Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases. Pancreatitis is the leading cause for gastrointestinal (GI) disease-related hospital admissions  and PDACIntravital imaging (IVI) enables the visualization and analysis of tumour cell dynamics in live animals and in real time. Previous research has used IVI to observe benign and malignant diseases of skin ,5, breaset al. [To overcome these limitations, previous studies have used different implantable imaging window designs. Ritsma et al.  were theet al. . While tet al. .et al. [To address this issue, Park et al.  developeDuring our recent experience developing an implantable imaging window for thyroid tumours , we foun. 2. 2.1G12D/p53fl/+/Pdx1-Cre/RosaYFP  [fl/+/RosaYFP ; and (6) mixed: Pdx1-Cre/RosaYFP . Mice were housed in static microisolator cages under specific pathogen-free conditions in a temperature- and humidity-controlled environment. All mice were provided with 1/8 inch corn cob as bedding material and Nestlets as nesting material. All experiments were performed during the daytime of a 12-hour day/night cycle. KPCY, PCY and CY mice were used between 16 and 20 weeks of age, when they develop palpable tumours. All others were born and raised within in the same animal housing facility and used between 8 and 12 weeks of age.Six variants of transgenic or autochthonous mice were used for intravital imaging: (1) MMTV-iCre/CAG-CAC-ECFP (ECFP labelled epithelia) used for imaging the mammary gland or the healthy pancreas; (2) FVB: MMTV-iCre/CAG-CAC-ECFP × c-fms-GFP  (ECFP latumours) ; (5) mix. 2.2−1. Selection occurred for two weeks with 600 µg ml−1 G418 in.complete media, changing media every 2 days. After reaching confluency, the top 10–15% Dendra2 green expressing cells were collected using flow assisted cell sorting (FACS).KPC cells  were cul. 2.3ad libitum. 50 µg kg−1 of cerulein in 100 µl sterile 1× DPBS was injected intraperitoneally at one-hour intervals for up to eight injections. Control mice received an equal volume of 1× DPBS. Mice were sacrificed 24 h after first injection. Pancreata were collected and fixed for 72 h in 10% formalin and paraffinized.The mice were fasted for 14–18 h with water given . 2.4Deparaffinized sections were blocked in 5% donkey serum for 1 h at room temperature, incubated with primary antibodies for overnight at 4°C. Sections were washed with PBST and incubated with secondary antibodies for 1 h at room temperature. Primary antibodies were chicken anti-GFP  at 1:250 and rabbit anti-cleaved caspase 3 . Lastly, sections were washed with PBST and mounted with Aquamount and DAPI. Slides were imaged on 3DHistech's Pannoramic 250 Flash II slide scanner.. 2.56 cells/50 µl PBS and kept on ice.KPC-dendra2 cells were cultured in McCoy's 5A medium  supplemented with 10% fetal bovine serum, 1% MEM non-essential amino acids , 1% sodium pyruvate  and 1% penicillin and streptomycin. Single-cell suspensions were prepared at a concentration of 1×10et al. [The orthotopic injection protocol was adapted from Erstad et al. . AnaesthA 10–15 mm longitudinal incision was made in the left flank of the abdomen of the mouse, just below the left costal margin. The spleen, along with the attached tail of the pancreas, was brought out of the incision using cotton swabs. 50 µl of KPC-dendra2 cell suspension was slowly injected using a 28-gauge needle  while holding the edge of the pancreas with atraumatic forceps. Successful injection was confirmed by the presence of a well-defined bubble within the pancreas. To prevent leakage, the needle was inserted parallel to the pancreas and slowly withdrawn over 30 s. The spleen and pancreas were returned to their anatomical position by gently retracting the skin and muscle flaps. Skin and musculature were closed using interrupted 5-0 silk suture. Surgical adhesive  was applied over the incision and sutures. Mice were observed throughout recovery. SWIP implantation occurred when tumours were approximately 0.5–1 cm.. 2.6a) was passivated before each use, as previously published [The custom-made reusable stainless steel SWIP window a was pasublished .Figure . 2.7All imaging was performed on a previously described, custom-built, inverted two-laser multiphoton microscope . All ima−1 155kD tetramethyl-rhodamine dextran. A thin coating of petroleum jelly was applied to the underside of the microscope stage plate to prevent wicking of water from the objective lens [et al. [−1 PBS was administered to prevent dehydration. Physiological temperature was maintained using a stage top environmental chamber. Vasculature of the tissue was identified by observing flowing erythrocytes and the tissue of interest identified by fluorescence. Vitals were monitored during imaging . A minimal amount of image processing was performed to optimize brightness and contrast of images.Mice were anaesthetized and ophthalmic ointment applied. Mice were injected retro-orbitally with 100 µl of 20 mg mlive lens . A previive lens . The fix [et al. . 