BRCA1 and BRCA2 (BRCA1/2) genetic testing of tumour tissue is being completed for all newly diagnosed high-grade serous ovarian cancer (HGSOC) in the province of Ontario, Canada. The objective of this study was to measure the psychological impact of tumour genetic testing among individuals with a new diagnosis of HGSOC.Reflex (automatic) BRCA1/2 tumour genetic testing as a component of their care. Eligible individuals were recruited from two oncology centres in Toronto, Canada. One week after disclosure of tumour genetic test results, consenting participants were asked to complete a questionnaire that measured cancer-related distress, dispositional optimism, knowledge of hereditary breast/ovarian cancer, recall of tumour genetic test results, satisfaction, and the psychological impact of receiving tumour genetic test results. The Multidimensional Impact of Cancer Risk Assessment (MICRA) questionnaire was used to measure the psychological impact of tumour genetic testing.Participants had a new diagnosis of HGSOC and received reflex p = 0.02), who received treatment with primary surgery (p = 0.02), and had higher reported cancer-related distress (p < 0.001). Higher dispositional optimism (p < 0.001) and increasing age (p = 0.03) were associated with lower total MICRA scores. Most (83.5%) participants reported being satisfied/highly satisfied with having tumour testing completed; however, 40.8% could not accurately recall their tumor test results.76 individuals completed the study survey; 13 said they did not receive their tumour test results. Of the remaining 63 participants, the average MICRA score was 26.8 (SD = 16.3). Higher total MICRA scores were seen among those with children (BRCA1/2 tumour genetic testing in women newly diagnosed with HGSOC. Increased dispositional optimism provided a protective effect, while increased cancer-related distress increased the psychological impact of tumour genetic testing. Educational resources are needed to help increase patient understanding and recall of tumour results, particularly when tumour genetic testing includes analysis of genes that may have implications for hereditary cancer risk. Additional research is required to better understand the patient experience of reflex tumour genetic testing.This study is the first to assess psychological outcomes following reflex  BRCA1 or BRCA2 genes (herein: BRCA1/2); however, additional ovarian cancer risk genes have also been identified [An estimated 20–30% of high-grade serous ovarian cancers (HGSOC) are attributed to a pathogenic or likely pathogenic variant (PV), colloquially known as ‘mutation’, in a cancer-risk gene –3. PVs centified .For women with an inherited (germline) PV in an ovarian cancer risk gene, there is a 50% chance of passing the risk on to each of their children. While screening for ovarian cancer has not been shown to improve outcomes –8. Thus,BRCA1/2 [BRCA1/2 PVs are somatic and would be missed by standard germline (i.e. blood) genetic testing [BRCA1/2 tumour genetic testing for all newly diagnosed HGOSC patients.Recent advances in the treatment of ovarian cancer, has prompted the widespread use of genetic testing for women with HGSOC. Treatment of ovarian cancer with poly(ADP-ribose) polymerase inhibitors (PARPi) has resulted in improved survival for individuals with a germline or somatic PV in BRCA1/2 , 11. Tum testing –14. In ABRCA1/2 PV is identified via tumour genetic testing [Reflex tumour genetic testing refers to tumour testing that is completed automatically as part of standard pathology review protocols. It can detect both somatic and germline PVs, although the type of PV (germline or somatic) can not be distinguished. Unlike other tumour genetic tests where PV may be unlikely to confer a hereditary cancer risk, additional germline testing is required when a  testing , 16. By  testing , 18.BRCA1/2 represents a major shift from the traditional germline genetic testing model for HGSOC patients, and the potential psychological impact for patients needs to be considered. Traditionally, HGOSC patients access germline genetic testing following a ‘pre-test’ appointment with a genetic counsellor where the benefits, limitations, and possible results of genetic testing are reviewed so an individual can make an informed decision about testing. Numerous alternative models of genetic service delivery have been implemented to increase patient access to germline genetic testing [BRCA1/2 genetic testing among newly diagnosed ovarian cancer patients.Reflex tumour genetic testing of  testing ; ovarian testing –22. Whil testing , these sBRCA1/2 tumour genetic testing reported among newly diagnosed HGSOC patients. Secondary objectives were to 1) identify factors associated with patient reported psychological outcomes; 2) measure the level of hereditary breast/ovarian cancer knowledge among HGSOC patients; and 3) determine the level of satisfaction with reflex BRCA1/2 tumour genetic testing reported among HGSOC patients. By reporting on the psychological impact of BRCA1/2 tumour genetic testing for ovarian cancer in Ontario, we hope to inform the successful implementation of tumour genetic testing at other institutions and for additional tumour types.The primary objective of this study was to measure the psychological impact of reflex A cross-sectional survey study of newly diagnosed HGSOC patients was conducted at two academic centers in Toronto, Ontario. Eligible participants were recruited from the University Health Network (UHN) from December 2019–August 2021 and from Sunnybrook Health Sciences Centre from December 2020 to August 2021.BRCA1/2 tumour genetic testing results reported in the past 3 months. Individuals were excluded if they had already received results of germline genetic testing for hereditary ovarian cancer risk or were unable to complete study surveys due to language barriers or cognitive capacity.Individuals with a new diagnosis of HGSOC were eligible to participate if they had: 1) pathology confirmed diagnosis of HGSOC; and 2) reflex BRCA1/2 tumour genetic testing results. Agreeable patients were approached by the study team to obtain informed consent. Consenting participants indicated their preferred method of completing the study survey . Surveys were issued 1 week after disclosure of tumour genetic testing results; a reminder call or email was sent 1 and 2 weeks after the survey was provided.Eligible participants with an upcoming appointment were flagged for their gynecologic oncologist (one of the nine participating surgeons), who disclosed Relevant clinical data  were abstracted from the medical record. Survey items were used to obtain additional socio-demographic data  as well as participant recall of their tumour genetic testing results.Dispositional optimism was evaluating using the LOT-R, which is a 10-item scale consisting of five-point Likert scale questions to measure dispositional optimism . The sixThe IES was used to measure cancer-related distress. The scale consists of 15 four-point response items to measure subjective distress following a specific event or stressor . In the FACIT.org). Per FACIT instructions, mean imputation was used to account for item-level missing data. Due to an initial error in survey development, one item of the MICRA questionnaire “Being uncertain about what my tumour genetic test result means about my cancer risk” , was missing from the survey of 20 participants. Analyses involving the MICRA questionnaire were run with and without this item and its inclusion did not significantly impact study findings. The total scale had an acceptable internal consistency (α = 0.86); internal consistencies were also calculated for distress (α = 0.85), uncertainty (α = 0.76) and positive experience (α =0.59) subscales. Due to the low internal consistency in our cohort, scores for the positive experience subscale are not reported.The MICRA questionnaire was developed to measure the psychological impact of genetic testing result disclosure . It is cBRCA knowledge scale. Developed by the National Center for Human Genome Research, the BRCA knowledge scale consists of 11 true/false items measuring four aspects of hereditary cancer: the prevalence of BRCA1/2 mutations, the patterns of inheritance, cancer risks associated with BRCA1/2 mutations, and risk management options for women with BRCA1/2 mutations [Participant knowledge of hereditary breast/ovarian cancer was assessed using a modified utations . Four trParticipant reported satisfaction was assessed using a five-point Likert scale question.Descriptive statistics were used to provide a summary of study variables. Variables were reported using means and standard deviations or frequencies and percentages, for continuous and categorical variables, respectively. To address the primary objective of describing the psychological impact of tumour genetic testing, MICRA mean scores, along with standard deviations, were calculated for the total score and two subscales .p values were reported. Participant knowledge and satisfaction were reported using descriptive statistics.Secondary objectives were addressed as follows: Univariable and multivariable analyses were undertaken to determine factors associated with all MICRA scores . MICRA scores were compared across levels of categorical variables using an independent t-test. Linear regression was used to measure the association of continuous variables with MICRA scores. Ordinary Least Square regression was used to undertake multivariable analysis, where included variables , age at diagnosis, having children (yes/no), positive tumour genetic testing results (yes/no), and cancer related distress  were selected a priori based on literature review and consultation with clinicians. Primary treatment (surgery/neoadjuvant chemotherapy) was added to the multivariable models post-hoc given its highly significant association with multiple MICRA outcomes. Hierarchial regression was completed where cancer-related distress was included in a second regression block. A minimum of 60 participants were required for analysis as 10 participants per predictor variable is considered appropriate for regression models with more than six predictor variables . RegressStatistical analyses were completed using IBM SPSS Statistics for Windows, version 28  and statistical significance was reported using a two-tailed α = 0.05. Given the exploratory nature of this study, statistical corrections for multiple testing were not completed.A total of 112 HGSOC patients met study inclusion criteria and were invited to participate. Of these, 84 (75.0%) consented and76 (80.5%) completed the study survey, for an overall response rate of 67.9%. The mean age at diagnosis was 62 years; most (88.2%) were diagnosed with stage III/IV disease and just over half (56.6%) were treated with primary surgery. Participant demographics are summarized in Table BRCA1/2 was identified in 23 (30.3%) of HGSOC tumours. Thirteen (17.1%) individuals reported that they never received their tumour genetic testing results. Another 16 (21.1%) recalled receiving their tumour results but were unsure what their results were, 7 (43.8%) of whom had a PV in their tumour. Of 47 individuals who recalled a result, 2 (4.2%) reported their results incorrectly (1/13 with positive and 1/34 with negative results).A PV in The mean LOT-R score for the total study cohort was 14.2 (SD = 5.2).n = 75). The mean intrusion and avoidance scores were 11.0 (SD = 7.6) and 15.0 (SD = 8.9), respectively. The mean total IES score was 26.0 (SD = 14.2); 39 (52.0%) individuals scored ≥26, indicating at least a moderate level of cancer-related distress and 9 (12.0%) scored ≥44 indicating a severe level of cancer-related distress.Intrusion, avoidance, and total cancer-related distress scores were calculated for all participants who completed the IES , with subscales scores of 6.0 (SD = 6.8) for distress, and 13.3 (SD = 9.0) for uncertainty. In addition to scored items, the MICRA questionnaire includes 2 unscored items related to coping with cancer and 2 unscored items related to children . Six (9.5%) individuals sometimes/always felt their tumour genetic testing results made it harder to cope with their cancer, and 36/61 (59.0%) sometimes/often felt results made it easier to cope with their cancer. Of 33 