Androgens are known to be an essential regulator of male health. Androgen receptor (AR) is widely expressed throughout the adrenal cortex, yet the wider role for androgen signalling in the adrenal remains underexplored. To investigate AR-dependent and AR-independent androgen signalling in the adrenal, we used a novel mouse model with a specific ablation of androgen receptor in the adrenal cortex with or without reduction of circulating androgen levels by castration. Our results describe AR expression in the human and mouse adrenal and highlight that the mouse is a viable model to investigate androgen signalling in the adrenal cortex. We show androgen signalling via AR is required for X-zone regression during puberty. Furthermore, cortex measurements define differences in X-zone morphology depending on whether circulating androgens or AR have been removed. We show androgens promote both cortical cell differentiation and apoptosis but are dispensable for the formation of the definitive cortex. Additionally, investigation of aged mice with AR ablation reveals severe cortex disruption, spindle cell hyperplasia and X-zone expansion. The data described herein demonstrates AR-signalling is required to facilitate X-zone regression, cell clearance and to protect against adrenal degeneration during ageing. A decline in circulating androgens has been associated with co-morbidities such as obesity2 cardiac disease3 and metabolic syndrome4. Androgen production in men occurs predominantly in the testis (~90%) with a small proportion produced in the zona reticularis (ZR) of the adrenal cortex6. Androgens primarily exert their effects through their cognate androgen receptor (AR), a nuclear receptor to testosterone and dihydrotestosterone. Upon binding, AR undergoes translocation into the nucleus to regulate gene transcription7. Androgens and AR have also been shown to act independently of each other to regulate cellular processes8.Androgens are essential regulators of male health, primarily in the maintenance and development of male sexual characteristics9. However, whilst AR is abundantly expressed in the adrenal cortex of both rodents11, and humans13, surprisingly little is known about androgen action on the adrenal cortex itself. This gap in our understanding is at least in part due to the perceived lack of suitable animal models. Mice have largely been overlooked as a model system for the human adrenal as mouse adrenals are unable to produce androgens due to the silencing of Cyp17a1 early in development. This lack of expression results in no 17α hydroxylase and 17, 20 lyse activity and consequently the rodent adrenal does not have a zona reticularis15. The fetal adrenal is thought to give rise to the adult adrenal cortex in both humans and mice17, so understanding the mechanisms that underpin its regulation is essential. In the mouse, these fetal cells are maintained for a period postnatally and regress in a sex dependent manner. In the human, this zone is known as the ‘fetal zone’, with the mouse homologue termed the ‘X-zone’19. Historical studies using castrated mice show that removal of circulating androgens leads to the redevelopment of an additional cortex zone known as the transient X-zone20. The redevelopment of the X-zone following castration and the abundant expression of AR in the mouse adrenal suggests that androgens are important regulators of the adrenal cortex and that despite not producing adrenal androgens, the mouse has utility for dissecting the role of androgen signalling within the adrenal. Previous global knockouts have ablated AR from the adrenal21, however, the caveats of this, like many global knockout models, is that the adrenals both produce and respond to multiple endocrine stimuli. For these reasons, it is difficult to attribute any phenotype to perturbed endocrine signalling locally rather than disruption of the wider hypothalamic-pituitary-axis.Previous research has focussed upon the body-wide impact of adrenal androgens22, permitting investigation of adrenal AR signalling. Due to the ability of AR and androgens to act independently of each other, additional models of castrated wild-type and castrated AR-ablated animals were investigated in parallel with the Ad-ARKO model, allowing us to independently dissect both androgen and AR action in the adrenal. We show that androgen signalling via the androgen receptor required for X-zone regression, furthermore, that androgens and androgen receptor also work independently to regulate multiple aspects of adrenal cortex function and morphology.For these reasons, we generated an adrenal-specific androgen receptor knockout model (Ad-ARKO) that ablates AR from the adrenal cortex in fetal and adult life23, however AR localisation in the adrenal cortex in human and mice has yet to be defined. To establish AR location within the adrenal cortex, human and mouse adrenals were examined using immunohistochemistry. Expression of human AR can be seen in the definitive cortex in fetal adrenals and in the zona reticularis of the adult adrenal. Unexpectedly, despite AR being a nuclear receptor, localisation of AR in human tissue is observed in the cytoplasm  show no differences in Ad-ARKO mice when compared to controls , an X-zone specific marker28, at d12, d21 and d35 shows that the X-zone in Ad-ARKO mice is more advanced in developmental stage than controls before, during and after puberty  C57BL/6J (Bl6) ‘BL6 castrated’, and ‘Ad-ARKO castrated’, cohorts in our study to determine receptor and ligand-independent effects on adrenal regulation in young adult mice (d80). These additional cohorts also show a significant increase in adrenal weight compared to controls . Ablation or mutations in Sf1 results in adrenal agenesis, and adrenal failure in Dax1 ablation models, so determining if these are impacted following loss of androgen signalling is essential35. Furthermore a recent study has shown ablation of Prkar1a in aged mice results in the maintenance of an X-zone in adulthood in males and following a pregnancy in females36. Due to a similar phenotype being observed in Ad-ARKO mice, we investigated transcript levels of Sf1, Dax1 and Prkar1a. Transcript analysis of Sf1 shows a significant increase in all experimental cohorts when compared with controls at d80  and d80 Fig. . Dax1, sice Fig. . Resultsols Fig. . In addiDue to failed regression of X-zone cells from the cortex during puberty, we investigated whether there is any disruption to normal adrenal cell apoptosis via immunostaining of cleaved caspase 3. Immunostaining of cleaved caspase 3 shows that in control mice, cortical cells undergo apoptosis at the cortex-medulla boundary, conversely, BL6 castrated and Ad-ARKO mice show no cleaved caspase-positive cells. However, Ad-ARKO castrated mice show aberrant apoptosis throughout the cortex Fig. . However37, adrenal Mc2r expression38 and circulating pituitary ACTH concentrations39. Corticosterone analysis reveals no changes in any cohort compared with controls  reveals differential staining patterns in the X-zones of all experimental cohorts. BL6 castrated mice have 3β-HSD positive X-zones, suggesting these cells have come from a definitive cortex lineage. Ad-ARKO mice have sporadic 3β-HSD cells in their X-zone, however, following castration, no positive cells can be seen in the X-zone. This suggests that some cells in the X-zone of Ad-ARKO mice are steroidogenic and lose this capability following removal of androgens Fig. . This coPrkar1,  is essential for adrenal formation, confirming that understanding the mechanisms that control the X-zone is essential to understanding adrenal regulation. Furthermore, they hypothesised that there is still an unidentified adult adrenal enhancer that activates during a key developmental stage to switch development from fetal to adult cells. The challenge surrounding understanding X-zone control is undoubtedly due to a number of genes identified as potential regulators. A study conducted by Sahut-Bernola et al. showed that the X-zone remains when Prkar1a is ablated from the adrenal cortex36. Consistent with their model, we note that Ad-ARKO mice also have X-zone maintenance that expands through the cortex with age and development of lesions in the outer cortex. However, we do not see the development of Cushing’s syndrome nor the switching on of Cyp17a1 in the adrenal cortex. Interestingly, we do note a significant downregulation in the transcript of Prkar1a. This suggests a relationship between AR and Protein Kinase A (PKA)43. The same group has recently extended these studies to look at castration and DHT treatment in their Prkar1a ablation models and found that androgen signalling is able to inhibit the PKA pathway via the RSPO/WNT/β-catenin pathway in the adrenal44.The current paradigm regarding X-zone regression suggests that the surge in testosterone at puberty drives X-zone involutionDax1 in the adrenal cortex also results in maintenance of the X-zone34. It was for this reason that Dax1 was thought to control the switch between fetal and adult cells. With a similar phenotype being observed in Ad-ARKO mice, demonstrates that the switch from adult to fetal cells is a complex mechanism and that Dax1 could be involved in the same pathway as androgens and PKA to drive adult cortex cell differentiation and X-zone regression. Additionally, Dax1 ablation results in eventual adrenal failure as the pool of adrenocortical progenitor cells cannot be replenished. This is not something that is observed in our Ad-ARKO or castration models suggesting that AR does not regulate definitive cortex differentiation. However, unlike the Dax1 ablation model, our Ad-AKRO model does not target AR in the adrenal capsule22, which is essential for coordination of ZG to ZF transition and maintaining the pool of progenitor cells46, so the authors cannot comment on the rolf of AR in this cell population.Ablation of Sf1 transcript in all experimental cohorts was also observed. It has previously been shown that androgens act as a repressor of Sf1 (52), an important mechanism to regulate Sf1 and its target genes (52). Sustained elevated expression of Sf1 has been implicated in conditions such as childhood adrenocortical tumours (53). Whilst it is speculation at this stage, our data imply that perturbed androgen signalling could be a contributory factor in Sf1-associated adrenal disease, this requires further