groups, although significant differences had been found between two laser platforms. Finally, as an uncommercial model, Femto LDV Z8 has not been accessible in clinical practice. Hence, the current findings would be helpful for improving this laser platform to a better situation.In conclusion, Femto LDV Z8 showed promising performances as a novel SMILE equipment and would be a hopeful choice for the correction of myopia. It has special and unique features for SMILE procedures, which needs refractive surgeons a slightly new learning curve. With its low-energy high-frequency nJ-level laser system, the Femto LDV Z8 provided smoother surface of lenticule than VisuMax. Further studies are suggested to evaluate the clinical influence caused by the difference in lenticule surface roughness created by these two laser platforms.


Domestic pigs express high phylogenetic similarity to humans and are often used as a compatible model in biomedical research. Porcine tissues are used as an accessible biomaterial in human skin transplants and tissue architecture reconstruction. We used transcriptional analysis to investigate the dynamics of complex biological system of the mucosa. Additionally, we performed computer analysis of microscopic images of cultured cells in vitro. Computer analysis of images identified epithelial cells and connective tissue cells in in vitro culture.Transcriptional analysis and live-cell imaging are a powerful tool to investigate the dynamics of complex biological systems. In vitro expanded porcine oral mucosal cells, consisting of populations of epithelial and connective lineages, are interesting and complex systems for study via microarray transcriptomic assays to analyze gene expression profile. The transcriptomic analysis included 56 ontological groups with particular focus on 7 gene ontology groups that are related to the processes of differentiation and development. Most analyzed genes were upregulated after 7 days and downregulated after 15 and 30 days of in vitro culture. The performed transcriptomic analysis was then extended to include automated analysis of differential interference contrast microscopy (DIC) images obtained during in vitro culture. The analysis of DIC imaging allowed to identify the different populations of keratinocytes and fibroblasts during seven days of in vitro culture, and it was possible to evaluate the proportion of these two populations of cells. Porcine mucosa may be a suitable model for reference research on human tissues. In addition, it can provide a reference point for research on the use of cells, scaffolds, or tissues derived from transgenic animals for applications in human tissues reconstruction. Domestic pigs express high phylogenetic similarity to humans and are considered as compatible model in biomedical research ,2. RecenOur research focuses on the porcine oral mucosa, consisting of two distinct layers (epithelium and fibrous connective tissue layer), with remarkable capacity for repair ,14,15. TAdditionally, we analyzed microscopic images of cells cultured in vitro. Current approaches in cell imaging require many hours of manual adjustments and curation, sparking the need for development of new, supervised machine learning techniques. Live-cell imaging is a powerful tool in the investigation of complex biological systems at a single-cell resolution ,24,25. NMathematical modeling of cellular data is an important new tool aimed at the task of understanding the biological mechanisms of cellular differentiation.Thirty young gilts of the local Landrace, used in the experiment, were raised on a local commercial farm, and exhibited a mean age of 155 days (140–170) and weight of 100 kg (95–120). The housing and feeding conditions of the pigs were identical. All of the experiments performed in the study were approved by resolution 32/2012 (30.06.2012) of Local Animal Ethics Committee of the Poznan University of Life Sciences, Poland.−1 streptomycin, 10 U mL−1 penicillin, 25 µg mL−1 amphotericin B). The excised tissue was washed twice in Dulbecco’s phosphate buffered saline . The buccal mucosa was surgically removed using sterile surgical blades. The tissue fragments were minced and incubated with 1 mg mL−1 collagenase type I  and 2 mg mL−1 dispase II  for 2 h at 37 °C in a shaking water bath. The cell suspension obtained from this digestion was centrifuged at 300× g for 8 min. Supernatant was removed and pellet was resuspended in 0.25% trypsin solution  for 10 min. Fetal bovine serum  was used to neutralize trypsin. The cell suspension was filtered through mesh to remove non-dissociated tissue fragments, and then isolated cells were centrifugated at 300× g for 8 min. The final cell pellet was dissolved in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 10 U mL−1 penicillin G, 10 mg mL−1 streptomycin, and 25 μg mL−1 amphotericin B . Cell viability was 85% to 95% as determined using a cell counter Adam-MC . The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Once the cell cultures attained 70–80% confluency, they were passaged by washing with PBS , digested with 0.25% trypsin solution , neutralized using the same volume of FBS , centrifuged (300× g for 8 min), and resuspended at a seeding density of 2 × 104 cells cm−2. The culture medium was changed every three days.Samples of buccal mucosa were obtained within 40 min of slaughter and transported to the laboratory in the transportation medium . In the periods of 7, 15, and 30 days, half of the cells were collected to isolate RNA and to perform microarray and real-time quantitative polymerase chain reaction (RT-qPCR) analysis.