individuals who answered questions related to having children, 27 (81.8%) reported they were sometimes/often worried about the possibility of their children getting cancer and 22 (66.7%) sometimes/often felt guilty about the possibility of passing on a cancer risk to their children. Of these 22, 10 individuals had a positive tumour result, 5 of whom accurately reported this information.Univariate analyses of factors associated with subscales and total score of the MICRA Tables  and 3, wp = 0.002) and higher total cancer-related distress (p < 0.001). Significantly lower MICRA distress scores were seen in individuals with a prior diagnosis of breast cancer (p = 0.004) and higher disposition optimism (p = 0.03).Several factors were associated with higher MICRA distress scores, including treatment with primary surgery (p = 0.006), treatment with primary surgery (p = 0.009), and higher cancer-related distress (p < 0.001). Lower uncertainty scores were noted in those with an older age at diagnosis (p < 0.001) and higher dispositional optimism (p = 0.001).Higher MICRA uncertainty scores were seen among individuals with children (p = 0.02), treatment with primary surgery (p = 0.02), and higher cancer-related distress (p < 0.001) were associated with higher total MICRA scores. Older age at diagnosis (p = 0.03) and higher levels of dispositional optimism (p < 0.001) were associated with lower total MICRA scores.Having children (p = 0.04). Higher levels of dispositional optimism (p < 0.001) and increased age at diagnosis (p < 0.001) were associated with lower uncertainty scores, whereas having children was associated with higher uncertainty scores (p = 0.01). As for the overall psychological impact to tumour genetics testing, higher levels of dispositional optimism (p < 0.001) and increased age at diagnosis (p = 0.01) were both associated with lower total MICRA scores. With the addition of cancer-related distress (Block 2), higher cancer-related distress was associated with higher genetic testing-related distress (p = 0.002). Higher levels of dispositional optimism (p = 0.03) and increased age at diagnosis (p = 0.01) remained significantly associated with lower uncertainty scores. Having children (p = 0.04) and increased cancer-related distress (p < 0.001) were associated with higher uncertainty scores. Regarding the overall psychological impact of tumour genetics testing, increased dispositional optimism was associated with lower total MICRA scores (p = 0.03) and increased cancer related distress was associated with higher total MICRA scores (p < 0.001).A summary of multivariable models is presented in Table BRCA knowledge items (65.8% correct) and questions about tumour testing (69.7% correct). Individual items and the percentage of correct responses are presented in Additional file 2.Average knowledge score among our participants was 66.8%. Scores were similar for BRCA1/2 tumour genetic testing completed as part of their cancer care. Among 23 patients with a positive tumour result, 18 (78.2%) reported they were satisfied/highly satisfied.Seventy-three participants ranked their level of satisfaction; 61 (83.5%) reported being satisfied/highly satisfied with having reflex BRCA1/2 genetic testing among HGSOC patients. Partients’ reported level of cancer-related distress had the most significant association with levels of genetic testing-related distress, uncertainty, and the overall psychological impact of tumour genetic testing. Higher optimism scores and increased age at diagnosis reduced the psychological impact of reflex tumour genetic testing in our cohort; however, when controlling for cancer-related distress, these protective effects decreased. Despite ensuring that all patients had tumour results disclosed by their gynecologic oncologist, many could not accurately recall their results; nevertheless, most participants were satisfied with having reflex BRCA1/2 tumour genetic testing completed as part of their cancer care.This is the first study to investigate the psychological impact of reflex BRCA1/2 PV, it is possible that receiving positive BRCA1/2 tumour genetic testing result may be perceived as ‘good’ news. The mean psychological impact of tumour genetic testing among ovarian cancer patients in this study was measured as 26.8 via the MICRA questionnaire. Though there is no cut-off to define a “high” MICRA score, this score is higher than recently published Norwegian (mean score = 17.7) [Previous research suggests that ovarian cancer patients have favourable attitudes towards germline genetic testing –32, and  = 17.7) , 34 and  = 17.7) , 34 coho = 17.7) . While tp < 0.001). Many ovarian cancer patients experience distress, particularly after initial diagnosis [p < 0.001). Individuals with higher dispositional optimism are expected to have a ‘glass half full’ outlook and may focus on the benefits of genetic information, such as identifying targeted therapies. Yet, the average level of dispositional optimisim reported in our cohort  was lower than published scores among ovarian cancer patients [When evaluating the psychological impact of genetic testing, results must be considered in the context of an individual’s personal experience. Baseline distress has been cited as the biggest risk factor for immediate psychological distress following germline genetic testing , and repiagnosis , 38. Ourpatients  and indipatients ; thus, lBRCA1/2 tumour genetic testing in our cohort. Consistent with published literature of germline genetic testing [p = 0.03). This may be due to the increased levels of psychological distress experienced by younger individuals following a diagnosis of ovarian cancer [p = 0.02). Of interest, a personal history of breast cancer was associated with significantly lower genetic testing related distress (p = 0.004) in our cohort. For these individuals, genetic testing may be of minimal concern when facing a second cancer diagnosis; however, only six individuals has a previous diagnosis of breast cancer completed the full study survey and definitive conclusions cannot be made on such small numbers. Finally, treatment with primary surgery, as opposed to neoadjuvant chemotherapy, was associated with a higher psychological impact of tumour genetic testing in our study (p = 0.02). Since reflex BRCA1/2 tumour genetic testing is typically arranged on a surgical specimen, this may be a function of decreased time from diagnosis to receipt of tumour genetic testing results. Individuals who were treated with primary surgery may also be starting adjuvant chemotherapy treatments at the time of result disclosure, which may contribute to increased distress. Additional studies are needed to better interpret these findings.Several additional factors were associated with the psychological impact of reflex  testing , 33, 34,n cancer . YoungerBRCA1/2 PV in their tumour could not correctly recall their genetic testing result, which likely translates to an inaccurate evaluation of the psychological effect of receiving a positive BRCA1/2 tumour genetic testing result.Previous research suggests that positive genetic test results are associated with a higher psychological impact of genetic testing , 34, 41.BRCA1/2 tumour testing in order to better understand and recall their results. Although 40.8% of participants in our study had met with a genetic counsellor prior to completing the study survey, it is unknown whether BRCA1/2 tumour results were available or discussed during their appointment.Overall, a large proportion (40.8%) of HGSOC patients in our study could not accurately recall their tumour genetic testing results. Likewise, in a study of individuals with advanced-stage solid tumour malignancy, 25% could not recall disclosure of their tumour genetic test results, despite documentation that such a discussion had occurred . It is pBRCA1/2 PV variants seen in tumour tissue were always inherited from a parent. In a qualitative study of individuals with advanced cancer who consented to a large tumour genetic testing program in Australia, participants acknowledged their level of knowledge was poor, but stated this did not impact their decision have tumour genetic testing [BRCA1/2 tumour genetic testing completed as part of their cancer care, available data suggest gaps in knowledge about tumour genetic testing may be of minimal concern for cancer patients. Nevertheless, additional education or post-test genetic counselling may be of benefit to ensure that cancer patients understand their results, particularly when testing genes like BRCA1/2 where many PV are germline in orgin and confer a hereditary cancer risk.The difference between tumour and germline genetic testing, including the types of information gleaned, is a complicated concept for many patients to understand. Previous literature states that cancer patients have moderate to poor knowledge of tumour genetic testing and most are unaware of the difference between tumour and germline genetic testing . Most in testing . CombineThe results of this study should be considered in context of its strengths and limitations. The provincial implementation of reflex tumour genetic testing for all HGSOC patients in the province of Ontario allowed us to evaluate the ‘real world’ experience of a diverse cohort of individuals. At the same time, the pragmatic approach of this study introduced variability in the information provided, as tumour genetic testing were disclosed by one of nine gynecologic oncologists. Such variation cannot be accounted for without a much larger sample size. Similarly, a larger sample size would allow for the inclusion of additional potentially relevant variables in our multivariable model and to identify factors which may have small effects on psychological outcomes. Our study is also limited in its cross-sectional design, and future studies, including longitudinal analyses, qualitative studies and direct comparisons of the psychological response to tumour and germline genetic testing, may provide better insight into the patient experience and areas where additional support is required. Using the MICRA questionnaire, which is designed to measure the psychological impact of germline genetic testing, may have given an inaccurate representation of experiences with tumour genetic testing. Additionally, the MICRA positive experience subscale had poor internal consistency in our cohort and we were unable to conduct reliable analyses for this measure. Further evaluation of the MICRA questionnaire in a tumour genetic testing context is required to determine whether the development of new measures is required. Finally, this study was conducted during the global COVID-19 pandemic and results should be interpreted with the knowledge that ovarian cancer patients experienced high levels of distress during this time .BRCA1/2 tumour genetic testing. The psychological impact of reflex tumour genetic testing in our cohort was higher than published data of ovarian cancer patients undergoing germline genetic testing; however, this may be a function of receiving a recent cancer diagnosis. Increased dispositional optimism provided a protective effect, but higher levels of cancer-related distress had the most significant association with psychological outcomes following tumour genetic testing. Knowledge scores and poor recall of tumour genetic testing results suggest that HGSOC patients may benefit from educational resources and/or post-test genetic counselling following receipt of BRCA1/2 tumour genetic test results. Additional research, including qualitative studies, are needed to better understand the patient experience of reflex BRCA1/2 tumour genetic testing.This study provides a first-look into the experience of newly diagnosed HGSOC receiving reflex