investigation. To determine the exact nature of the relationship between these signalling pathways, it would be pertinent to look at the androgen response elements in the promoter regions of these genes to determine if AR is directly upstream or if the changes observed are an off-target effect caused by general disruption to the adrenal cortex. The genes investigated in this study following AR ablation are by no means an exhaustive list of known cortex and X-zone regulators. We have in the first instance, focused on a sub set of key regulators, however, following the results of this study and demonstrating the importance of AR in the adrenal, it would be essential to investigate other known regulators. There is avenue for future research into the role of AR and their interactions with TRβ147 shown to be important in X-zone maintenance but not development, and activin, shown to promote X-zone apoptosis49. Further study would also seek to examine cohorts castrated at additional time points. This study examined absence of circulating androgens from the point of castration onwards in acute (14 days) and chronic (6 months) androgen loss, post puberty. A potential caveat of this is that there is still the potential for stimuli from steroid hormones. This has been shown to occur through memorized histone modification and DNA methylation51. This may be particularly pertinent for long terms castrated mice that will have had circulating androgens for 6 months. Future work would seek to perform castration before the onset of puberty to determine is this could be a potential confounder.A significant upregulation in 48. Protein localisation of Cleaved caspase 3 showed no apoptotic cells in BL/6 castrated or Ad-ARKO mice, suggesting androgen signalling is important for cell clearance from the adrenal through apoptosis. This phenotype has been documented previously in a global AR knockout model, however, this was thought to be a result of the loss of AR signalling from the pituitary21, our results show that this is a within-adrenal role for androgen-signalling.The maintenance of the X-zone in adulthood in Ad-ARKO mice brings into question cell clearance from the adrenal cortex. Under normal conditions adrenocortical cells migrate towards the cortex-medulla boundary and undergo apoptosiset al. described an increase in severity in adrenal cortex disruption with age in Prkar1a knockout mice, showing X-zone expansion and the development of primary pigmented adrenocortical disease (PPNAD) with age36. Due to the overlapping phenotype, we hypothesised that the severity of the phenotype in our androgen manipulation models would progress with age. Again, we noted a disparity between our models depending on whether AR or androgens have been targeted. Both castrated cohorts show no major adverse phenotypes, there is no increase in corticosterone, cortex disruption or X-zone expansion. X-zone measurements show X-zone regression, phenocopying what is observed in an ageing female, this phenotype is well described in the literature54. Conversely, the Ad-ARKO shows X-zone expansion and spindle cell hyperplasia. This result is of particular interest as it demonstrates that AR but not androgens are needed to protect against age-related degeneration of the cortex. Furthermore, that androgens are potentially still able to stimulate the adrenal in Ad-ARKO mice and without the presence of AR to bind to, could result in the aberrant stimulation of off target pathways. AKR1B7 and 3βHSD staining and X-zone measurements taken in our d80 cohorts further strengthen this. Measurements of the X-zone demonstrated significant morphological and size differenced depending on whether, androgens or AR has been targeted. Furthermore, AKR1B7 localisation is still observed in Ad-ARKO mice compared to castrated cohorts with no AKR1B7 expression. Additionally, 3βHSD showed localisation in Ad-ARKO adrenals but not in either castrated cohorts. This suggests that again, without the presence of AR to bind to, there is activation of off target signalling pathways in the adrenal cortex that requires further investigation.Sahut-Bernola The presence of AR in both human and mouse adrenal suggests that the mouse could provide a suitable model to investigate human conditions, however, due to the differences in localisation of adrenal AR in the human and rodent this could potentially suggest that although present in both, could have varying roles. This would need to be taken into consideration in future studies regarding adrenal androgen signalling and would be an avenue for future research.In conclusion, this study not only highlights many new roles for androgen signalling in the adrenal but shows that the mouse has utility as a model to explore androgen action within the adrenal, opening up a novel entry point towards further increasing our understanding of adrenal function. Through cortex measurements, we demonstrate androgens and AR can act independently of each other and show that AR in mouse and human display similar spatiotemporal expression. Furthermore, we demonstrate that androgens act via AR to regress the X-zone and influence both cortical cell differentiation and apoptosis. We highlight that AR is potentially upstream of key adrenal-developmental genes but is dispensable in the formation of the definitive cortex. Finally, we demonstrate that androgens acting via AR protects against age-related degeneration of the adrenal cortex, with implications for our understanding in preventing adrenal degeneration and tumour development.In Vivo Experiments) guidelines55. Ethical approval was obtained for the use of archived human fetal adrenal tissue from the East Scotland Research Ethics Committee (Reference Number: LREC08/S1101/1). Ethical approval was obtained for the use of archived human adult adrenal tissue from the Danish regional ethics committee (Reference Number: H-1-2012-007).All mice used in experiments were under a strict standard of care and experimental planning covered by licensed approval from the UK Home Office (License Number 80/7704) and adhere to the ARRIVE . An amplicon of 102 bp indicated the inheritance of the Cre recombinase transgene.Mice were genotyped for the inheritance of Cre recombinase as previously described2 until unconscious, followed by cervical dislocation. These animals were termed ‘BL6 castrated’ and ‘Ad-ARKO castrated’.Isoflurane was administered via inhalation. A single 1 cm incision was made into the scrotum and testes exposed and removed. Following removal of testes, the site of incision was closed with sterile sutures. Mice were injected subcutaneously with Buprenorphine 0.05 mg/kg, whilst anaesthetized, and allowed to recover whilst being monitored. Mice were closely monitored over 24 hours for any welfare problems, and twice daily from then onwards. At the end of the experiment, mice were culled by inhalation of COTwo castration time points are used in this study, short term and long term. Short-term castrations were performed on postnatal day 64, allowed to age for 14 days and collected at postnatal day 80. Long-term castrations were performed on 6-month-old mice, left to age another 6 months and collected at 12 months old.Body weight was measured, and adrenals were removed and weighed. Reproductive organs were also examined, removed and weighed. Tissues were fixed in Bouin’s fixative  for 4 hours . Bouin’s-fixed tissues were processed and embedded in paraffin wax, and 5 µm sections were used for histological analysis. Sections of adrenals were stained with haematoxylin and eosin using standard protocols and examined for histological abnormalities.58 a single or double antibody tyramide fluorescent immunostaining method, as described previously59, or automated Bond immunostaining method, as described previously58. Exceptions to this protocol are the H2O2 concentration washes performed at 3% H2O2 TBS. Antibodies used are listed in Table Immunolocalization was performed either by a single antibody colourimetric (DAB) immunostaining method, as described previouslyActb .RNA was obtained from frozen adrenals from n = 8 d80 BL6 castrated, n = 8 Ad-ARKO, n = 8 Ad-ARKO castrated and n = 8 control mice, using the RNeasy Mini extraction kit with RNase-free DNase on the column digestion kit  according to the manufacturer’s protocol. RNA yield was quantified using a NanoDrop 1000 spectrophotometer . Random hexamer primed cDNA was prepared using the SuperScript VILO cDNA synthesis kit (Life Technologies) according to manufacturers’ protocols. Quantitative PCR was performed on d80 BL6 castrated, Ad-ARKO, Ad-ARKO castrated and control adrenals for the genes of interest listed in Table Immediately after culling, blood was collected from mice via cardiac puncture with a syringe and needle, blood was collected in EDTA coated tubes to prevent coagulation. Mice were collected at 10:00 am following minimal handling. Handling, culling and blood collection was performed in under two minutes. Plasma was separated by centrifugation and stored at −80.Corticosterone in 12 m aged mice was measured using a mouse corticosterone ELISA kit (KO14-H5) according to manufacturer’s instructions. ACTH was measured using a mouse ACTH ELISA kit (ABIN415571) according to manufacturer’s instructions. All samples were run as a single assay for each hormone.60, without modifications. However, since the steroid concentration in several of the mouse samples were higher than the normal analytical range for steroids in human serum, the linearity of calibration curves was investigated by preparing calibration materials in synthetic serum expanded in the high concentration area by including a total of 23 different dilutions of the standard stock solution. The linear range of calibration curves for regression analysis are shown in Supplementary Tables 60, the linear range for each individual calibration curves, and the inter-day validation. All chemical analyses were performed at the Dept. of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital.Serum analysis of d80 corticosterone and circulating androgens was achieved through the use of a new and sensitive isotope-dilution TurboFlow-LC-MS/MS method. Using this method, we are able to quantify androstenedione, testosterone, 17α-hydroxyprogesterone (17-OHP) progesterone, and corticosterone in human and mouse serum as previously describedComparison of adrenal cortex zones was performed