® WT Expression Kit). The resulting cDNA was biotin labelled and fragmented using the Affymetrix GeneChip® WT Terminal Labeling and Hybridization . cDNA fragments prepared in that way (5.5 μg) were hybridized to the Affymetrix® Porcine Gene 1.1 ST Array Strip (48 °C/20 h) . The microarrays were then subjected to washing and staining based on the protocol of the Affymetrix GeneAtlas Fluidics Station. The scanning of the array strips was performed using the Imaging Station of the GeneAtlas System . The following preliminary analysis was conducted with the use of the Affymetrix GeneAtlasTM Operating Software . The gene expression data quality was evaluated based on the quality control criteria included in the software. The resulting CEL files were imported into further software for downstream data analysis.The in vitro cultured cells were collected and suspended in the TRI reagent . The RNA isolation was based on the procedure described by Chomczynski and Sacchi . Samplesp < 0.05 and expression fold change higher than |2|.All of the graphs and analysis presented in the manuscript were compiled using Bioconductor 3.11  and R 3.6.3 programming language . Each of the CEL files was merged with a corresponding description file. The Robust Multiarray Averaging (RMA) algorithm was used for result standardization, with moderated t-statistics of the empirical Bayes method performed to enable identification of statistically significant differences. Furthermore, Benjamini and Hochberg’s false discovery rate was used to correct the obtained p-value for multiple comparisons. The cutoff value for differentially expressed gene selection was assumed at Single–organism cellular process  and single–multicellular organism process  represent processes that is carried out at the cellular level. Regulation of cellular component organization  includes processes involved in the formation or disassembly of cell structures. Biological adhesion , which includes the processes responsible for cell attachment to a substrate or another cell. Other processes responsible for the transition from proliferation to development and differentiation, negative regulation of cell proliferation , developmental process , and anatomical structure development .Differentially expressed genes were subjected to selection by examination of genes involved in cell migration regulation. The differentially expressed gene list (separated for up and downregulated genes) was uploaded to DAVID software  , where gString 10.0 software  was used for extraction of interactions between differentially expressed genes/proteins belonging to the analyzed gene ontology (GO) terms . InteracFinally, the REACTOME FIViz application, a part of the Cytoscape 3.7.2 software , was used to investigate the functional interactions between genes belonging to the chosen GO BP terms. The Reactome FIViz app  is used to examine pathways and network patterns related to cancer and other types of diseases. REACTOME accesses the pathways stored in the Reactome database, allowing to perform pathway enrichment analysis of a set of genes, visualize hit pathways using manually laid-out pathway diagrams directly in Cytoscape, and investigate functional relationships among genes in hit pathways. The application can also access the Reactome Functional Interaction (FI) network, a highly reliable, manually curated pathway-based protein functional interaction network covering over 60% of human proteins.® Green I serving as a detection dye, and target cDNA quantified with the use of the relative quantification method. The relative abundance of CCL8, CXCL2, DACH1, DUSP5, FABP5, IL6, PLK2, PPARD, PTGS2, and SPP1 was standardized to the internal standards ACTB and PBGD. For amplification, 2 µL of cDNA solution was added to 18 µl of QuantiTect® SYBR® Green PCR  and primers , with SYBR primers . One RNAhttp://scikit-image.org/) [The mucosal cell cultures were visualized during their first 7 days of growth with differential interference contrast microscopy (DIC), using Olympus IX70 microscope . After the microarray analysis, the morphological dynamics were investigated by analyzing the DIC images. The collected DIC images during in vitro culture were