Ophiopogon japonicus is widely used as a tonic herb in China. According to the origins, MaiDong of Chinese materia medica can be classified as Zhe MaiDong (Ophiopogon japonicus in Zhejiang), Chuan MaiDong (Ophiopogon japonicus in Sichuan), Duanting Shan MaiDong (Liriope muscari), and Hubei MaiDong (Liriope spicata). In terms of quality control, polysaccharides-based evaluations have not yet been conducted. In this study, microwave-assisted extraction (MAE) was used for the preparation of polysaccharides from 29 batches of MaiDong. HPSEC-MALLS-RID and HPAEC-PAD were employed to investigate their molecular parameters and compositional monosaccharides, respectively. The ability to scavenge ABTS radicals and immune promotion abilities, in terms of nitric oxide releasing and phagocytosis on RAW 264.7 macrophages, were also compared. The results showed that polysaccharides in different MaiDong varied in molecular parameters. All polysaccharides mainly contained fructose and glucose with small amounts of arabinose, mannose, galactose, and xylose. For polysaccharides of Zhe MaiDong and Chuan MaiDong, the molar ratio of Fru to Glc was roughly 15:1 and 14:1, respectively. Zhe MaiDong exhibited better antioxidant and immune promotion activity, and so did that of fibrous roots. The pharmacological activity, however, did not account for the variation in growth years. Finally, indicators for quality control based on multivariate statistical analysis included: yield, antioxidant activity, the content of fructose, and RI signal. It was concluded that MaiDong’s fibrous roots had similar components to the root, and their quality was not significantly affected by growth age. This may provide some guidance for the cultivation and use of MaiDong. Daodi in Chinese) medicinal materials, TCMs with regional characteristics grown in a specific ecological environment, are thought to be of higher quality, according to traditional Chinese medicine theory [The demand for traditional Chinese medicines (TCMs) continues to grow alongside their modernization and globalization. The contradiction between the supply and demand of TCMs could be effectively alleviated by artificial cultivation ,2. Studie theory ,7.Ophiopogon japonicus  is a tonic of the same origin as medicine and food and has a long history of use in China. It is widely used in clinical practice because it has the effect of nourishing the Yin and moistening the lung. Modern pharmacological research has demonstrated the effects of MaiDong, which include anti-oxidation, anti-tumor, anti-inflammatory, and cardiovascular protective properties [Zhe MaiDong) and Sichuan (Chuan MaiDong) provinces are two major authentic geographical origins of O. japonicus that originates in China. Chuan MaiDong can typically be harvested after growing for one year for medicinal purposes; however, Zhe MaiDong must grow for three years before harvest, resulting in greater cultivation expense. Therefore, their quality differences are the major concern in clinics [Zhe MaiDong and Chuan MaiDong, saponins, isoflavones, and other small molecules have been compared [MaiDong from various sources, the total homoisoflavonoid concentration [Zhe MaiDong are usually higher than those of Chuan MaiDong.operties ,10,11,12 clinics . To assentration , antioxintration , and antntration  were evaMaiDong. However, few studies have focused on the difference between Zhe MaiDong and Chuan MaiDong because of their complexity. In this study, physicochemical and pharmacological properties of polysaccharides from 29 batches of Zhe MaiDong and Chuan MaiDong, as well as Duanting Shan MaiDong (Liriope muscari) and Hubei MaiDong (Liriope spicata), were investigated and compared, which could be helpful to improve their quality control.Polysaccharides, with anti-glycemic, immunomodulatory, and gastrointestinal protective effects ,18,19,20O. japonicus, L. muscari (named as Shan MaiDong), and L. spicata (named Hubei MaiDong) are listed in Raw materials of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from International Laboratory . Potassium persulfate was purchased from Fluka . Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), and phosphate-buffered saline (PBS) were purchased from Gibco-Invitrogen . Cell counting kit 8 (CCK8) was purchased from MCE . Griess reagent, fluorescein isothiocyanate-dextran (FITC-Dextran), and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich . An endotoxin detection-specific Limulus test kit was purchased from Bioendo Technology . Trifluoroacetic acid (TFA) was purchased from Fisher Scientific . Nylon membrane filters (0.22/0.45 μm) were purchased from Millipore . Deionized water was prepared using a Millipore MilliQ-Plus system . All the other reagents were of analytical grade.g for 10 min , and the supernatant was collected. The extracted residue was dried above a hot water bath . Then a microwave-assisted extractor  was used to extract polysaccharides from samples. The low molecular weight compounds of crude polysaccharides were removed using ultra centrifugal filters  by centrifugation at 2500× g. After ultrafiltration, the fraction with a molecular weight of more than 1 kDa polysaccharides was obtained and stored in a 4 °C refrigerator for subsequent analysis after freeze-drying.All samples with sticky nature were stored in a −80 °C refrigerator for 24 h, mixed with diatomaceous earth of equal amounts to avoid clogging during grounding, and sieved to a fine powder (250 μm ± 9.9 μm). Each sample (1.0 g) was immersed in 20.0 mL of 80% methanol, then refluxed in a Syncore parallel reactor  for 2 h at 65 °C. Subsequently, the extract solution was centrifuged at 4000 × HPSEC-MALLS-RID was employed to detect the fractions with molecular weight and content of polysaccharides according to our previous report . HPSEC-M+, Dionex, MA, USA) was used for the analysis of compositional monosaccharides according to a previous method with minor modification [HPAEC-PAD , and used for evaluating the effects of polysaccharides. Cells were cultured in DMEM supplemented with 10% FBS and 1% P/S at 37 °C in a humidified atmosphere of 5% CO3 cells/well) were cultured in 96-well microplates overnight, then treated with LPS (0.4 μg/mL) and a series of concentrations of sample polysaccharides for 24 h, respectively. An equal volume of culture medium was used as blank control. Subsequently, the original culture medium was discarded and stained with 100 μL of culture medium containing 10% CCK8 for 2 h in dark. The absorbance values were read at 450 nm, and the cell viability was calculated as the ratio of absorbance values between the sample and vehicle control group.RAW 264.7 cells (5 × 104 cells/well) were seeded in 96-well microplates overnight, and then cells were treated with a series of concentrations of sample polysaccharides and LPS (0.4 μg/mL) for 24 h, respectively. An equal volume of culture medium was used as vehicle control. Subsequently, 75 μL of supernatants were collected and mixed with an equal volume of modified Griess reagent at room temperature for 15 min. The absorbance was measured at 540 nm. NO production was expressed as the ratio of absorbance values between the sample and the LPS-treated group.RAW 264.7 cells (5 × 104 cells/well) were cultured in 96-well plates overnight, and then incubated with culture medium, LPS (0.4 μg/mL), and a series of concentrations of samples polysaccharides for 18 h, respectively. Then, the cells were treated with FITC-dextran (0.1 mg/mL in culture medium) and incubated at 37 °C for an additional 1 h in dark. After incubation, the cells were collected with cold PBS after being washed three times. A BD Accuri™ C6 Cytometer  was used to analyze. The percentage of phagocytosis was expressed as the ratio of phagocytic rate between treatment and control cells.RAW 264.7 cells  was used for Pearson correlation analysis. Origin Pro 2022b  was used for cluster analysis.GraphPad Prism 8.0.2  was used to analyze and process the data. Data were presented as mean ± SEM from at least three independent experiments for each sample. Statistical significance between the experimental groups was determined by Student’s Microwave-assisted extraction parameters were optimized by using sample OJZ7. Single-factor investigation was carried out on liquid-solid ratio, extraction power and time. All sample extracts were made up to 100 mL in volumetric flasks and filtered through a 0.45 μm filter membrane before analysis. HPSEC-MALLS-UV-RID profile of OJZ7 polysaccharides mainly had two peaks, Peak1 and Peak2, based on RI detection d. The efMaiDong had some differences in the RID signal profiles; however, the RID signal profiles were similar when compared within the same source samples. Furthermore, all polysaccharide samples from fibrous roots had higher RID response values at Peak2 than those from the root. The Mw analysis revealed that OJZP, OJCP, and LMP had similar molecular weight distributions, with LSP having a larger molecular weight than LMP. Meanwhile, polysaccharides in OJZ8, OJZ9, and OJZ10 had higher molecular weights than other OJZP. However, polysaccharides in OJC8, extracted from the fibrous root, did not significantly differ in terms of molecular weight from other OJCP. Similarly, growth years had no consistent effect on polysaccharide molecular weight. The molecular weight of the polysaccharides, the RID signal, the laser signal, and the UV signal were all considered for inclusion in the quality control indicator, after combining the above information.All the sample polysaccharides were dissolved in HPSEC mobile phase at 2 mg/mL and filtered through a 0.45 μm filter membrane before analysis. HPSEC-MALLS-UV-RID chromatograms of representative samples are shown in MaiDong have a consistent monosaccharide composition, but with minor differences in content, which was similar to other research [MaiDong are removed for medicinal purposes, but the results of this study suggested that the polysaccharides in the fibrous roots may be complementary to those in the root, to some extent. At the same time, because the growth years did not result in significant changes in monosaccharide composition, it may be possible to shorten the growth years to save cultivation costs.After complete acid hydrolysis, monosaccharide compositions of polysaccharides were determined by HPAEC-PAD. research ,25. NormMaiDong was thought to have antioxidant properties [MaiDong to scavenge ABTS radicals. MaiDong, and their 20% maximal inhibitory concentration (IC20) for ABTS scavenging ability is listed in 20 values of 1.81, 1.87, 1.40, and 2.30 mg/mL, respectively, whereas the IC20 values of the polysaccharides extracted from the root were all greater than 3 mg/mL. The fibrous roots have a substantially lower molar ratio of Fru and Ara in Zhe MaiDong and Chuan MaiDong than that of their tuberous roots, which might contribute to the varied antioxidant activity. In general, the antioxidant capacity of OJZP was comparable to that of OJCP in terms of ABTS scavenging rate and IC20 value, and both were much higher than that of LMP and LSP, with LMP slightly higher than LSP. It is interesting to note that the antioxidant capacity of OJZP did not change consistently over the growth years, according to the results of the ABTS radical scavenging test (MaiDong, the ABTS radical scavenging capacity should be used as an indicator for evaluating the quality of polysaccharides in different origins of MaiDong.operties . Modern operties . ABTS raoperties . In thising test . The ratMacrophages play an indispensable role in the innate and adaptive immunity of the human body . StudiesO. japonicus in Zhejiang with different growth years, all samples had a strong ability to promote NO release except OJZ13. Meanwhile, among the polysaccharides extracted from the root of O. japonicus in Sichuan, L. muscari and L. spicata with one-year-growth, not all samples promoted NO production, and showed no significant difference from the control group at the concentrations of 5, 10, and 20 μg/mL, except for OJC3. OJZ9 and OJC8 were both polysaccharides extracted from fibrous roots, and they were found to be more effective at promoting NO release than polysaccharides from the root. It has been reported that polysaccharides rich in β-1,4-D-Manp may help to activate the immune system by attaching to the mannose receptor on macrophages [We selected a series of sample concentrations to determine the effect of cell viability on the results of subsequent experiments, and finally chose concentrations of 5, 10, and 20 μg/mL. All samples in this series neither significantly promoted RAW 264.7 cell proliferation nor had cytotoxicity; the results are shown in rophages . As a reMaiDong were consistent with the results of NO release, reflecting the following overall trends (MaiDong, and more samples are needed for further