by serially sectioning the whole adrenal at 5 µm, which produced on average 50 slides, per one adrenal, with one section per slide. To account for the shape of the adrenal and allow for consistent measurements, counts were performed on slides 15, 25 and 35 in Ad-ARKO and BL6 castrated animals. X-Zone depth was measured from the medulla boundary to the outer edge of the X-zone, with 5 measurements per slide, across sections 15, 25 and 35 in 5 castrated, 5 Ad-ARKO and 5 Ad-ARKO castrated samples. X-zone depth was measured by placing a 5 × 5 square grid over an ×40 magnification image of the X-zone. Five 2000 µm squares were chosen at random and cells within each box were counted across sections 15, 25 and 35 in 5 castrated, 5 Ad-ARKO and 5 Ad-ARKO castrated samples. Zona Fasciculata/Zona Glomerulosa (ZF/ZG) depth was measured from the X-zone boundary to the capsule, with 5 measurements per slide, across sections 15, 25 and 35 in 5 castrated, 5 Ad-ARKO and 5 Ad-ARKO castrated samples. Overall capsule depth was measured from the medulla boundary to the capsule, with 5 measurements per slide, across sections 15, 25 and 35 in 5 castrated, 5 Ad-ARKO and 5 Ad-ARKO castrated samples. All individual counts can be seen in Supplementary Fig. Statistical analysis was performed using GraphPad Prism  using a two-tailed unpaired t-test (if comparing two groups), a one-way ANOVA with Tukey’s post-hoc test (if comparing multiple groups groups), or a two-way ANOVA with Tukey’s post-hoc test (if comparing multiple groups and variables) Values are expressed as means ± S.E.M.Sup Figures 1-4 and Sup Tables 1-3


Regarding the individual professional, knowledge about available SSR-templates was lacking , and only 44% (n = 94) of the respondents agreed that using SSR facilitates the most accurate diagnosis. Related to social setting, support from the multidisciplinary team members was lacking . At organization level, SSR leads to extra work  because of its incompatibility with other information systems . Main facilitators of SSR were incorporation of speech recognition  and improvement in communication during multidisciplinary team meetings . Both barriers and facilitators existed in various domains. These factors can be used to develop implementation tools to encourage SSR usage.Standardized structured reporting (SSR) enables high-quality pathology reporting, but implementing SSR is slow. The objective of this study is to identify both barriers and facilitators that pathologists encounter in SSR, in order to develop tailored implementation tools to increase SSR usage. We used a mixed method design: a focus group interview helped to identify barriers and facilitators in SSR. The findings were classified into the following domains: innovation, individual professional, social setting, organization, and economic and political context. We used a web-based survey among Dutch pathologists to quantify the findings. Ten pathologists participated in the focus group interview, and 97 pathologists completed the survey. The results of both showed that pathologists perceive barriers related to SSR itself. Particularly its incompatibility caused lack of nuance  contains supplementary material, which is available to authorized users. Discussion of each patient in multidisciplinary team (MDT) meetings is fundamental to optimal treatment decisions . The comThe use of standardized structured reporting (SSR) in pathology instead of the traditional narrative, free-text reporting (NR) improves exchange of diagnostic and treatment information . As a coThe use of SSR in pathology varies widely across laboratories and tumor types –23. In tThe adoption of innovations such as SSR is laborious, particularly when innovation requires people to change their daily habits . OfferinWe used a mixed method design. We used a qualitative focus group interview to identify barriers and facilitators that Dutch pathologists encountered in SSR. We used a web-based survey to quantify the findings: to identify possible determinants and to assess the importance of the barriers and facilitators.PALGA, the nationwide network and registry of histo- and cytopathology in the Netherlands, was established in 1971 and achieved nationwide coverage in 1991, which means that all 46 Dutch pathology laboratories are connected to the PALGA network. The PALGA database contains the reports of all pathology tests in the Netherlands. This database is used for supporting patient care, for evaluating and monitoring the population screening programs, and for scientific research purposes . More dePALGA introduced SSR by providing their nationally available SSR-templates . The DutOther important pathology associations are the Dutch Society of Pathology (NVVP) and the Dutch Society of Pathology Residents (LPAV). Both pathologists and residents are members of the NVVP, whereas the LPAV only allows residents. At the time of data collection, 395 pathologists  and 105 One of our researchers (CS) purposively e-mailed 30 pathologists. We aimed to include pathologists from university and non-university hospitals with different lengths of experience, as well as the range of opinion leaders, middle majority, and stragglers regarding SSR. The participating pathologists filled in a short questionnaire consisting of questions about the pathologists’ characteristics, clinical setting, and SSR usage. They also signed informed consent forms for recording the focus group interview and for anonymously analyzing the data obtained.www.palga.nl/nieuws), on the PALGA LinkedIn page, and in the newsletters from the Dutch Society of Pathology Residents and the Dutch Society of Pathology for 4 consecutive weeks. The introduction of the questionnaire informed the respondents about the research question and the anonymous data analysis. Completing the questionnaire took 10 to 15 min.We invited and reminded pathologists to complete the survey via the PALGA network. The PALGA liaisons distributed the survey to their pathologist colleagues. Further, a call to complete the survey was posted on the PALGA webpage  independently extracted barriers and facilitators in SSR-implementation from the transcribed interview. The factors identified were allocated to the five domains. The two researchers discussed any discrepancies until they achieved consensus.α) and component analyses to confirm the reliability of the questionnaire. We turned negative items around to calculate Cronbach’s α and mean scores. We found high Cronbach’s α’s in all domains or sub-domains; credibility (.85), feasibility (.89), compatibility (.76), knowledge and skills (.95), attitude (.75), social setting (.80), and organizational factors (.74). We deemed the questionnaire reliable by these results. We used conventional content analysis to analyze the qualitative comments in the survey [We used IBM SPSS Statistics 24 to analyze the survey results. We included questionnaires with at least 50% of the answers completed . We usede survey .α to check the consistency of the domain and calculate the mean score for each domain. Further, we employed multivariable linear regression analyses to investigate the existence of possible determinants of barriers and facilitators identified [p ≤ 0.20) in the multivariable linear regression analyses.In analyzing possible determinants at domain level, we used Cronbach’s entified . Based oTen pathologists participated in the focus group interview—five men and five women—with an average of 14.9 years of experience (3–30 years). Three pathologists worked in a university hospital and seven in a non-university hospital.The survey yielded 119 responses, of which 97 had at least the minimum number of answers required (82%). Table n = 97) frequently used SSR. On average, 61% of the SSR-templates were used. The main reasons for not using SSR-templates were non-availability for specific tumors, the teaching setting of the laboratory, lack of awareness of available templates, lack of nuances in SSR, and the perceived extra time necessary for SSR. Most respondents  preferred SSR, 18% (n = 93) preferred NR, 4% (n = 93) preferred a local template, and 10% (n = 93) had no opinion. Furthermore, 28% (n = 97) lacked knowledge about the available SSR-templates. Most pathologists received information about updates of SSR-templates via their PALGA liaison .Of the survey respondents, 82% . However, 82% (n = 97) of the respondents acknowledged the added value of SSR, since it facilitates a uniform  and complete  pathology report.Pathologists mentioned influencing factors related to the credibility, feasibility, and compatibility of SSR Fig. . First, n = 97), for reporting revisions or consultations , and for reporting biopsies . However, SSR-templates are feasible for 90% of the routine diagnostics , including large resections , and, in contrast to the focus group findings, even after neoadjuvant therapy . SSR could be used by all pathologists, but are more attractive to pathologists without a sub-specialization . Furthermore, SSR-templates are supportive tools, either a checklist  or a calculator for cut-off points .Second, feasibility of SSR depended on the type of specimen. SSR-templates are inadequate for the reporting of multiple specimens per patient , lacked nuances in these pathology reports , and showed poor report readability . In addition, the faltering of follow-up questions when clicking was undesirable . However, 83% (n = 94) stated that SSR were compatible with current practices. Specifically, SSR should not include only mandatory items  because it was helpful to be able to add extra information to non-mandatory items . The pathologists also agreed with these mandatory items . If speech recognition was added to the SSR, 54% (n = 94) would use it more often.Third, SSR provided the respondents with difficulties in identifying relevant slides in colleagues’ standardized structured pathology reports  and had sufficient knowledge  and skills  for using SSR. With regard to attitude, only 44% (n = 94) agreed that the use of SSR facilitated the most accurate diagnosis, and 46% (n = 97) agreed that black-and-white conclusions in generalized terms are undesirable. However, all respondents were in favor of guidelines, and 97% (n = 94) were also in favor of SSR and motivated to use them . Uniformity of the pathology report is important .Figure n = 94) of the survey respondents lacked support from MDT-members