analyzed using a sequence of image processing and analysis operations, based on the collected data followed by quantitative assessment of selected relevant morphological features and their distributions. All methods described below were implemented using Python 3.5  and image processing libraries OpenCV 4.2.0   and SciKge.org/) .regions (segments) from the input image. The interpretation of regions depends on the class of images with the goal to obtain exactly one separate region for each cell. It has been a historical challenge for one segment to accurately represent each individual cell using image analysis software if a cell is divided into two or more regions, it is referred to as over-segmentation; if two or more cells are merged in one region, this implies under-segmentation.Representative images obtained during a 7-day in vitro culture were used to perform the analysis . The prowatershed segmentation technique [seed (a.k.a. marker). Feeding the algorithm with carefully selected seeds is thus essential for the quality of the outcome. Two methods for determining seed locations are either the Bespoke or the Feineigle method. With these two methods it was possible to determine seed locations. The Bespoke method first removes the pixels that are almost certain to belong to the background, with is achieved by normalizing image brightness, calculating image gradient using the Sobel filter, and then applying Otsu’s thresholding method [f of the examined specimen, its integration should lead to the reconstruction of f, which is realized by formulating an optimization problem in a form of a set of equations that are subsequently solved using Gauss-Seidel relaxation.DIC images were analyzed using the echnique  and the echnique . The watg method . The Feig method  attemptsIn both above scenarios, the resulting segmentation undergoes further postprocessing. In particular, small artifacts (impurities) are removed, which we achieve by setting a magnification-specific minimum threshold of cell’s area .k parameter (the number of clusters) set to 3, which was applied to all 6080 cells gathered from all images. Additionally, the isomap dimensionality reduction technique was used to verify whether the obtained clusters are compact and thus adequately define classes of regions [The next stage of the analysis was the clustering process using k-means algorithm with the  regions .For morphological analysis and separation of keratinocyte and fibroblast populations of cells cultured in vitro, the following data were collected from each cell: Logarithm of the area, eccentricity, perimeter, major and minor axis length , and solidity (the ratio of the cell area and the area of its convex hull). To enable aggregation and comparison of measurements obtained from images of different resolution, we adopted the common measurement unit of 100 µm. There are 6 features that are calculated for each segmented cell individually . For an image that contains n such regions, the features are gathered in an n × 6 array.To capture the potentially multimodal characteristics of the cells in a given sample, we conduct cluster analysis on those arrays using a family of k-means algorithms.−1 of 4′,6-diamidino-2-phenylindole (DAPI), mounted on an anti-fade medium on glass slide, and examined by an LSN 510 confocal system with an Olympus Fluoview 10i microscope .In 7, 15, and 30 days of in vitro culture, porcine buccal mucosal cells were collected and fixed in acetone-methanol solution (1:1) for 10 min at −20 °C. In the next steps, cells were washed, and to block non-specific binding, samples were incubated in 3% BSA with 0.1% Tween-20 for 30 min at room temperature (RT). Porcine oral mucosal cells were incubated at 1 h with a mouse monoclonal anti-cytokeratin 8 + 18 + 19 antibody . The antibodies were diluted 1:500 in PBS with 1.5% BSA and 0.1% Tween-20. After washing, the samples were incubated with MFP488-labeled goat anti-mouse IgG  diluted 1:500 in PBS with 0.1% Tween-20 for 1 h at RT. A similar labeling process was carried out for fibroblasts isolated from porcine mucosa. Fibroblasts were used as a negative control. After incubation, samples were washed, and cells were suspended in 0.1 mL of mineral oil containing 1.5 μg mL® Porcine Gene 1.1 ST Array Strip was used to investigate the expression of 12,257 transcripts. Genes with fold change higher than abs (2) and with corrected p value lower than 0.05 were considered differentially expressed. This set of genes consisted of 130 different transcripts. Up and downregulated gene sets were uploaded to the Database for Annotation, Visualization and Integrated Discovery (DAVID) search separately, with only the gene sets with adjusted p value lower than 0.05 selected. The analysis showed that differently expressed genes can be classified to 56 gene ontology groups. This manuscript focuses on anatomical structure development, biological