investigation; though MaiDong produced in Zhejiang should be collected at the third year. As can be seen in the typical phagocytosis flow charts (The results of phagocytosis experiments of polysaccharides from l trends c. First,w charts , the majw charts b and phaw charts c more efScientific research has revealed that the biological activity of polysaccharides is frequently linked to their structure . Using PPearson correlation analysis is used to reflect the linear correlation of variables, and the Pearson correlation coefficient is a measurement of the linear correlation between two sets of data . A PearsMaiDong are recorded in MaiDong polysaccharides, had a significantly negative relationship to antioxidant and immunological activity of the polysaccharides. Meanwhile, the Mw at Peak1 and UV at Peak2 reflected no correlation with antioxidant activity. For immune activity, the content of Man also had the highest correlation value 0.817, followed by that of Ara at 0.713, while the UV signal at Peak2, and Mw at both Peak1 and Peak2, did not show a correlation with immune activity. The correlation coefficients between the pharmacological activity and physicochemical properties of polysaccharides in MaiDong. In conclusion, seven indicators were chosen for polysaccharide quality assessment: yield, Mw, RI, laser, UV, monosaccharide composition, and ABTS scavenging activity.In addition to the molecular parameters, monosaccharide composition, and pharmacological activity of polysaccharides, the yield of polysaccharides is also an important factor to evaluate their quality . The higO. japonicus in Zhejiang from other MaiDong and could distinguish the difference between fibrous roots and root. L. muscari and L. spicata were also distinguished from O. japonicus in Sichuan. However, the growing years did not show an influence on the results. MaiDong produced in Sichuan usually can be collected in the first year, but MaiDong produced in Zhejiang should be collected in the third year. The difference might be mainly derived from the climate, and cultivation conditions should also be carefully investigated in the future. Nevertheless, chemical variation [Cluster analysis, by considering the seven indicators together, results in a spectrum plot as shown in ariation ,14,15,16Through the Pearson correlation analysis, we found that Man, Ara, and Xyl showed significantly high correlation values for antioxidant capacity and immune activity, while the signal of UV and Mw showed low correlation values. It has been shown that the structure of mannose backbone or side chain modifications in polysaccharides has a relevant link to their mediated biological activities, such as immunomodulatory activities . This maMaiDong. However, most likely due to the nature of the samples themselves, the properties of polysaccharides in L. muscari, L. spicate, and O. japonicus in Sichuan were closer.Finally, through the cluster analysis , the yieMaiDong were comprehensively compared, and the results showed that different MaiDong had similar molecular parameters and compositional monosaccharides. O. japonicus in Zhejiang had better antioxidant and immune-promoting activities than O. japonicus in Sichuan. There was no effect of growing age on the pharmacological activity of O. japonicus in Zhejiang. The fibrous root had better pharmacological activity than the root. The polysaccharides yield, content of Fru, antioxidant activity, and RI signal can be employed as quality indicators of MaiDong, which is helpful to the cultivation, selection, use, and quality control of MaiDong.In this study, the physicochemical properties and pharmacological activities of different sources of


The coefficient of variation (CV) of intra-assay and inter-assay were <10 and 15%, respectively. A comparative test of 66 clinical samples showed that the coincidence rate was 93.9% between the indirect ELISA and a commercial varicella-zoster virus IgG ELISA kit. Thus, the indirect ELISA kit developed here may be useful for achieving rapid, sensitive, and specific detection of anti-VZV antibodies.Varicella-zoster virus (VZV), a highly infectious agent that causes varicella (chickenpox), can also cause zoster (shingles), a disorder that is frequently associated with severe neuralgia. A reliable serological VZV diagnostic assay would be useful for identifying unprotected individuals and for surveilling post-vaccination immunoprotection status. Toward this goal, VZV membrane glycoprotein E (gE), the immunodominant VZV protein, served as target antigen in an indirect ELISA kit developed here to detect anti-VZV antibodies in clinical samples. For target antigen preparation, Chinese hamster ovary (CHO) cells were modified to express and secrete the VZV gE ectodomain, which was subsequently purified and used as coating antigen in an indirect ELISA. Ultimately, the optimal purified gE coating antigen concentration was determined to be 2 μg.ml Varicella-zoster virus (VZV) is a human-specific virus and infection does not occur, or is highly restricted, in other species . It usuaalpha Herpesviridae family, genus Varicellovirus. Five phylogenetic VZV clades have been identified, but the most disparate still have 99.8% sequence conservation  quickly evaluating the serological status of patients and hospital personnel when nosocomial varicella breaks out ; (b) evaA variety of methods for the antibody detection to VZV have been described. Fluorescent antibody to membrane antigen assay (FAMA) is an accepted gold standard assay for the indicator of immune status to VZV . HoweverThe main objects of FAMA assay and gpELISA are the specific VZV antibodies to envelope glycoproteins, and the gE protein is the major and most immunogenic glycoprotein of VZV. Therefore, CHO cells were used to express VZV gE ectodomain in this study. Then, we developed an indirect ELISA kit coated with gE protein for rapid detection of VZV antibodies. It might can be used for the auxiliary diagnosis of VZV infection, vaccine efficacy studies, and seroepidemiological surveillance activities.DH5α competent cells were purchased from Genscript . Human embryonic kidney 293T (HEK293T) cells were obtained from ATCC  and cultured with Dulbecco’s modified Eagle’s medium  with 10% (v/v) fetal bovine serum . CHO cells were obtained from Invitrogen  and maintained with SMM CHO-SI medium . The pLVX-IRES-ZsGreen1, pMD2.G, and psPAX2 plasmids were stored in our laboratory. The anti-VZV positive and negative sera were purchased from National Institutes for Food and Drug Control of China. The clinical serum samples were from healthy volunteers. All chemical reagents used in the experiments were obtained from commercial sources and of analytical grade.EcoRI-F, 5′-CCGGAATTCATGGGAACAGTGAATAAGCCTGTGGTGGGC-3′ and gE-XhoI-R, 5′-CCGCTCGAGTCAGGCCAGGCCGCCGGTCCAAG-3′) was designed to amplify the VZV gE ectodomain gene . Then the gene was cloned into the pLVX-IRES-ZsGreen1 lentiviral expression vector to construct the recombinant expression plasmid pLVX-gE-IRES-ZsGreen1. The connection product was transformed into DH5α competent cells, and the single clone was selected from the LB culture plate containing ampicillin. The pLVX-gE-IRES-ZsGreen1 recombinant plasmid was extracted and further confirmed to be constructed correctly via digestion with restriction enzymes EcoRI (NEB) and XhoI (NEB) followed by DNA sequence analysis.The optimized VZV gE protein gene (GenBank No. MF898328.1) according to the codon bias of CHO was synthesized by Sangon Biotech Co., Ltd . A pair of primers , psPAX2 (2.94 μg), and pMD2.G (1.61 μg), were mixed with 12 μl jet PRIME®  and subsequently incubated in 200 μl jet PRIME® buffer for 10 min at room temperature. Then, the mixture was added dropwise to HEK293T cells. The fluorescence was observed at 24 and 48 h post-transfection under the inverted fluorescence microscope (Olympus), respectively. Finally, the supernatant containing the pLVX-gE-IRES-ZsGreen1 lentivirus was collected.5 cells/well) in 12-well plates were incubated with lentivirus  diluted in SMM CHO-SI Medium. The fluorescence was observed at 72 h post-transduction and the cells were subcloned via a limiting dilution method in 96-well cell plates. Finally, the CHO-gE cell with highest expression of gE was selected by sandwich ELISA. Briefly, 96-well ELISA plates  were pre-coated with commercial gE mAb  diluted in 0.05 M carbonate–bicarbonate buffer  and incubated at 37°C for 2 h. After washing with PBST (PBS containing 0.05% Tween-20), the plates were saturated with 200 μl of PBST containing 5% skim milk at 37°C for 2 h. Then, 100 μl of the cell supernatant was added to each well and incubated at 37°C for 1 h. After washing, 100 μl/well of anti-VZV positive serum (1: 500 dilution in PBS) was incubated at 37°C for 1 h. Then the wells were washed again, 100 μl/well of HRP-conjugated goat anti-human IgG  diluted in PBST containing 5% skim milk was incubated at 37°C for 1 h. After washing, the reactions were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) for 5 min and stopped by 2 M H2SO4. Finally, the OD450 nm was measured by a plate reader (Bio-Rad).Chinese hamster ovary cells were transduced with the pLVX-gE-IRES-ZsGreen1 lentivirus according to methods described previously . Brieflyg for 10 min, the gE protein in the supernatant was collected and subsequently purified by Q-Sepharose® Fast Flow column described previously  every day. After centrifugation with 500 eviously . The purPurified gE protein was resolved by 12% SDS-PAGE gels and transferred to PVDF membrane. The PVDF membrane was saturated with 5% skimmed milk at 4°C overnight. Then, the membrane was incubated with the anti-VZV positive serum (1: 500 dilution in PBS) at 37°C for 1 h. After washing with PBST three times, the membrane was incubated with HRP-conjugated goat anti-human IgG  at 37°C for 1 h. After washing three times, the blots were developed by AEC .−1, 100 μl/well) diluted in 0.05 M carbonate–bicarbonate buffer  and incubated at 37°C for 2 h. Plates were washed with PBST three times and saturated with 5% skim milk (200 μl/well) at 4°C overnight. After washing three times, the plates were incubated with the clinical serum samples  at 37°C for 30 min. After washing seven times, the wells were added with HRP-conjugated goat anti-human secondary antibody  and incubated at 37°C for 30 min. After washing seven times, the reactions were developed using tetramethylbenzidine (TMB) (100 μl/well) for 5 min and stopped with 2 M sulfuric acid (100 μl/well). The absorbance was measured immediately at 450 nm on a microplate reader. When the OD450 nm was >0.286, the sample was positive. Otherwise, the sample was negative.Indirect ELISA kit was developed for rapid detection of varicella-zoster virus antibodies. In the indirect ELISA, optimal dilutions of antigen, serum, and HRP-conjugated goat anti-human secondary antibody were studied by the checkerboarding. The optimal indirect ELISA was as follows. Around 96-well ELISA plates  were coated with gE protein , separately.EcoRI and XhoI. The pLVX-gE-IRES-ZsGreen1 recombinant plasmid was extracted from DH5α cells and the sequencing result was correct. As shown in EcoRI and XhoI. The results indicated that we successfully constructed the pLVX-gE-IRES-ZsGreen1 recombinant plasmid.The truncated VZV gE gene was amplified with the length of 1,656 bp  and clonThree plasmids pLVX-gE-IRES-ZsGreen1, psPAX2, and pMD2.G were co-transfected into 293T cells to obtain lentivirus. The fluorescence and cells were observed at 24  and 48 hChinese hamster ovary cells were transduced with lentivirus to gain CHO-gE. The stable CHO-gE cell line was screened by subcloned three times using the limiting dilution method, and the CHO-gE cell with high expression of gE was screened by sandwich ELISA. The fluorescence and cells of selected CHO-gE cell line were observed as shown in −1 measured by a BCA protein assay kit and the expression quantity was 400 mg.L−1.Chinese hamster ovary-gE cells were shaking cultured, the supernatant was collected and the gE protein was purified by Q-sepharose fast flow column. As shown in The optimal conditions and repeatability of indirect ELISA were evaluated as shown in alpha-herpesvirus. It can cause varicella and HZ. During HZ, VZV multiplies and spreads within the nerve ganglia, resulted in intense inflammation, neuronal necrosis, and severe PHN. The incidence and severity of HZ and PHN increase with age, which is of particular concern in the case of aging society.Varicella-zoster virus is a ubiquitous neurotropic human B has been approved for the prevention of HZ, and it could reduce the incidence of HZ by 97.2% in adults  than previous baculovirus system .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2022.897752/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