to use SSR. However, SSR facilitated communication within the multidisciplinary team . The focus group participants mentioned that clinicians receiving the standardized structured pathology report had to become familiar with this new way of reporting and did not universally agree with the SSR format. The survey respondents stated that superiors  and colleagues in the pathology department  encouraged SSR and considered it useful in MDT-meetings .Forty-five percent (n = 93), SSR was considered inappropriate for the education of residents. Both the focus group interview and survey respondents  said that using SSR leads to more work because data must be entered into multiple systems. In addition, SSR was incompatible with other departmental software systems . However, the quality systems of the laboratories did not affect the use of SSR . Sufficient financial resources were available to the laboratories to use SSR , and using SSR also had high priority in the respondents’ pathology departments . Furthermore, PALGA delivered appropriate information about changes after a SSR update and development of new SSR .According to 33% of the respondents (n = 94) reported that a mandatory SSR-template, such as for the national screening program for bowel cancer, is a facilitator.The participants did not name any barriers in the economic and political context during the focus group interview. The respondents , career stage and type of hospital (p = .034 and p = .090), and being a specialist or not and type of hospital (p = .138 and p = .182), respectively. Additionally, determinants for compatibility and attitude were found. These were being a specialist or not (p = .056) and gender (p = .151), respectively. After multivariable analyses (Table p = .034); pathologists’ career stage had an influence on the knowledge and skills of pathologists to work with SSR.The mean scores for all domains and sub-domains were above 2.1   of the respondents represent the Dutch population of pathologists and pathology residents. In addition, the results showed that the respondent preferences were diverse, and even when pathologists preferred SSR, they still mentioned barriers. Moreover, only pathologists were included in this study. The pathology report readers, who are clinicians such as oncologists, surgeons, and radiologists, should be included to obtain a complete overview of the possible influencing factors. These clinicians are part of the MDT that uses the standardized structured pathology reports to take treatment decisions.In conclusion, this study identified barriers to and facilitators for implementing SSR among pathologists in the Netherlands. Implementation tools based on these factors could increase the extent and speed of SSR-implementation. We would recommend investigating barriers to and facilitators for SSR-implementation among clinicians receiving pathology reports such as oncologists, surgeons, and radiologists to further increase the use of SSR.Additional file 1General information about the PALGA foundation (PDF 7 kb)Additional file 2translated from Dutch to English) (PDF 448 kb)Screenshots of the Dutch PALGA SSR-template of breast cancer biopsy (


IntroductionVentricular septal defect (VSD) is one of the more common congenital heart defects, and aortic regurgitation (AR) is its major complication if it remains unrepaired. We aim to determine the AR incidence in various types of VSD, its immediate and intermediate six to 12-month post-VSD repair outcomes of AR.MethodsWe conducted a retrospective review of medical records of all children aged 18 years or younger who were diagnosed with single VSD at our institution from 2016 to 2018. VSD was classified according to its location and relation to the tricuspid annulus and semilunar valve. AR severity grading was done according to the American Society of Echocardiography, and vena contracta width (VC) was taken as the main parameter for severity. We defined trivial-to-mild AR as VC width less than 0.3 cm, moderate AR was 0.3-0.6 cm VC width, and severe AR was VC width of more than 0.6 cm. Immediate and intermediate outcomes of surgical closure, such as residual VSD and AR, were observed.ResultsOne hundred ninety patients with isolated single VSD were included in the study. Of those, 114 patients had perimembranous VSD (60.0%), 64 patients had muscular VSD (33.7%), and 12 patients had supracristal VSD (6.3%). The median age of our study cohort was six months, with a male to female ratio of 1.3:1. Aortic valve prolapse  and AR  were the most common findings on echocardiographic evaluation of VSD patients. Most cases of VSD with AR had trivial-to-mild AR, . AR was most commonly seen in supracristal VSD  followed by perimembranous VSD . VSD closed spontaneously in 34 patients (17.9%) and 98 patients (51.6%) patients underwent surgery. Residual VSD after surgical closure was present in 57.1% (56) and 17.3% (17) of the patients immediate postoperatively and six- to 12-month postoperative follow-up, respectively. Similarly, residual AR after surgical closure of VSD was present in 32.7% (32) and 15.3% (15) of the patients immediate postoperatively and six- to 12-month postoperative follow-up, respectively.ConclusionThe incidence of AR with VSD was very high in our study; AR was most commonly associated with supracristal VSD. After surgical repair, mild AR decreased with time. Early corrective surgery of VSD can prevent this complication and help improve outcomes. Of all congenital heart disease (CHD), ventricular septal defects (VSDs) are the most common form -2. VSDs The natural history of an untreated or undiagnosed VSD that does not spontaneously close is associated with several complications like hemodynamic disturbances, congested cardiac failure, growth failure, right ventricular or left ventricular outflow tract obstruction, aortic valve prolapse (AVP), and subsequent development of aortic regurgitation (AR) -5. AR isThe incidence of CHD in Pakistan is highly underestimated due to the popular practices of home deliveries and quick discharge of neonates from hospital deliveries. VSDs are estimated to be the most common cardiac defects in Pakistan, responsible for 25%-45% of all congenital cardiac defects according to some hospital-based studies -9.VSDs are divided into three types: perimembranous (pm), muscular, and doubly committed subarterial (DCSA)/Supracristal . AlthougRegarding the type of VSDs, the limited data from Pakistan show slightly contrasting results. One study showed DCSA as the least common type of VSD, and another study showed muscular VSDs as the least common; both, however, concluded pm-VSD as the most common . AR is pWe retrospectively reviewed the medical records and echocardiographic findings of all patients aged 18 or younger with a single VSD diagnosis via transthoracic echocardiography at our institution from 2016 to 2018. Approval of the ethical review committee was obtained (ERC-27/2019). The study included children who had at least one presurgical echocardiographic study and two postsurgical studies (within 24 to 48 hours and six to 12 months postoperative). Children were excluded if they had complex congenital heart disease or multiple VSDs or VSD with any other associated CHD and if they died immediately postoperative. The study ultimately included 190 patients. VSDs were classified according to location and relation to the tricuspid annulus and semilunar valve, as described by Sutherland et al. . Aortic AR severity grading was done according to the American Society of Echocardiography, and vena contracta width (VC) was taken as the main parameter for severity. AR was defined as trace-to-mild if VC width was less than 0.3 cm, moderate AR was defined as VC width of 0.3 cm to 0.6 cm, and severe AR was defined as VC width more than 0.6 cm .Statistical analysis was performed by using IBM SPSS Statistics for Windows, Version 21.0. . Results were presented as mean and standard deviation for continuous variables  and frequency and percentage for categorical variables . The chi-square test was applied, and a p-value of ≤0.05 was considered significant.pm-VSD was the most common type (60%) in our study population. AVP was noted in 55 patients (28.9%), while AR was noted in 44 (23.2%) patients. Demographic characteristics of the patients, types of VSD, number of spontaneously closed VSDs, and number of patients who underwent surgery with residual VSD or AR are shown in Table Patients with supracristal VSD had the highest incidence of AR , and patients with muscular VSD had the lowest incidence of AR , as shown in Table Among the patients with preoperative AR, most of the patient had trivial-to-mild AR , as shown in Table AR severity also improved; of the 30 children who had trivial-to-mild AR preoperatively, 23 retained it in the immediate postoperative period, and 10 still had it at the six- to 12-month follow-up. Of the 11 children with moderate AR who needed aortic valve repair, eight children still had AR in the immediate postoperative period, and five had AR at the six- to twelve-month follow-up. All patients with severe AR underwent aortic valve replacement. One of them had severe AR as an immediate complication due to paravalvular leakage for which reoperation was performed. None had severe AR at the 12-month follow-up  patients in our study underwent surgical closure, and 56 (57.1%) had a residual VSD immediately following the procedure. Of those, 39 cases (69.6%) of residual VSD closed spontaneously within one year and remained patent in 17 (30.4%) patients. Bol-Raap et al.  reportedThe reduced AR due to surgical valve repair we noted in our study signifies the beneficial effects of timely surgery on chances of complete resolution of AR. AR resolves or improves in up to 84% of patients postoperatively, and younger patients tend to experience better results ,30.Our study was limited by its retrospective nature, and we did not review the original echocardiogram findings. Our study was also limited in that we used a short follow-up period of one year.While pm-VSD is the most common type of VSD, DCSA/supracristal is most important due to its association with the development of AR. Postoperatively, AR decreases in severity and may completely disappear. Early surgical correction is advisable to prevent the occurrence and progression of AR to reduce morbidity and mortality associated with VSD complications.