adhesion, developmental process, negative regulation of cell proliferation, regulation of cellular component organization, single−multicellular organism process, and single−organism cellular process GO BP terms. To present the genes as heatmaps the gene sets were subjected to hierarchical clusterization procedure and presented as heatmaps (CEBPA), for which the level of expression is similar to the control day. In the case of the 30th day of in vitro culture for the CCL2, ITGB3, TGFB1, and PLK2 genes, the expression level is higher than on day 15 and is close to the control level. Numerical values corresponding to the data presented in heatmaps was presented in single−organism cellular process GO BP term, and on the other hand, the groups negative regulation of cell proliferation, biological adhesion, and regulation of cellular component organization were represented by a smaller number of genes. LYN, TGFB1, and ETS1 were present in all analyzed ontological groups. Genes SPP1, PDPN, FCER1G were present in all ontological groups except negative regulation of cell proliferation GO BP term. LIF was presented in all GO groups except biological adhesion GO BP term. STRING interaction network was generated to visualize relations between the differentially expressed genes belonging to the selected GO BP terms. Using such prediction method provided us molecular interaction network formed between protein products of studied genes  during the seven-day in vitro culture. To confirm the presence of keratinocytes in further days of in vitro culture, the distribution of cytokeratin 8 + 18 + 19 (pCK) in cells was detected using an immunohistochemistry and confocal microscope. The results obtained from this analysis are shown in Oral mucosa is an accessible tissue with remarkable capacity for repair. Composed of lining epithelium and supporting connective tissue, it allows to obtain two types of cells, keratinocytes and fibroblasts, in primary in vitro cultures. Interactions between these two different cell populations are important for the homeostasis of the tissue. Keratinocytes are described as small-sized (10–20 μm in diameter) polygonal cells that tend to change their morphology and size when undergoing differentiation process. During the differentiation process, they lose their ability to proliferate, and express irregular size and shape. In long-term in vitro conditions, these cells were reported to reach up to 50 μm in diameter .The fibroblasts exhibit significantly diversified morphology, which depends on the location and different environmental conditions . The tisimage segmentation in computer vision and pattern recognition, was particularly challenging, as the boundaries of cells render rather faintly in DIC imaging . The object separation stage, known as  imaging . Moreoveentation , with laentation , proved entation . Computeentation . Researcentation . Jaccardentation .Some problems had to be solved during the computer analysis of DIC images. For higher magnification (40×), the Bespoke method does not perform satisfactorily, making it necessary to use the Feineigle technique. However, images at 10× magnification following the Bespoke segmentation method provided results that were visually more compelling than the Feineigle method for higher magnifications. Additionally, lower magnification allows the analysis of a larger numbers of cells and thus arrive at more significant statistical differences. Therefore, it is better to use images with a magnification of 10× for analysis.Our algorithm applied a colored patch to each designated region. Most regions delineate individual cells, though occasionally the segmentation algorithm groups a few cells together into one region, or misinterprets various image artefacts as cells. However, the algorithm usually manages to identify individual cells even in the later stages of culture growth, when the density of cells is high and most of them are directly adjacent to other cells.The algorithmic approach allowed the assessment of morphological types of cells that are present in the in vitro culture, providing the possibility for quantitative comparison between different morphological types of cells. Analysis of DIC images permits the determination of the percentage share of particular morphological types, which were followed by microarray analysis. As a result of the morphological assessment, two types of cells were selected. Some smaller and oval cells (blue color) that correspond to keratinocytes and second fiber-like cells (red color) that correspond to fibroblasts . The thinegative regulation of cell proliferation ontology group, associated with processes that reduce cell proliferation during our transcriptomic analysis. It is also important to analyze the ontological groups such as regulation of cellular component organization that consists of genes involved in formation or disassembly of cell structures, including