Aim of this study is to test the predictive value of Pulse Wave Transit Time (PWTT) for fluid responsiveness in comparison to the established fluid responsiveness parameters pulse pressure (ΔPP) and corrected flow time (FTc) during major abdominal surgery.Forty patients undergoing major abdominal surgery were enrolled with continuous monitoring of PWTT  and stroke volume . In case of hypovolemia  a fluid bolus of 7 ml/kg ideal body weight was administered. Receiver operating characteristics (ROC) curves and corresponding areas under the curve (AUCs) were used to compare different methods of determining PWTT. A Wilcoxon test was used to discriminate fluid responders (increase in stroke volume of ≥ 10%) from non-responders. The predictive value of PWTT for fluid responsiveness was compared by testing for differences between ROC curves of PWTT, ΔPP and FTc using the methods by DeLong.p = 0.029). No difference in predicting fluid responsiveness was found between best-PWTT and ∆PP , or best-PWTT and FTc .AUCs (area under the ROC-curve) to predict fluid responsiveness for PWTT-parameters were 0.61 (raw c finger Q), 0.61 (raw c finger R), 0.57 (raw c ear Q), 0.53 (raw c ear R), 0.54 (raw non-c finger Q), 0.52 (raw non-c finger R), 0.50 (raw non-c ear Q), 0.55 (raw non-c ear R), 0.63 (∆ c finger Q), 0.61 (∆ c finger R), 0.64 (∆ c ear Q), 0.66 (∆ c ear R), 0.59 (∆ non-c finger Q), 0.57 (∆ non-c finger R), 0.57 (∆ non-c ear Q), 0.61 (∆ non-c ear R) . Hence, the highest AUC to predict fluid responsiveness by PWTT was achieved by calculating its respiratory variation (∆PWTT), with a pulse oximeter attached to the earlobe, using the R-wave in ECG, and correction by Bazett’s formula . ∆PWTT was sufficient to discriminate fluid responders from non-responders .The online version contains supplementary material available at 10.1186/s12871-023-02016-0. Perioperative fluid therapy has a significant impact on patients’ outcome –4. UnforConsequently, targeted fluid management needs adequate monitoring. Although different approaches to monitor fluid status have been described , assessiPulse wave transit time (PWTT) is the time period from the R-wave in ECG to the upstroke of the pulse plethysmographic waveform or the arterial pressure curve. Its respiratory variation is a flow-based monitoring parameter and was shown to predict fluid responsiveness in sedated ICU patients . FurtherThe role of PWTT in guiding intraoperative fluid management has not been established. We therefore conducted a prospective monocentric observational clinical study to first, identify which way of assessing PWTT would achieve the highest AUC for prediction of fluid responsiveness (best-PWTT). Second, we compared best-PWTT to difference in pulse pressure (∆PP) and corrected flow time (FTc), both validated fluid responsiveness monitoring parameters.clinicaltrials.gov .With approval from the State ethics committee of Rhineland-Palatinate, Germany (authorization number 837.004.16) and after obtaining written informed consent, we enrolled patients at the Department of Anesthesiology of the University Medical Center Mainz, Germany. We conducted the study in accordance with the declaration of Helsinki 1996 and ICH guideline E6  and registered the protocol prior to enrolling the first patient on This manuscript adheres to the applicable TRIPOD guidelines .2, scheduled for major abdominal surgery with an expected higher intraoperative fluid turnover. Exclusion criteria were symptomatic vascular disease, cardiac rhythm other than sinus, symptomatic cardiac valve disease, restrictive lung disease, and sepsis. Also, patients suffering from esophageal disease of any kind were excluded.We enrolled non-pregnant patients with an ASA-PS 1–3 and a BMI < 35 kg/mWe placed all patients on standard non-invasive monitors. Anesthesia was induced according to the standard operating procedures [SOP] of our hospital using 1.5–2.5 mg/kg body weight (BW) Propofol, 0.2–0.5 µg/kg BW Sufentanil and 0.5–0.6 mg/kg ideal body weight (IBW) Atracurium or 0.6 mg/kg IBW Rocuronium at the discretion of the attending anesthesiologist. Maintenance of anesthesia was accomplished using Sevoflurane adjusted to age-corrected mean alveolar concentration. Analgesia was adapted to meet patient individual demands using additional Sufentanil boluses of 5–10 µg. A non-opioid analgetic was administered 30 min before the end of the procedure for early post-operative analgesia.max and PPmin are the maximum and minimum pulse pressure during one respiratory cycle. ΔPP measurements were calculated from beat-to-beat arterial pressure values and reported as the average value of the last 32 s.An arterial cannula was placed in the radial artery for continuous, invasive blood pressure monitoring . Invasive blood pressure monitoring was calibrated by placing the pressure transducer at the level of the right atrium and venting the transducer to atmospheric pressure for reference. ∆PP was calculated asStroke volume was measured by esophageal doppler monitoring . The tip of the doppler probe was placed in the pars thoracica of esophagus at the level of the descending aorta. Stroke volume was calculated by the product of stroke distance (area of the doppler derived velocity–time waveform) and aortic cross-sectional area .−1 and fraction of inspired oxygen (FiO2) was set to keep oxygen saturation above 95% according to departmental standard operating procedures. All settings and target values were continuously adapted to patient characteristics and clinical situation using repetitive blood gas analyses.Patients were mechanically ventilated with a tidal volume ≥ 8 ml/kg IBW using pressure-controlled ventilation. Positive end-exspiratory pressure was kept between 5–8 mbar, respiratory rate was set to 12–16 minIn case of possible hypovolemia a fluid bolus of 7 ml/kg IBW was administered at the discretion of the attending anesthesiologist.Difference in pulse pressure [∆PP] ≥ 9% , 14 and/corrected flow time [FTc] ≤ 350 ms Trigger for fluid bolusing were:An increase in stroke volume ≥ 10% following the fluid bolus was considered ‘fluid responsive’.Pulse wave transit time (PWTT) was assessed with a 6-poled-ECG and pulse oximeter probes placed at the fingertip and the earlobe .Beginning of PWTT was either defined by the R- wave (R) or the Q-wave (Q) in ECG.End of PWTT was assessed by the upstroke of the plethysmography wave of a pulse oximeter attached either to the fingertip (finger) or the earlobe (ear).Measurements were corrected (c) according to Bazett’s formula  or left Respiratory variation of PWTT (∆PWTT) was calculated or PWTT was analysed as measured (raw).For the purposes of this study, we assessed PWTT based on 16 different combinations of the following variables:Simultaneous measurements of all 16 PWTT parameters in addition to ∆PP, FTc, stroke volume (SV), cardiac output (CO), mean arterial pressure (MAP) and heart frequency (HF) were recorded immediately before and 1 min after completion of each fluid bolus.max and PWTTmin are the maximum and minimum values of PWTT within 20 s and PWTTmean is the average value of PWTT within the last 20 s.Pulse oximetry waveforms and ECG waveforms were recorded with sampling periods of 8 ms and 4 ms, respectively. The rising point of the plethysmography waveform representing pulse wave arrival was defined as the point where the differentiated signal reaches 30% of the peak value of the derivative . The resThis study was an exploratory study aiming at obtaining tentative signals which PWTT parameters might be useful to predict fluid response. Sample size was primarily guided by feasibility. The number of patients to be included during the course of one year was expected to be about 40, providing about 80 measurements with subsequent bolus. Assuming that fluid response would occur in roughly half of the patients, we would expect approximately 40 responses and 40 non-responses. To test whether the area under the curve (AUC) of an obtained receiver operating characteristics (ROC) curve is higher than 0.5 a one-sided Wilcoxon Mann Whitney test can be applied. With 40 responses and 40 non-responses it is possible to reject the null hypothesis at the 5% significance level with 80% power if the true AUC is at least 0.662. Thus, the planned sample size was sufficient to identify possibly useful PWTT parameters.We performed a complete case analysis including only datasets without missing data on the variables needed for analysis of the primary endpoint (prediction of fluid responsiveness). However, multiple imputations were calculated to prevent bias by analysing complete cases only .The proportion of missing data ranged from 5.6% (raw non-c finger R) to 26% (∆ non-c ear R). There were no obvious systematic patterns in missing data. Estimates obtained using multiple imputation deviated by only 3.3% on average. When restricting the analysis to complete cases only, the same PWTT-parameter achieved the highest AUC for prediction of fluid responsiveness (best-PWTT) as deviation of best-PWTT after multiple imputations from corresponding mean in complete case analysis was 0.18% only.The ability to predict fluid responsiveness for the 16 different PWTT parameters assessed in this study was described by the area under receiver operating characteristic curve (AUC). Differences between ROC curves were analysed using the methods by DeLong . A paramDifferences in stroke volume change after fluid bolusing between responders and non-responders were assessed using a two-sided Wilcoxon test.After identifying which way of assessing PWTT achieved the highest AUC for prediction of fluid responsiveness (best-PWTT), we compared AUC of best-PWTT with AUCs of ∆PP and FTc using the methods by DeLong  to test Positive predictive value was calculated for all fluid responsiveness parameters investigated, i.e. 16 PWTT-parameters as well as ∆PP and FTc. As both, administering no fluids despite they are needed as well as to do so but at the wrong time can be harmful, only positive predictive values with sensitivity of at least 80% are displayed in this analysis. Furthermore, we calculated the F1-Score thus balancing the importance of sensitivity and positive predictive value. We used precision recall curves to illustrate relation of sensitivity and positive predictive value Fig. 4 .SAS 9.4  was used for all statistical analyses. Boxplots were generated using Excel 2016 .Eighty patients were scheduled for major abdominal surgery with an expected higher intraoperative fluid turnover. 40 patients did not meet the inclusion criteria. The remaining 40 patients consented and were enrolled in the study. 