Artemisia amygdalina D. The results indicated that leaf explants cultured on solid Murashige and Skoog (MS) media supplemented with 1.0 mg/L α- naphthalene acetic acid (NAA) and 4% sucrose instigated the higher AR induction frequency (90  ±  4.25) and maximum AR biomass (fresh biomass: 17.7 g/L). Furthermore, in the AR when transiently elicited with different elicitors for different time periods, methyl jasmonate (Me-J: 0.5 mg/L) resulted in the higher production of total phenolic content (TPC: 3.6 mg), total flavonoid content (TFC: 2.3 mg) and phenylalanine ammonia-lyase (PAL: 4.8 U/g×FW) activity, respectively. Nonetheless, considerable levels of the major bioactive compounds such as α-thujene (6.8%), α-pinene (8.3%), 1,8-cineole (16.2%), camphor (8.4%) and verbenole (10.2%) were recorded in the Me-J treated AR. Thus, a feasible protocol for production of essential oils through AR in A. amygdalina was established, which can be exploited for commercial production of the industrially important terpenes.The limited production of bioactive essential oils in natural plants does not meet the increasing worldwide market demand. Plant cell culture technology can be used for the higher production of industrially important essential oils. In the present study, a suitable method for production of essential oils was developed through establishment and elicitation of adventitious roots (AR) in a medicinally important plant  Artemisia of family Asteraceae has 500 species worldwide, majority of the plant species are medicinally and economically important [Artemisia amygdalina D. is one of the vital plants, reported for its diverse pharmacological and ethno medicinal uses by people from different communities. It is found across Asia, Europe and North America. In Pakistan, it is found in the subalpine region of Khyber Pakhtunkhwa and Kashmir, where locally it is known as Veer Thethven [A. amygdalina is a 1.5 m tall, stout stemmed leafy, aromatic perennial herb with simple serrate leaves that are hoary tomentose beneath and glabrous green above, flower heads of 2–3 mm, bearing short axillary racemes [A. amygdalina are the terpenes, p-cymene, 1,8-cineole and artemisinin [A. amygdalina plants in their natural habitat have been over-harvested and exploited, not only by the local people but also by different pharmaceutical industries for making different herbal products. Furthermore, deforestation has drastically eroded this plant species in its natural reservoirs and it has been listed in the critically endangered plant species [A. amygdalina through seeds is limited due to the higher mortality rate of seedlings in early stages of growth. Biotechnological methods, especially plant cell culture technology, has provided suitable platforms for the production of healthy plant material and useful secondary metabolites year-round in any season under aseptic growth conditions in a limited space and shorter time [A. amygdalina. Furthermore, notable elicitors such as Me-J, melatonin and PAA were applied in vitro to enhance the secondary metabolite profiles and production of essential oils in AR.Herbal medicines are getting resurgence for their worldwide recognition in the treatment of many diseases, especially in the era of antibiotic resistance, where many commercially available synthetic antibiotics are becoming impotent against several human diseases ,2. The tmportant . AmongstThethven ,7. A. am racemes . The pla racemes ,10. The  racemes ,12. The emisinin . Howeveremisinin . Due to ter time ,15. Adveter time ,17. Althter time . Elicitater time . It is ater time ,21. Seveter time ,22. The Artemisia amygdalina were obtained from the seed bank at the plant cell culture laboratory, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan. Viable and healthy seeds were selected for germination. Surface sterilization of seeds was employed by washing in tap water for 25 min, followed by treatment in 70% ethanol for 5 min and with 0.1% (w/v) solution of mercuric chloride (HgCl2) for 5 min, and finally, the seeds were washed with sterile distilled water five times. Sterilized seeds were placed on filter paper to dry and were subsequently cultured on Murashige and Skoog (MS) [−2 sec−1, 25 ± 1 °C temperature and 70% relative humidity. Young leaf sections (~1.0 cm) were collected from 50 day-old germinated plants and were used for establishment of adventitious roots in the subsequent experiments.Seeds of oog (MS)  medium, w/v) HgCl2 for 5 min and finally rinsed with sterile distilled water three times. For optimization of AR cultures, leaf explants were cultured on MS solid media, supplemented with different auxins such as indole-3-butyric acid (IBA), α-Naphthalene acetic acid (NAA) or indole-3- acetic acid (IAA) at different levels . The media also contained 3.0% sucrose and was solidified with 0.8% agar. The pH of media was maintained at 5.8, following autoclaving for sterilization at 121 °C (105 kPa) for 18 min. For control treatment MS, media containing no auxin was used. All the culture flasks were placed under growth room conditions of 16 h light and 8 h dark photoperiod with light intensity of ~40 μM m−2 sec−1, 25 ± 1 °C temperature and 70% relative humidity. After a four-week culture period, the data were recorded as percentage of AR induction , days to root initiation, number of AR per explant (mean), AR length (cm) and biomass (fresh biomass: g/L).Leaf explants were surface sterilized before inoculation in the culture growth media. Briefly, leaf segments were first treated with sterile distilled water for 5 min, then treated with 0.1%  and augmented with the different concentrations (1–6%) of sucrose. The media composition and the growth conditions were maintained the same as used in the initial AR optimization experiments. Media supplemented with 3% sucrose was considered as control treatment. After a four-week culture period, the data were recorded as percentage of AR induction , days to root initiation, number of AR per explant (mean), AR length (cm) and biomass (fresh biomass: g/L).−2 sec−1, 25 ± 1 °C temperature and 70% relative humidity. The data were recorded as proliferation of AR (%) and AR fresh biomass (g/L) in response to each elicitor treatment.AR grown over solid media containing 1.0 mg/l NAA and 3% sucrose were taken and pretreated with different elicitors for different time periods (transient elicitation). Elicitors used were melatonin (Mel), methyl jasmonate (Me-J) and phenylacetic acid (PAA). The AR were treated with each elicitor at concentration of 0.5, 1.0 or 1.5 mg/L for 30, 60 or 90 min, respectively, and after elicitor treatments, approximately 2 g/L of AR were transferred into liquid MS media containing 1.0 mg/L NAA and 3% sucrose, following incubation on rotary shaker at 110  rpm for a 30-day culture period. The rotary shaker conditions were adjusted at 16 h light and 8 h dark photoperiod with light intensity of ~40 μM mDifferent samples of Adventitious roots (AR) established in vitro in the present study were selected on the basis of maximum growth in the respective culture treatments and were subjected to different assays for phytochemical analysis. AR samples were annotated as NAA-AR (AR grown over solid MS media containing 3% sucrose and 1.0 mg/L NAA), Sucrose-AR (AR grown over solid MS media in the presence of 4% sucrose and 1.0 mg/L NAA), Mel-AR (AR pretreated with melatonin following culturing in liquid MS media containing 3% sucrose and 1.0 mg/L NAA), MeJ-AR (AR pretreated with methyl jasmonate following culturing in liquid MS media containing 3% sucrose and 1.0 mg/L NAA) and PAA (AR pretreated with phenylacetic acid following culturing in liquid MS media containing 3% sucrose and 1.0 mg/L NAA). All the AR samples were compared with the in vitro raised plants (control plant sample) for the estimation of secondary metabolite profiles.For extract preparation, all the selected samples were dried in a hot air oven at 50 °C for two days and were powdered using mortar and pestle. Each sample weighing dried two grams were taken separately in 50 mL and was mixed with 10 mL of 50% methanol , placed on a shaker , sonicated (30 min), vortexed (5 min) and sonicated again (15 min). It was then centrifuged  and the supernatant was syringe filtered and transferred to Eppendorf tubes. The plant extract was diluted to a final concentration of 10 mg/mL for a uniform analysis. The extract solution was stored at 4 °C until used.2CO3 (6 g/mL) was added to all the wells and mixed properly. DMSO (20 μL) was taken as a negative control, while 20 μL (4 mg/mL methanol) of Gallic acid was used as a positive control. The plate was incubated for 90 min at 25 °C. OD was measured at 630 nm using microplate reader .The results were expressed as gallic acid equivalent (GAE) mg/g DW of extracts.The Folin-Ciocalteu assay was employed for the estimation of TPC in the selected samples, according to the method of Velioglu et al. . The ass2O) and aluminum chloride (10% w/v). The final volume of the reaction mixture was raised by addition of 160 μL of distilled water and was adjusted at 200 μL. Following incubation in dark condition for approximately 30 min, the absorbance of reaction mixture was taken at 450 nm on  microplate reader. Quercetin was used as positive and methanol as negative control. The results were expressed as mg quercetin equivalent (QAE) mg/g DW of extracts.Total flavonoid content was determined by using the established protocol of Chang et al. . In a 96−1 phenylalanine and 0.5 mL of sample at pH 8.8. The reaction mixture was incubated at 30 °C and further 0.2 mL HCl was added to stop the reaction. Once the reaction was stopped, after 30 min, absorbance was recorded at 290 nm. The quantity of synthesized trans-cinnamic acid was calculated using its 9630 M−1 cm−1 extinction coefficient. The increase in absorption was reported depending on the quantity of the product produced. In particular, one unit of PAL operation (U) was described as the quantity of