the plasma membranes, and developmental process ontology group that consists of genes involved in the process of cell development and differentiation. We have also analyzed genes related to development, marked as anatomical structure development group. The other three groups, single–organism cellular process, single–multicellular organism process, and biological adhesion are related to cell adhesion and communication processes.Following the evaluation of DIC images, a long-term culture in vitro was carried out. Long-term cultures are usually associated with a reduction in cells’ proliferative capacity and the transition to differentiation. This highlighted the importance to analyze the CEBPA) interacts with CDK2 and CDK4 and reduces cell proliferation by inhibiting these kinases [CEBPA can be expressed in basal keratinocytes. Increased co-expression of CEBPA and CEBPB was demonstrated in keratinocytes, that leave the basal layer and undergo terminal differentiation [After comparing the obtained results, only three genes belonged to the ontological group containing genes responsible for stopping or reducing the proliferation process. The CCAAT/enhancer-binding protein alpha , which is a nuclear receptor that plays an important role in glucose and lipid homeostasis [The eostasis . In the eostasis ,53. It weostasis  and suggeostasis .CEBPA was at a similar level with IL6 and PPARD showing slightly lower level of expression. In the last period of long-term culture, the three genes described above were downregulated. Increased CEBPA expression after 7 and 15 days is consistent with previously reported results by Lopez et al. [Three genes were upregulated after seven days of in vitro culture. After 15 days of in vitro culture, the expression of z et al. . It appeSingle-organism cellular process ontology group contains genes that influence processes carried out on cellular level. One of the genes present only in this ontological group is ATPase type 13A3 (ATP13A3), which is expressed in epithelial cells [STEAP1) [STEAP1 expression correlates with the increase in cell adhesion to the culture dish [al cells . The higal cells ,55. Anot[STEAP1) . The proure dish .TGFB1) was downregulated after 15 days of in vitro culture and was upregulated after 7 days and 30 days of in vitro culture [ETS1), regulates cell development. ETS1 is normally expressed in the proliferative layer of epithelium. It was presented that ETS1 can block terminal differentiation of keratinocytes and promote their proliferation [LYN). The expression of LYN has been previously reported in the mouse keratinocytes [SMPDL3A), may be functionally related to other sphingomyelinases. It was investigated that SMPDL3A expression is regulated by liver X receptor (LXR) and may be species-specific [SMPDL3A expression in immortalized cell lines obtained from kidney, liver, and skin fibroblasts [Among the analyzed ontological groups, only three genes were represented in all groups. Transforming growth factor beta 1  encodes a protein that is responsible for elongation DNA and was described in an earlier study [RFC4 in skin cells were presented by Phatak et al. [Replication factor C subunit 4 , confirming the multi-regulatory function of IL6. A large number of interactions with REL generated by STRING is related to the fact that this factor belongs to the NFKB family. Lorenz et al. described REL as a key regulator of the growth and death of keratinocytes that may be playing an important role in epidermal carcinogenesis [REL expression, decreased proliferation was observed. These cells were very small and did not form colonies in culture [Based on the analysis of the STRING interaction network and information contained in databases and comparison of text information from published scientific articles, there is a correlation between ETS1 and CEBPA. Since the increased expression of ogenesis . Anotherogenesis . In the  culture .Standardizing and normalizing primary cultures is essential to achieve repeatable results and to create a model that allows reliable analysis, which could bring us closer to translational research on oral mucosa regeneration.The DIC images analysis algorithm that we used to extract cell images, and that further enabled us to map the different morphological populations among the cells obtained from porcine oral mucosa, is a great tool that could offer significant opportunities in future biomedical research. The use of machine learning allows to obtain more precise results, as well as to automate the entire process through the use of appropriate software. Automated image analysis can significantly reduce the costs associated with the identification of cell types and eliminate human errors. Further transcriptomic studies that followed the analysis obtained by DIC complemented our aim to characterize the cellular identities of oral mucosa in long-term in vitro conditions.