1 patient was excluded due to not receiving a fluid bolus and 1 patient because of incomplete documentation.In total, data of 38 patients were analyzed. Patients’ demographics are shown in Table All procedures were performed in laparotomy. Patients receiving a laparoscopy respectively the use of pneumoperitoneum were not included in the study.Out of 85 fluid boluses administered to 38 patients, 74 datasets were eligible for complete case analysis. We identified 28 out of 38 patients as fluid responders, fluid responsiveness rate was 67.6% (50 out of 74 fluid boluses given).Hemodynamic measurements before and after fluid bolus separated for fluid responders and non-responders are shown in Table p = 0.029).Of all 16 different ways to assess PWTT, a measurement of ∆PWTT with use of the R-wave in ECG, plethysmography at the earlobe, and correction by Bazett’s formula performed best to predict fluid responsiveness , and was able to discriminate fluid responders from non-responders .Both ∆PP  and FTc  obtained results that were close to ‘best-PWTT’ for prediction of fluid responsiveness. Using the methods by DeLong  for compIn this prospective clinical study we compared multiple ways of measuring and calculating the non-invasive monitoring parameter PWTT:Earlobe vs Fingertip Even though several studies [ studies , 21 atta studies .Q- vs. R-Wave Both, Q-Wave [, Q-Wave  as well , Q-Wave , 17, 21.Corrected vs. non-corrected Not surprisingly, a correction to compare measurements at different heart rates improves the performance of a hemodynamic monitoring technology. The current clinical standard is the most widely used Bazett formula [ formula .Respiratory variation A number of clinically established parameters to assess fluid responsiveness is based on heart–lung interaction [eraction . So it heraction  and PWTTeraction . Howevereraction  do withoConsequently, we first identified one ‘best-PWTT’ approach achieving the highest AUC for prediction of fluid responsiveness, i.e. measuring PWTT by a pulse oximeter attached to the earlobe and using the R-wave in ECG, correcting by Bazett’s formula, and calculating the respiratory variation of PWTT (∆PWTT).In the present study, the area under the ROC curve for ‘best-PWTT’ was 0.66 . Accordingly, PWTT showed only poor ability as a diagnostic tool to discriminate fluid responders from non-responders intraoperatively. We hence were not able to confirm previous positive results from an ICU population  in a cliSecond, after identifying ‘best-PWTT’ we compared the new flow-based monitoring parameter to established alternatives: The performance of ∆PP  and FTc  was not better when compared with best-PWTT.Although several clinical studies in major abdominal surgery show an improved outcome of intraoperative fluid management monitored by esophageal doppler , the repThis study has several limitations. First, all patients being studied had to have a stable sinus rhythm being mandatory for an assessment of PWTT. However, this applies also to ∆PP. Second, stroke volume measurements by esophageal doppler are affected by substantial variability between different observers . To miniIn conclusion, PWTT showed only poor ability to predict fluid responsiveness intraoperatively (AUC 0.66). Interestingly, established fluid responsiveness parameters ∆PP and FTc did not perform better in the present study population. Furthermore, F1-Scores suggest ∆PWTT to be a well-balanced parameter not leaning too much on sensitivity or positive predictive value alone. Being non-invasively measured in real time and without the need for cost-intensive additional monitoring devices PWTT comes along with well needed attributes in clinical routine. This is crucial, as even though at least one hemodynamic parameter to predict fluid responsiveness would be available in more than 90% of cases performing volume expansion, they are roughly used in a third . Thus, cAdditional file 1. Best-PWTT dataset legend.


Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion.18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry.In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by in vivo apoptosis and decreased microvessel density.The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted max of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVMechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. A standard curve was then plotted according to the concentration of the standards and the corresponding OD values, and the amount of sVEGFR-2 and VEGF was calculated using a regression equation. The lower limit of detection for sVEGFR-2 was 300 ng/L and that for VEGF was 12.5 ng/mL. The ELISA reader and washer used in this study were Stat-Fax 2100 and Stat-Fax 2600 (USA), respectively.Pleural tumour tissues were stained using a TUNEL kit  according to the manufacturer’s protocol, and imaged and analysed using Image J in a blinded manner. The TUNEL-positive apoptotic cells were counted in at least five random fields (magnification: 200×), and the relative percentage was calculated. The experiment was repeated thrice.p values less than .05 were considered statistically significant.All statistical analyses were performed using SPSS software version 23.0  and GraphPad Prism software version 7.0 . All tests were repeated three times or more. Quantitative data are expressed as mean ± standard deviation. Multiple groups were compared by one-way analysis of variance (ANOVA), and the average number of pairwise comparisons was determined by Tukey’s test. Spearman’s rank correlation coefficient test was used to determine the association between two variables. Survival duration and rate were evaluated by the Kaplan–Meier method, and the log-rank test was used to compare survival curves. For all tests, two-sided The effects of BEV, CDDP, APA and their different combinations were tested in an LLC-induced MPE model. The grouping, therapeutic regimens and subsequent analyses are outlined in p < .01). Combining CDDP with either APA (21.00 ± 0.16 g) or BEV (20.80 ± 0.28 g) further improved the body weight of tumour-bearing mice (p < .01 compared to control), although no significant difference was observed between the CDDP + APA and CDDP + BEV groups (p = .67). The highest average body weight was observed in the CDDP + APA + BEV group (21.53 ± 0.28 g). Consistent with this finding, the drug triad also significantly prolonged the median survival of tumour-bearing mice to 30 days compared with only 20 days observed in the control group (p < .01 compared to Control), although the individual drugs were less effective compared to their combination (p < .01). Finally, pleural effusion volume was also significantly lower in the CDDP + APA + BEV group (0.59 ± 0.06 mL) compared with that in the CDDP + BEV (0.78 ± 0.04 mL), CDDP + APA (0.79 ± 0.04 mL), APA (1.05 ± 0.11 mL), BEV (1.01 ± 0.08 mL) and CDDP (1.32 ± 0.06 mL) groups. CDDP + APA and CDDP + BEV had a similar effect on MPE as that observed with body weight (p = 1.00). Taken together, the combination of BEV, APA and CDDP significantly decreased tumour growth and pleural effusion and prolonged the survival of the tumour-bearing mice compared to the individual drugs.As shown in ol group . Further18F-FDG PET/CT , CDDP (4.03 ± 0.18) and APA (3.36 ± 0.17) monotherapies were significantly lower than that for the control group (4.49 ± 0.29). The different combinations of APA, BEV and CDDP further lowered the SUVmax values, with the lowest was observed for CDDP + APA + BEV (2.41 ± 0.33). No significant differences were observed between the CDDP + APA and CDDP + BEV groups . Taken together, the combination therapy inhibited the growth of tumour nodules in the pleural cavity by effectively reducing glucose metabolism.The metabolic effects of different therapies were evaluated by G PET/CT  with SUVe uptake . The SUV+ staining in the tumour tissues compared to the monotherapies and untreated control. Consistent with this, the MVD in the pleural tumour nodules was significantly decreased in the CDDP + APA + BEV (2.80 ± 0.83), CDDP + BEV (7.60 ± 0.87) and CDDP + APA (7.04 ± 1.12) groups compared to that in each of the monotherapy groups and the control group (21.13 ± 1.55). The MVD was highest in the untreated controls, followed by the CDDP, BEV, APA, CDDP + BEV, CDDP + APA and CDDP + APA + BEV groups in that order. Taken together, BEV and APA in combination with CDDP synergistically inhibited tumour angiogenesis, resulting in significant tumour regression.Tumour growth and angiogenesis were also evaluated in terms of the MVD. As shown in + apoptotic tumour cells was highest in mice treated with the drug triad, followed by CDDP + APA, CDDP + BEV, BEV, APA and CDDP in that order (p < .01). Furthermore, the apoptosis rate was significantly higher in the CDDP + APA + BEV group compared to the rest . The combination of APA or BEV with CDDP increased apoptosis compared to either monotherapy (p < 0.01). Therefore, the combination of an angiogenesis blocker with CDDP can synergistically induce apoptosis in the tumour cells.As shown in at order  and werep < .01 vs. all) and pleural exudates  of mice in the CDDP + APA + BEV group were the lowest compared to that in other treatments groups. Consistent with this, the level of sVEGFR-2 was the highest in the untreated control group in the serum and pleural fluid, followed by the CDDP, BEV, APA, CDDP + BEV, CDDP + APA and CDDP + APA + BEV groups in that order. There was a significant difference between other groups with CDDP + APA + BEV (p < .05). Taken together, CDDP + APA + BEV achieved simultaneous inhibition of VEGF and sVEGFR-2 in the serum and pleural fluid, which decreased neo-angiogenesis and growth.To gain further insights into the mechanisms underlying the anti-angiogenic effects of BEV, APA and CDDP, we next analysed the levels of VEGF and sVEGFR-2 in the serum and pleural fluid of the tumour-bearing mice. As shown in in situ levels of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 proteins compared the control group (p < .01), as well as the monotherapy groups (p < .01). Interestingly, HIF-1α, VEGF, VEGFR-2 and MMP-2 expression was similar between the CDDP + APA and CDDP + BEV groups (p > .05). These findings indicate that BEV, APA and CDDP inhibited angiogenesis in LLC xenografts by blocking the MAPK/ERK and Ras/Raf/MEK/ERK pathways.The MAPK pathway is a critical oncogenic pathway and the target of multiple chemotherapeutic drugs . TherefoThe aim of our study was to analyse the possible synergistic effect of the combination of BEV, APA and CDDP against NSCLC complicated with MPE. The combination therapy induced apoptosis in LLC cells and reduced the volume of pleural effusion in tumour-bearing mice, which significantly prolonged their survival. Mechanistically, the drug triad suppressed NSCLC growth and angiogenesis by inhibiting the VEGF-induced MAPK/ERK signalling pathway. This is the first study to identify the therapeutic value of combining BEV, APA and CDDP in NSCLC.via autophosphorylation and endothelial cell signal transduction [VEGF plays a vital role in promoting angiogenesis and the pleural invasion of VEGF-overexpressing tumour cells initiates MPE formation . At pressduction ,58. Thussduction ,59. Studsduction ,60. Furtsduction .18F-FDG uptake, which is indicative of lower glucose metabolism and better prognosis in vivo [max of tumour masses to slower tumour growth and a better clinical outcome [18F-FDG uptake by the tumours compared to the monotherapies.The decrease in MPE relieves the squeezing of lungs and reduces severe hypoxia, which increases food intake in mice ,61,62. P in vivo . Studies outcome ,69. We fet al. [Soluble VEGFR-2 (sVEGFR2) contains the extracellular domain of the VEGFR-2 receptor but lacks the tyrosine kinase domain and is activated in the hypoxic tumour microenvironment ,71. W. Xet al.  found thThe combination of BEV, APA and CDDP inhibited neo-angiogenesis in the LLC xenografts by blocking the MAPK/ERK and Ras/Raf/MEK/ERK pathways, and had a more pronounced effect on HIF-1α and MMP-2 than either drug alone. HIF-1 is the key activator of hypoxia-induced angiogenesis and is regulated by the tumour suppressor gene von Hippel Lindau (VHL). Under normoxic conditions, HIF-1α is rapidly degraded by the ubiquitin-proteasome pathway under the control of VHL. However, hypoxia or VHL deletions/mutations results in the heterodimerization of HIF-1α and HIF-1β, which then translocates to the nucleus and activates VEGF transcription . MMP-2 iin vitro studies have shown that inhibition of VEGF signalling effectively stimulates anti-tumour immunity and enhances the efficacy of TKIs and immune checkpoint blockade [The present study has some limitations that ought to be considered. The small sample size and short study duration may have affected the results. In addition, we did not analyse the molecular and cellular mechanisms underlying the synergistic action of BEV, APA and CDDP. Further studies are needed to determine the long-term and short-term toxicity profiles of these drugs, as well as the mechanistic basis of their action. Some blockade ,79,80. Fblockade . Simultablockade . At the In summary, the combination of BEV, APA and CDDP prolonged survival of LLC tumour-bearing mice by reducing MPE and glucose metabolism. A better understanding of this combination will define new approaches to enhance drug efficacy in the treatment of NSCLC patients with MPE by possible moderation of VEGF/MAPK/ERK pathways.