absorbance at 0.01 variation. The assay was repeated thrice and the mean value of the ΔA270 nm/min was obtained using the maximum linear rate for both the test sample and blank.The method adopted by Khan et al.  was utilDPPH  based assay was employed for the determination of antioxidant potential in the selected plant samples according to the method of Abbasi et al. . In a reThe free-radical scavenging activity of the sample towards DPPH radical was calculated using the equation:−1 and mass scan range at 30–600 a.m.u. Finally, the mass spectrum profiles of the VOCs obtained for each plant sample were compared with spectra of several standard reference compounds in the plantcyc library (http://pmn.plantcyc.org/). Comparison of the retention indices (RI) of the VOCs in the mass spectral chromatograms of the samples with standards selectively identified the different metabolites in each sample [The selected plant samples were subjected to hydro-distillation in a Clevenger type apparatus for extraction of essential oils. For this purpose, fresh biomass (∼15 g) from the cultures of each sample was added with 300 mL of distilled water in a round-bottom flask. After three hours of distillation, anhydrous sodium sulfate was added to the mixture to initiate the drying of volatile compounds. For this purpose, anhydrous sodium sulfate was used as desiccation agent. Essential oil in each plant sample was calculated on the basis of accumulation of dry matter as mL/100 g × dry matter. The extracted samples were kept in air tight vials under dark at 4 °C until further processing. GC-MS apparatus was used for the analysis of volatile organic compounds (VOCs) in the selected samples. A dimethylpolysiloxane-coated capillary column  was attached to the Shimadzu GC–MS-QP 2020 system. The n-hexane extract (0.2 μL) was injected to the apparatus in split ratio (1:5) with helium as a carrier gas at the rate of 1 mL/min. The mass spectrometer (MS) was configured with different operating conditions such as injection port temperature at 280 °C, column temperature at 50 °C for 2 min followed by an increase to 300 °C at a rate of 3 °C/min, a detector with a temperature of 310 °C, with an ion source 280 °C, ionization energy of 70 eV, solvent delay at 8.0 min, EV voltage of 3000 V, scan speed with 2000 ush sample .P = 0.05).The data were statistically analyzed by one way analysis of variance (ANOVA) test using the statistical software SPSS 16.0 and mean values in each data set were compared using Duncan’s multiple range test procedure  sucrose and incubated for a culture period of four weeks. The results indicated that among all the auxins tested in vitro, the highest growth parameters  were recorded in the explants cultured in the presence of 1.0 mg/L NAA  and growth parameters (mean number of AR per explant: 16.6 and AR fresh biomass: 25.2 g/L) in the leaf explants of A. scoparia grown over solid MS media [Artemisia vulgaris cultured on MS solid media supplemented with1 4.9 μM IBA in combination with 1.4 μM IAA [The primarily objectives of the current research work were to optimize a feasible protocol for the establishment of adventitious roots (AR) and production of bioactive metabolites in the medicinally important and critically endangered plant mg/L NAA . It is aMS media . Moderat4 μM IAA .L. pumila, at 5 mg/L of IBA [In another study, the highest AR induction frequency (72.4 ± 9.3%) and biomass production were recorded in leaf explants of L of IBA . In thisOrthosiphon stamineus [Contrarily, IAA addition to MS media at 3 mg/L, resulted in the highest AR induction frequency (88.3%) and biomass production (0.060 g DW) in leaf explants of tamineus . Auxins tamineus . All thetamineus ,37.A. amygdalina, which prevented the in vitro contamination. Moreover, the stringent sterilization protocol that we have developed for the surface sterilization of explants in a variety of medicinal plants those we have cultivated in vitro in our research laboratory also promoted the aseptic AR growth in present study. Contamination during in vitro cultures is troublesome and can be a direct threat to the plant cellular growth, resources and time in optimization of any micro propagation protocol for any medicinally important plants. The source of explants is a critical factor in controlling any sort of in vitro contamination in culture flasks. Explants taken from the plant germplasm raised in vitro under aseptic growth conditions can reduce significantly the level of contamination during in vitro plant cell growth as compared to the explants taken from the wild grown plants [In the present study, the rate of necrosis and contamination in the explants forming AR was negligible . This is because the leaf explants used for establishment of AR were taken from the in vitro raised plantlets of n plants ,39.w/v) resulted in a prominent declination in AR induction and biomass formation in leaf explants of Gynura procumbens. However, optimal AR growth (fresh biomass: 13.8 ± 1.60 g) was observed in the media augmented with 2% w/v sucrose [w/v sucrose induced the maximum root biomass (fresh weight: 121.71 g/L) in the adventitious roots of Hypericum perforatum L [Sucrose is one of the important carbohydrates and act as carbon source to fulfill the energy requirements of plant cells during in vitro growth. Sucrose has an important function in AR induction and development through the instant availability of its hydrolytic products such as glucose and fructose in the culture media. The later monosaccharides can easily be transported to the plant cell mainly by active transport and partially by passive transport, where they influence root growth and development . During  sucrose . In anotoratum L . SucroseA. amygdalina grown over solid media containing 1.0 mg/L NAA and 3% sucrose were taken and were transiently elicited (pre-treatment) with different elicitors for different time periods; after that, approximately 2 g/L of AR pretreated with each elicitor were transferred into liquid MS media containing 1.0 mg/L NAA and 3% sucrose, following incubation on rotary shaker for a 30-day culture period.Shake flasks cell culture using liquid media facilitate the efficient bioprocessing of plant tissues. The prospects of bioprocess technology include production of biomass and enhancement of medicinally active metabolites; those may not be found in the counterpart natural plants. Moreover this technology provides a suitable model system to study and understand the plant cell cycle, effects of plant growth regulators or elicitors on the growth, physiological responses and secondary metabolism of plant cell during in vitro growth ,31,43. IA. amygdalina; however, all the elicitors employed in the current study have been well explored for their effects on growth in different plant cell culture studies in a variety of medicinal plants [A. scoparia; a medicinally important Artemisia plant species [A. bracteosa and S. rebaudiana [Ajuga bracteosa [The data in l plants ,31,44. Ul plants ,44. As e species . Contrarracteosa ,31.Stevia rebaudiana, when leaf explants were pre-treated with 0.5mg/L Me-J for 45 min and 1.0mg/L PAA for 15 min, respectively. Moreover, callus explants of Fagonia indica when treated with 0.5 mg/L Me-J and with 1.0 mg/L PAA for 2 h, maximum, resulted in higher AR induction frequencies of 88% and 80% respectively, in the cultures [In context of time period and concentration of each elicitor tested, different responses in AR growth were observed, for instance, AR pretreatment with Mel at 0.5 mg/L for 60 min, at 1.0 mg/L for 30 min and at 1.5 mg/L for 30 min, which resulted in higher growth response and AR biomass formation a,b. Furtcultures . In thiscultures ,30,31. FCompared with data on AR biomass formation on solid media, no significant increase in biomass was observed in AR suspension cultures, established in response to pretreatment of AR with elicitors. On liquid media, maximum AR biomass (18 g/L) was observed at PAA (1.0 mg/L) treated cultures; this was followed by Mel, producing 17 g/L of fresh AR, when treated at 1.5 mg/L. Lower values in biomass formation were observed for Me-J treated cultures b. LiquidArtemisia species are well known to harbor sufficient amount of antioxidant metabolites including phenolic acids and flavonoids. The wild grown A. amygdalina has been profiled for the presence of phenolic and flavonoid compounds; however, production of these medicinally important secondary metabolites through plant in vitro cultures has not been well explored. In this study, the effects of elicitation on production of antioxidant potential in AR samples, harvested from the respective growth treatments, were evaluated through calorimetric tests. The results showed that AR pretreated with elicitors significantly enhanced the production of total phenolic content (TPC), total flavonoid content (TFC) and pheyl alanine ammonia lyase (PAL) activity in the AR grown in liquid media  and TFC (7.6 mg), respectively. No significant differences in the levels of TPC, TFC and PAL activity were observed in the Mel and PAA treated AR samples, respectively. However, AR grown over solid MS media in presence of 4% sucrose and 1.0 mg/L NAA, were found to accumulate higher levels of TPC (2 mg), TFC (1.4 mg) and PAL (3.8U/g × FW) when compared with AR established in the presence of 3% sucrose and 1.0 mg/L NAA on the same media, showing production of TPC (1.5 mg), TFC (1.3 mg) and PAL (3.4 U/g), respectively , while 7% sucrose was optimal for the enhanced production of total flavonoid content (1.45 mg), respectively [Similarly, Ali et al.  studied ectively a. Sucrosectively . It can ectively .A. amygdalina grown under continuous light and dark conditions, respectively [Stevia rebaudiana [Elicitation is one of the most successful strategies used in vitro for the enhancement of the production of plant secondary metabolites in a lot of medicinal plants, in order to meet the higher industry demands for making pharmaceutical