The aim of this article is to investigate the exact solution by using a new approach for the thermal transport phenomena of second grade fluid flow under the impact of MHD along with exponential heating as well as Darcy’s law. The phenomenon has been expressed in terms of partial differential equations, then transformed the governing equations in non-dimentional form. For the sake of better rheology of second grade fluid, developed a fractional model by applying the new definition of Constant Proportional-Caputo hybrid derivative (CPC), Atangana Baleanu in Caputo sense (ABC) and Caputo Fabrizio (CF) fractional derivative operators that describe the generalized memory effects. For seeking exact solutions in terms of Mittag-Leffler and G-functions for velocity, temperature and concentration equations, Laplace integral transformation technique is applied. For physical significance of various system parameters on fluid velocity, concentration and temperature distributions are demonstrated through various graphs by using graphical software. Furthermore, for being validated the acquired solutions, accomplished a comparative analysis with some published work. It is also analyzed that for exponential heating and non-uniform velocity conditions, the CPC fractional operator is the finest fractional model to describe the memory effect of velocity, energy and concentration profile. Moreover, the graphical representations of the analytical solutions illustrated the main results of the present work. Also, in the literature, it is observed that to derived analytical results from fractional fluid models developed by the various fractional operators, is difficult and this article contributing to answer the open problem of obtaining analytical solutions the fractionalized fluid models. Multiple products such as honey, soup, jelly, china clay, tomato sauce, artificial fibers, synthetic lubricants, concentrated fruit juices, pharmaceutical chemicals, paints and coal, etc. are some applied illustrations of such fluid. Systematic analysis of such fluid flow models have significantly important for theoretical studies and practical implementations in modernistic mechanization. Among such fluids, second grade fluid attracted special attention, which is the commonest non-Newtonian fluid due to its more extensive applications and substantial role in different fields serving as mechanical as well as chemical applications, bio engineering operations, metallurgy and especially in food processing industries. Lubricants that are used to lubricate the components of engine like gears, bearings, etc., are considered as differential type fluids. The study of second grade movement in the context of fluid mechanics, was explored by several mathematicians, scientists, researchers and engineers that depends upon various situations. Rajagopal et al.7 examined the influence of various applications of differential type fluid, for instance, in theological problems, in biological sciences, chemical, petroleum and geophysics field. The parabolic partial differential equations governing the flow in the presence of a magnetic field, Hall currents and the free stream velocity has been studied by Takhar et al.8 Modather et al.9 presented the analytical solution of the problem related to MHD mass and heat transfer of an oscillatory two-dimensional viscous fluid that is electrically conducting over an infinite vertical permeable moving plate which is embedded in a porous medium along with a chemical reaction and transverse magnetic field.The process of heat and mass transfer has a great importance from the industrial point of view. Many researchers and scientists concentrate on this area. In modern technologies and various industrial fields, the non-Newtonian fluid theory has extensive impact because Newtonian fluid model can not express many flow characteristics. A non-Newtonian fluid that obeys the nonlinear relationships between the rate of shear strain and the shear stress. The non-Newtonian fluid theory has significant utilization in modern engineering, especially in petroleum industry used to extract crude oil from different petroleum productions. The properties of Newtonian fluid in most of the cases are not valid but scientists desire to model the complex models for non-Newtonian fluid. The importance of non-Newtonian fluid has been enlarged from the last few decades, specifically in the research field. The non-Newtonian fluids have numerous ever-increasing applications in industrial sectors, but some specific are mentioned here, such as at large-scales reducing and enhancing heating/cooling systems, biochemical and process engineering, extrusion of molten plastic in industry, reducing oil pipeline friction, polymer processing, reducing fluid friction, well drilling, flow tracers, biological materials, biomedical flow analysis, plastic foam processing, lubrication processes, food processing industries, chemical processing, all emulsions, handling of muds, slurries and complex mixtures. Many researchers and scientists focused on non-Newtonian fluid while considering different fluid geometries. Therefore simulating and modelling the flow phenomena of non-Newtonian fluid that is facilitated and play the important role in human life. Researchers investigated different non-Newtonian fluid models regarding physical and computational characteristics such as second grade model, viscoplastic model, power law model, Bingham plastic model, Jeffery model, Oldroyd-B fluid model, Brinkman type model, Casson model, Walters-B fluid model and Maxwell model10 and Ali et al.11 analyzed the characteristics of upper-convected second grade nanofluid thin film flow over a time-dependent stretching sheet with variable thermal conductivity and Cattaneo–Christov double diffusion theory.Viscosity of fluid keeps its leading role in the biological fluids, polymer process, mayonnaise, melt solutions, colloidal suspensions and lubrication models. The Carreau nanofluid viscosity model can explain features of non-Newtonian fluids in the shear-thinning/thickening regions. Carreau viscosity model and thermal radiation under the influence of non-uniform heat source/sink transportation phenomenon of heat over the surface was discussed by Assad et al.12, Labropulu13, Fetecau et al.14, Tawari and Ravi15 and Islam et al.16 studied the unsteady second grade fluid, employed the method of separation of variables, to compute analytical solution. Rehman et al.17 elaborated the MHD second grade fluid flow to analyzed the effects of radiative thermal flux and computed the analytical solutions by employing Laplace integral transformation. Ali18 investigated free convection flow of an electrically conducting fluid along a vertical plate embedded in a thermally stratified porous medium in the presence of a uniform normal magnetic field. MHD mixed convection from a semi-infinite, isothermal, vertical and permeable surface immersed in a uniform porous medium in the presence of thermal radiation and Dufour and Soret effects are studied by Ali19. Assad et al.20 presented a comprehensive analysis of the second-order velocity slip phenomenon and stagnation point flow of cross nanofluid with a spectral relaxation approach over the geometry of porous medium. Some significant studies regarding second grade fluid having interesting facts are described by Rashidi et al.21, Baranovskii22, Arianna et al.23, Dinarvand et al.24 and Fetecau et al.25Some researchers and scientists are focused to investigate the flow regime of second grade fluid geometrically for configurations of many interesting features, because flow analysis of differential type fluids have wide practical applications and theoretically studies, having prominent effects in many industrial fields, for example, Erdogan26, multi step approach28, Riccati-Bernouli sub-ordinary diffrential equation Sub-ODE techniq (RBSODET)29,reproducing the kernel Hilbert space method31, simple equation modification method32, residual power series method33 and several others36. Mehmet et al.37 applied 43. These fractional operators having different type of non-singularized kernels, some of the kernels are mentioned here such as, Rabotnov exponential function, Exponential kernels and Mittag-Leffler functions. Mehmet et al.44 investigated the exact solution and a qualitative study for the fractional second-grade fluid described by a Caputo fractional operator. Aziz et al.45 has been discussed heat source impact on unsteady magneto-hydro-dynamic (MHD) flows of Prabhakar-like non integer second grade fluid near an exponentially accelerated vertical plate with exponentially variable velocity, temperature and mass diffusion through a porous medium. The analytical treatment to the bio heat transfer Pennes model via modern fractional derivatives are analyzed by Kashif et al.46 Comprehensive analysis of heat and mass transfer of MHD natural convection flow of water-based nano-particles in the presence of ramped conditions via Caputo-Fabrizio fractional time derivative are investigated by Aziz et al.47The fractional/differential calculus is an eminent mathematical field that growing immensely due to enormous significance investigates the non integer order behavior of integrals and derivatives as well as their applications and properties. The concept of differential calculus is old like classical calculus, first time in 1695, new idea about fractional calculus introduced when a letter from Leibniz to L’Hospital was written. This field attracted the attention of well known mathematicians, researchers and scientists that proposed and built different fractional integrals and fractional derivatives. The researchers faced too much difficulties to developed a real physical phenomenon by employing the traditional calculus techniques, then the fractional differential equations have great importance for mathematicians and researchers. Since it has been investigating numerous physical models in different scientific fields such as biology, physics, chemistry, acoustic waves, finance, control theory, fractal dynamics, signal processing, hydro magnetic waves, diffusion reaction process, anomalous transport, fluid flow problems, engineering processes, oscillation, dynamical processes and many other disciplines. The main reason for exploring the numerical or exact solutions due to its significance in various daily life. To gain the numerical or exact solutions, researchers and mathematicians have been implemented numerous techniques. For instance, unified method48 and Ying-Qing Song et al.49 discussed the flow of fractional version of differential type fluid model by using different fractional operators namely CF and ABC, respectively, and computed solution for each fractional model, but both respective studies presented work without analyzed the effect of mass diffusion. But in the literature fractional second grade fluid model with fractional operators CPC, CF and ABC, along with the set of non-uniform boundary conditions for velocity with exponential heating, saturated in porous media, are not investigated yet nor published. To