products and drugs . Similarectively . In a rebaudiana .In comparison to AR samples, lower values of TPC, TFC and PAL activity were observed in the in vitro raised plant (control sample). It is worth mentioning that liquid media significantly enhanced the production of these important secondary metabolites in AR than the AR established on solid media. The production of TPC and TFC, which basically represent the antioxidant compounds in a plant tissue, was found in correlation to the expression of PAL activity a. This dArtemisia scoparia [o free radical scavenging assay is preferred for its optimal performance and authentic results. Nonetheless, it is a robust, simple, easy and reliable method to test the antioxidant potential of a plant tissue [Interestingly, the antioxidant activity in the present study was observed at almost in a similar fashion in all the plant samples b. All thscoparia . In compt tissue . Antioxit tissue . These ct tissue . The bypt tissue . Upon act tissue . Generalt tissue . In suppt tissue ,31.A. amygdalina. Among all the selected samples, maximum total essential oil accumulation (96%) was observed in Me-J treated AR followed by Mel (79%) and PAA (73%), respectively. However, a moderate level (70%) was observed in the sucrose-induced AR and the lowest total essential oil (53%) was recorded for the in vitro raised plant , α-pinene (8.3%), 1,8-cineole (16.2%), camphor (8.4%) and verbenole (10.2%) in the Me-J treated AR. Accordingly, methyl jasmonate (Me-J) has been reported for its influential role in the maximum accumulation of terpene volatiles in cell suspension cultures of ectively ,55. Cont to Me-J .A. amygdalina, which are important volatiles used in different biological and pharmacological applications including antifungal, antibacterial, anti-inflammatory, antioxidant, anti-stress, anticeptive and analgesic activities [Compared with other cell lines, a higher level of α-terpineol (6.7%) was detected in the PAA treated AR. Furthermore, the maximum production of pinocarvone (8.9%) was recorded in the AR grown over solid media in presence of NAA . Sucrosetivities ,61,62. Btivities .A. amygdalina; a critically endangered medicinal plant. On solid MS media containing 1.0 mg/L NAA and 4% sucrose, higher AR induction frequency and growth were observed in leaf explants. Transient elicitation of AR followed by growth in liquid media resulted in the enhanced production of phenolic compounds and essential oil. Among the different elicitors, Me-J showed modulating effects on plant cell secondary metabolism and biosynthesis of terpenes. The present study suggests a suitable method for the production of industrially important essential oils. It further provides a sustainable method for production of AR and also provides a promising ex-situ conservation method for the protection of this critically endangered plant A. amygdalina. Genomic and transcriptomic studies are further recommended to enhance the essential oils production through AR cultures in A. amygdalina.For the first time, Adventitious roots (AR) were established in


A rector of one university has recently asked me: “I understand the connection between mathematics and physics, but what may be the connection between mathematics and biology?” Indeed, it is a common understanding that there is a relationship between mathematics and physics. After all, many universities offer a double major in mathematics and physics. Why is this so? Is mathematics really so distant from other natural sciences? If not, then why isn't it completely normal to say, for example, a mathematics and biology study program? Why are we separating these two natural sciences? Why are we doing so much to keep biology and biomedical sciences far away from mathematics' love hug? One of the possible answers lies in the dynamics of the development of individual natural sciences, ie, in different timing of their emergence from a set of natural phenomena descriptions. In other words, in the timing of quantification of certain natural sciences. Mathematics is by itself quantitative, whether it is algebra, algebraic topology, fractal geometry, or category theory. Physics became quantitative very early on. In fact, even in antiquity, at the very beginning of the discovery and description of physical phenomena, there was a need for their quantification. Unlike physics, chemistry became quantitative in the 17th century, mostly thanks to the work of Anglo-Irish chemist Robert Boyle. Biology began to quantify a century later. Although some authors consider the work of Fibonacci from the 12th century as the beginning of quantification, it was not until the 18th century, ie, until the works of Daniel Bernoulli, Thomas Malthus, and later Francois Verhulst, that biology as an exact scientific discipline emerged from the collection of descriptions of natural phenomena related to the living world. As Bernoulli and Malthus mainly quantified spreading of disease, they can also be regarded as the initiators of modern epidemiology. However, if someone searched for the interactions between mathematics and biology throughout history, they would find surprisingly many of them. The problems that could not be solved at the level of description drove the development of mathematical tools and theories. Biology has more or less influenced the development of statistical theories, stochastic process theories, dynamic systems theory, nonlinear partial differential and functional equations, topology geometry, etc. On the other hand, mathematical concepts have helped the development of contemporary biology and, consequently, biotechnology and biomedicine. One of the rare mathematical concepts whose name contains a biological component are genetic algorithms (GAs). Similar to how design and creation of many technical inventions  were inspired by nature, GAs were inspired and based on genetic phenomena and natural selection. GAs are inevitable when it comes to contemporary data usage and artificial intelligence, with their main advantage being the ability to solve far more complex problems than those that can be solved by traditional methods. In medicine, the application of GAs is not limited to image processing, but includes very complex use in radiology, oncology, cardiology, endocrinology, gynecology, pediatrics, surgery, pulmonology, infectology, radiotherapy, rehabilitation medicine, orthopedics, neurology and pharmacology – hence in almost every branch of medicine (The basic flow of GA consists of five parts . ThroughGAs usage in medicine can be explained using a very simplified case of therapy optimization. Let us suppose that for the treatment of a patient seven different therapies are available that can be combined and that do not interact with each other. Each therapy causes measurable damage to the organism. The chance of success of each therapy and the harm to the organism are shown in The ways to apply GAs in medicine are constantly being developed, especially in the areas where the use of GA has largely settled, as in the case of ECG interpretation . Further


However, the mechanisms underlying this effect have not been fully elucidated. Because 14‐3‐3ε functions as a molecular chaperone on cell cycle regulation, the interaction between 14‐3‐3ε and the target proteins is worth an in‐depth study. In this study, the use of targeting proteomics identified 352 14‐3‐3ε‐binding proteins in SGC‐7901 cells. Analysis of gene ontology (GO) was performed using PANTHER to annotate the biological processes, protein classes, and pathways of these proteins. In 25 cell cycle‐related proteins, filamin A was reduced following ATPR treatment, and this change was validated by immunoprecipitation. The cell cycle was arrested at the G0/G1 phase following ATPR treatment or filamin A silencing in SGC‐7901 cells. Furthermore, subcellular expression analysis showed that 14‐3‐3ε and filamin A were transferred from the cytoplasm to the nucleus after ATPR treatment. On the other hand, overexpression of 14‐3‐3ε, in SGC‐7901 cells, resulted in an increase in the total cellular level of filamin A and an increase in the subcellular localization of filamin A in the cytoplasm. ATPR treatment of the 14‐3‐3ε overexpression cells decreased the total level of filamin A and redistributed filamin A protein from the cytoplasm to the nucleus. Immunohistochemical analysis showed that the expression levels of 14‐3‐3ε and filamin A in gastric cancer tissues were significantly higher, with a predominant localization in the cytoplasm, compared to the levels in matched tissues. Taken together, our results suggest that ATPR can induce nuclear localization of filamin A by reducing the binding of 14‐3‐3ε and filamin A, which may be the mechanism of ATPR‐induced G0/G1 phase arrest.4‐amino‐2‐trifluoromethyl‐phenyl retinate (ATPR) was able to induce the G According to the World Cancer Report in 2014, there were approximately 952 000 new cases of gastric cancer diagnosed worldwide in 2012, and the mortality was ranked third, immediately following lung cancer and liver cancer.Cell cycle regulation is complex, and various factors participate in the process, in which molecular chaperones play an important role. As molecular chaperones were discovered, aberrant expression of molecular chaperones has been widely reported in cancer tissues and cells.0/G1 phase arrest. With the widespread use in clinical trials, the side effects and adverse reactions of ATRA, such as retinoic acid syndrome, drug resistance, and a high recurrence rate, affected its application in the clinical setting. ATRA has achieved remarkable effects in treatment of hematological malignancies, but for solid tumors (such as gastric cancer), its effect is far from being satisfactory. Therefore, developing safer and more effective drugs has become an urgent need.0/G1 arrest and inhibit proliferation in a variety of tumor cells, including gastric cancer cells.All‐trans retinoic acid (ATRA), a retinoid, is the first‐line