fill this gape a new fractional second grade fluid model developed by applying the definition of recently introduced a new fractional derivative operator, namely CPC, CF and ABC operators, under effectively applied conditions for concentration, velocity field and temperature distribution. For seeking exact solution expressions in terms of G-functions by employing Laplace integral transformation method to solve the fractional models developed for velocity, concentration and temperature distribution. For physical analysis the influence of parameters like as second grade parameter t, fractional parameter Gm, Magnetic number M, Prandtl number Pr, Schmidt number Sc, thermal Grashof number Gr are portrayed graphically by using Mathcad software. Furthermore, for validation the current result, also accomplish the comparative analysis with different published work.In the previous investigation, Sami Ul Haq et al.H(t) represented unit step function and y and t only.The following principal equations for second grade fluid under Boussinesq’s approximation, for velocity,concentration and energy transfer are obtained as51:Consider the MHD second grade fluid flow near an oscillating infinite vertical plate that is nested in a porous material. The plate is considered at mentclass2pt{minimg represents the fluid velocity, temperature, concentration, density, coefficient of volumetric thermal expansion, second grade parameter, thermal conductivity, kinematic viscosity, specific heat capacity, coefficient of volumetric expansion for concentration and gravitational acceleration respectively.With initial (ICs) and boundary (BCs) conditions are given below:Writing the proposed problem in terms of dimensionless form, the following dimensionless quantities are considered:When substituting the Eq.  into EqsThe fractional model for momentum, energy and concentration distribution are formulated by using Constant proportional-Caputo hybrid fractional derivative operator is described as:The fractional model for momentum, energy and concentration distribution are formulated by using Caputo–Fabrizio fractional derivative operator is described as:Also,The fractional model for momentum, energy and concentration distribution are formulated by using Atangana–Baleanu time fractional operator is described as:Laplace transformation of Constant proportional-Caputo hybrid time fractional operator is written as:Now, Laplace transformation of Caputo–Fabrizio time fractional operator is written as:Also, In this section, the analytical solution derived of the non-dimensional fractionalized second grade fluid model by employing the technique of Laplace transformation.Employing Laplace integral transformation to Eq.  with conTaking Laplace inverse which transform the above equation in time parameter, then exact solution of Eq.  is givenIt is not easy to find Employing Laplace integral transformation to Eq.  with conTaking Laplace inverse which transform the above equation in time parameter, then exact solution of Eq.  is givenIt is not easy to find Applying inverse transformation, we getEmploying Laplace integral transformation to Eq.  with conn in Eq.  used to Taking Laplace inverse which transform the above equation in time parameter, then exact solution of Eq.  is givenIt is not easy to find Applying inverse transformation, we getIt is not easy to find Applying inverse transformation, we getSolving Eq.  using 11, 12) a a12) andC from exponential form, but Laplace inverse can be find if Applying conditions given in Eq.  for concFor concentration solution, taking Laplace inverse of Eq. , so finaSolving Eq.  using 11, 12) a a12) andApplying conditions given in Eq.  for concC from exponential form, but Laplace inverse can be find if It is not easy to written the concentration solution For concentration solution, taking Laplace inverse of Eq. , so finaSolving Eq.  using 11, 12) a a12) andApplying conditions given in Eq.  for concC from exponential form, but Laplace inverse can be find if It is not easy to written the concentration solution For concentration solution, taking Laplace inverse of Eq. , so finaSolution of Eq.  with thethe Eqs.  and 59)68\documeApplying Laplace inverse to write the solution for momentum equation as:Employing Laplace inverse then it takes the form as:D is calculated as:Similarly, P, we haveNext consider R, so considerSimilarly, we can calculate Solution of Eq.  with thethe Eqs.  and 65)81\documeApplying Laplace inverse to write the solution for momentum equation as:Employing Laplace inverse then it takes the form as:Next consider R, so considerSimilarly, we can calculate Solution of Eq.  with thethe Eqs.  and 53)97\documeu, as we have computed for CF and ABC fractional derivative operators. The function Similarly, we find the Laplace inverse of 48 investigated with exponential heating by removing the term Gm, i.e, 49 investigated with exponential heating by removing the term Gm, i.e, For validation purpose of the current study, we recovered the same velocity field equations Eq.  as Sami ions Eq.  as Ying-Gr, Prandtl parameter Pr, Schmidt number Sc and mass Grashof number Gm on fluid velocity, concentration and temperature are evaluated and executed graphically in Figs. The unsteady second grade fluid with natural convective flow over flat plate of infinite length has been examined under exponential heating. Fractional model developed for non-dimensional velocity, concentration and energy equations by using CPC, CF and ABC fractional operators. Exact solution expressions obtained in terms of generalized G-function by simplifying the fractionalized model analytically with initial boundary conditions are provided for the proposed problem. For physical significance of various system parameters involved in the problem such as fractional parameter entclass1pt{minimamentclasspt{minimay via CF and ABC operator, for different values of Figures Sc on concentration profile of second grade fluid by taking its dissimilar values of Sc, where the values of fractional parameters have been supposed between 0 and 1. Physically, boundary layer of concentration is declined due to change of Schmidt number from small to large.Figures Gr and mass Grashof number Gm on second grade fluid flow against y via CF and ABC operator varying values of Gr describes the fraction of thermal buoyancy force to viscous force, but Gm describes the fraction of species buoyancy force to viscous force, that are acting on the fluid transportation, as a result, with an increasing in Gr or Gm cause a remarkable increasing impact on the second grade fluid velocity. Physically, an increasing the values of thermal or mass Grashof numbers that leadsto decrease in viscous hydrodynamic forces, and as a result, the momentum of the second grade fluid is higher.Figures y, for different values of Figures Sc on velocity curve is depicted, against y, for different values of Sc, at four different valuesof fractional parameter Sc. It is noticed that, momentum diffusivity is quicker than species diffusivity when Sc is greater than one Figures Figures Figure t, and significant effect noticed on fluid velocity curve together with different parameters. It is noteworthy to point out that the graphical view of the fluid velocity is higher. Further, it is observed that the same velocity field equations Eq. (48 investigated with exponential heating by removing the term Gm, i.e, 49 investigated with exponential heating by removing the term Gm, i.e, Figure ions Eq.  as Sami ions Eq.  as Ying-Pr has disclosed quick and thicker heat diffusivity;The temperature distribution corresponding to smaller and larger values of Sc increases, this is because of the relative thickness of the hydrodynamic layer.Mass concentration profile represented the decreasing behavior as the values of Sc that declines the velocity field.Increasing values of Gr and Gm stimulates the velocity field.The growing values of Pr causes to decline velocity contour.An increase in the values of It can be seen that fluid velocity graphs represents dual behavior for small and large time.It can be noticed that the second grade fluid velocity represents same behavior for small and large values of It is depicted that the fluid’s velocity for constant caputo-proportional hybrid derivative operator is greater than Atangana Baleanu and Caputo Fabrizio models respectively for small time but reverse order observed for large time.In comparison with Atangana Baleanu and Caputo Fabrizio fractional derivatives, the CPC fractional model is exceptionally suitable for simulating the history of velocity function.In this paper, unsteady second grade fluid flow under the effect of MHD along with exponential heating as well as Darcy’s law near an oscillating infinitely flat plate saturated in a permeable medium is analyzed. For the sake of better rheology of differential type fluid, developed a fractional model by employing the new definition of CPC, CF and ABC fractional derivative operators and exact solution obtained by using Laplace integral transformation. For several physical significance of various system parameters, the graphical representations of the analytical solutions illustrated the main results of the present work. Also, in the literature, it is observed that to derived analytical results from fractional fluid models developed by the various fractional operators, is difficult and this article contributing to answer the open problem of obtaining analytical solutions the fractionalized fluid models. Some essential major concluding observations obtained from the graphical analysis are summarized as follows:


Twitter has become a key platform for public health campaigns, ranging from mental health awareness week to diabetes awareness month. However, there is a paucity of knowledge about how Twitter is being used to support health campaigns, particularly for Alzheimer’s Awareness Month. This presentation aims to: 1) identify how Twitter was used to share dementia discourse during Canada’s Alzheimer’s Awareness Month in January; and 2) explore actions to enhance dementia awareness using Twitter for future Alzheimer’s Awareness Month campaigns. Tweets were collected from Twitter using the Twint application in Python from January 1 to January 31, 2022. Filters were used to exclude irrelevant tweets , and the remaining 1,289 tweets were exported to Excel. Tweets were divided among eleven coders and analyzed using inductive thematic analysis. Analysis revealed four main themes: dementia education and advocacy; fundraising and promotion; sharing experiences of dementia; and opportunities for future actions such as collaborative partnerships and educational tweets to correct stigmatizing language and dementia stereotypes. Increased educational content, collaborative partnerships, and evidence-informed research are essential to enhancing dementia awareness strategies on Twitter during Alzheimer’s Awareness Month in Canada. Further research is needed to develop, implement, and evaluate methods to improve dementia awareness on Twitter during Alzheimer’s Awareness Month and beyond.