drug for the treatment of acute promyelocytic leukemia (APL). The main mechanism of ATRA is through its binding to the receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR), to regulate the expression of the target gene, as well as to induce G0/G1 cycle arrest by reducing the binding of filamin A and 14‐3‐3ε and regulating the subcellular localization of filamin A. IHC analysis in this study shows that 14‐3‐3ε and filamin A proteins are both overexpressed in gastric cancer tissues compared to adjacent nontumor counterparts. Our results implied that downregulating the binding of 14‐3‐3ε to cell cycle‐related targeted proteins might have an antitumor effect.In this study, targeted proteomics was used to find the 14‐3‐3ε‐binding proteins in gastric cancer SGC‐7901 cells, and this process identified 25 cell cycle‐related proteins. In addition, the combination of 14‐3‐3ε and filamin A, an actin cross‐linking protein identified as a CDK1‐binding partner, was reduced after ATPR treatment. Therefore, we found that ATPR might play a role in G22.12. ATPR was synthesized by the School of Pharmacy, Anhui Medical University, with a purity of 99%. ATPR was prepared as a stock solution of 10−2 mol/L in dehydrated alcohol and kept at −20°C. Cells were treated with ATPR  for 48 hours and then collected. The vehicle group  was set up, and the cells were treated under the same conditions. Cell nuclear and cytoplasmic proteins were extracted using nuclear and cytoplasmic extraction reagents .Human gastric adenocarcinoma SGC‐7901 cells  were grown in DMEM containing penicillin and streptomycin (each 100 mg/L) and supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2.2g at 4°C. The supernatant was collected, and the protein concentrations were determined using a BCA Protein Assay Kit  with BSA as the standard.Total proteins were collected by centrifugation for 15 minutes at 14 000 g and washed three times with NP‐40. Finally, the supernatants were discarded. 2 × loading buffer was added to the beads with boiling water for 5 minutes. The obtained samples were subjected to vertical electrophoresis, and the gel was stained with Coomassie Brilliant Blue dye  or immunoblotted.For immunoprecipitation, the supernatants of all groups were first diluted to 2 mg/mL to take each of them to a volume of 2 mL for the next step. The anti‐14‐3‐3ε or antifilamin A antibody  was added to the diluted 2‐mL supernatant of the ATPR group. The 4‐mL supernatant of the vehicle group was divided into two equal parts, adding normal rabbit IgG  and anti‐14‐3‐3ε or antifilamin A antibody to each. The supernatant and antibody were incubated overnight at 4°C and then added to 80 μL Protein A/G‐plus agarose beads (Thermo Fisher Scientific). They were incubated 2 hours at 4°C. The beads were collected by centrifugation for 5 minutes at 200 For Western Blot, equal amounts of protein were separated on 12% SDS‐PAGE and transferred to a 0.45 μm PVDF membrane  followed by blocking in 5% skim milk in TBST at room temperature. The membranes were incubated overnight at 4°C with anti‐14‐3‐3ε , antifilamin A , anti‐GAPDH , and anti‐H3 . The membranes were washed in TBST and incubated with secondary antibody (1:10 000) for 1 hour at room temperature followed by exposure to electrochemiluminescence. Finally, ImageJ was used to measure the protein bands.2.3The protein bands were excised from the one‐dimensional Coomassie blue‐stained polyacrylamide gel. The bands were digested in the gel with an excess of sequencing‐grade trypsin .2.4http://pantherdb.org) classification systems.The raw data files from samples and BSA were analyzed using the SEQUEST  search engine and the Proteome Discoverer  using the human nonredundant peptide database obtained from the UniProt human database . The UniProt accessions of identified proteins were uploaded on PANTHER  and CY3‐conjugated goat anti‐mouse secondary antibody . DAPI  was used to counterstain the nuclei, and cells were visualized with a laser scanning confocal microscope .2.66 TU/mL 14‐3‐3ε gene‐lentiviral particles overnight, and three wells were transduced with empty lentiviral particles as the control.−5 mol/L, and cells were cultured for 48 hours.For overexpression of 14‐3‐3ε, SGC‐7901 cells were maintained in 1 mL of complete medium with 5 mg/mL Polybrene per well and were treated with 3 × 102.76/mL) were spun down and resuspended in PBS. After incubation with ribonuclease and propidium iodide at 37°C for 30 minutes, cells were filtered through a nylon mesh  before analysis on a flow cytometer .Specific procedures refer to the cell cycle kit  instructions. Cells were harvested and washed with PBS twice and then fixed with 70% ethanol for 12 hours. The fixed cells . Twenty paired human gastric cancer samples (GC) and their adjacent nontumorous gastric tissues (NT) were obtained from surgical resection performed at our hospital between March 2017 and June 2017. Patients who underwent primary surgical resection had not received radiotherapy and chemotherapy before surgery. The resected specimens were fixed in 10% formalin for paraffin embedding. Pathological examination was performed on all of the GC and the NT samples.2.9The immunohistochemical procedure was performed according to the general‐purpose SP kit  instructions. Sections were incubated with anti‐14‐3‐3ε or antifilamin A at 4°C overnight, and each section was incubated with secondary antibody at room temperature for 30 minutes. For negative controls, the primary antibody was substituted with PBS. Each experiment was repeated three times, and images were taken using a microscope . Semiquantitative integration method was adopted with modifications from Magara et al2.10t test and one‐way analysis of variance were used.Data presented in images are from one representative experiment. All statistical analyses were carried out using SPSS version 17  and GraphPad Prism (GraphPad Software). All data are shown as the mean ± standard deviation. Each experiment was performed at least in triplicate, and the measurements were performed in three independent experiments. To compare the two groups, Student’s 33.10/G1 phase arrest in gastric cancer cells by downregulating the total expression of 14‐3‐3ε and inducing its nuclear location.In our previous study, ATPR was shown to induce G3.2To fully understand the function of the 14‐3‐3ε‐binding proteins founded in targeted proteomics, the 352 co‐expressed proteins were annotated with GO terms using PANTHER. The biological processes, protein classes, and pathways involved are shown in Figure 3.3In 14‐3‐3ε‐binding proteins, filamin A was chosen as the first to study. To verify the results of proteomics analysis, immunoprecipitation and immunofluorescence double staining were chosen to analyze the binding of filamin A to 14‐3‐3ε. Immunoprecipitation showed a significant binding of filamin A with 14‐3‐3ε (Figure 3.40/G1 phase when it was ATPR‐induced or when filamin A was silenced (Figure 0/G1 phase distribution was not observably changed after ATPR treatment on siRNA‐filamin A cells (Figure To explore the role of filamin A in the cell cycle, we silenced the expression of filamin A in gastric cancer cell SGC‐7901 and then treated them with ATPR. As shown in Figure d Figure B. Notabls Figure B. This i3.50/G1 phase arrest was attenuated when 14‐3‐3ε was overexpressed (Figures To investigate the specific effects of ATPR on the binding of 14‐3‐3ε and filamin A, we analyzed the expression of filamin A in ATPR‐treated gastric cancer cells in the total protein level, cytoplasm, and nuclear fraction, respectively. Subcellular expression analysis showed that filamin A was transferred from the cytoplasm to the nucleus after ATPR treatment and showed a significantly high nucleus to cytoplasm ratio for 14‐3‐3ε and filamin A in the ATPR group, which suggested that ATPR was able to regulate the subcellular localization of filamin A Figure A. Accord Figures C. When 1 Figures D. Our re3.6The expression of 14‐3‐3ε protein was analyzed by immunohistochemical staining in 20 tumor tissues paired with the adjacent normal tissues Figure A. The re4Regulation of protein function requires specific protein‐protein interactions in many ways to achieve precise biochemical and cellular functions. The 14‐3‐3 protein family is a class of molecular chaperone proteins which interact with a wide range of proteins, including transcription factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, apoptosis factors, and tumor suppressors, thus playing key roles in many cellular processes.Researchers have used a variety of technical means to achieve a comprehensive understanding of the 14‐3‐3‐binding protein. Ye et alFilamin A is a well‐known actin cross‐linker protein. It involves multiple cell functions, such as cell signaling, cell migration, and adhesion.0/G1 phase arrest is waiting for further study. Furthermore, other 14‐3‐3ε‐binding proteins identified in our proteomics experiment need to be investigated, and the colocalization of the two target proteins in nuclear fraction should be analyzed so as to support our present results.In fact, filamin A was described as potential 14‐3‐3‐binding proteins in HEK293 cells and the activation of T cells recruited 14‐3‐3 proteins to the cell membrane and cytoskeleton to function in different T‐cell processes.In conclusion, our study analyzed the biological processes, protein classes, and molecular functions of 14‐3‐3ε‐binding proteins in gastric cancer cells before and after ATPR treatment. We found that the binding of 14‐3‐3ε and filamin A was decreased after ATPR treatment. ATPR can regulate the subcellular localization of filamin A by reducing the binding of 14‐3‐3ε and filamin A, which may be the mechanism of ATPR‐induced cell cycle arrest. Therefore, targeting 14‐3‐3ε and its binding proteins could be a promising strategy for cancer treatment.The authors have no conflict of interest. Click here for additional data file. Click here for additional data file.