50 µl h. 2.8Quantification of image drift and instability was performed using our previously published ROI_Tracker plugin for ImageJ  to mark . 3Given the compliant and delicate nature of the murine pancreas, as well as its relatively deep positioning compared to other abdominal organs, the pancreas can be challenging to immobilize for intravital imaging.et al. [et al.  braces the tissue and presses the pancreas against the cover glass so as to fill the entire clear aperture of the window, greatly increasing the volume of tissue available for observation. Upon testing this new window design, we indeed observed a markedly improved lateral stability. However, we still observed substantial axial drift over several hours as a result of tissue settling within the gap between the cover glass and the plate . Furthermore, although the integrated plate offered improved stability, its presence limited the ability to image solid pancreatic tumours, as their large size would not fit within the limited space between the plate and cover glass.In using the AIW, we found that, even with adhesive applied to the inner side of the frame, a large amount of axial and lateral drift could be observed , and, as was reported by Park et al. , the tis. 3.1a) that would allow stable time-lapsed imaging of both healthy and diseased pancreatic tissue, we developed the surgical protocol summarized in figure 1a). This window design, which we used previously for lung imaging [figure 1b) which mimics the body's use of ligaments to secure and stabilize organs. This soft basket  keeps the tissue apposed to the glass with constant axial pressure, preventing lateral and axial motion. For larger solid tumours, the support point and the direction of the cross stitch can be adjusted according to the size and position of the tumour for optimal support.In order to create a permanent, implantable optical-imaging window a that wo imaging , include imaging  for relo imaging . While t imaging  cross stc). The cross stitches (b) are placed with 5-0 silk suture where one end of each cross stitch is tied to the abdominal muscle and the free end left to form the ‘basket’ (d). The tail and body of the pancreas is then laid on top of the basket using sterile cotton swabs to avoid damaging the delicate tissue (e). A purse-string suture placed through the skin and abdominal wall secures the window frame to the abdomen (f). After placement of the SWIP frame on top of the pancreas, the skin and abdominal wall are placed in the groove of the window frame and the purse-string tightened. By ensuring that stitch steps are not greater than 5 mm, excessive skin folding can be avoided when tightening the purse string. The free ends of cross stitch are next tightened and tied in the groove. The open aperture of the window frame is gently dried with compressed air and a cover glass is affixed to the bottom of the bored recess of the frame using cyanoacrylate glue (g). This procedure reseals the abdominal cavity, prevents infection and effectively makes the window and the pancreas one monolithic unit (h). Using custom-made fixturing and stage plates [The surgery consists of a approximately 10 mm longitudinal incision made in the left upper quadrant of the abdomen. The skin and abdominal musculature are spread to expose the pancreas and spleen within a circular incision approximately 10 mm in diameter c. The crstitches b are pla‘basket’ d. The tae tissue e. A purs abdomen f. After ate glue g. This phic unit h. Using e plates  further . 3.2a and electronic supplementary material, movie 3. After this time, we observed an increased level of lateral and axial stability .In order to compare the performance of the SWIP to that of the AIW and the PIW, we implanted these windows into the abdomen of mice whose epithelia were transgenically labelled by cyan fluorescent protein  see Met. We obsefigure 2c–e, during the first hour, there was a period of ‘settling’ during which a high level of lateral drift was observed for the AIW and PIW (greater than 40 µm), while the lateral drift in the SWIP was slight (less than 16 µm). During the subsequent period, the PIW showed marked improvement in lateral stability and its drift was similar to the SWIP, while the AIW still performed poorly in immobilizing the tissue (figure 2f–h). Figure 2i–k shows the comparison of axial stability between these three kinds of imaging window. Though all windows exhibited the need for settling time, the SWIP showed the lowest level of drift overall and is suitable for long-term (up to the 12 h protocol limit) imaging .To directly compare the amount of drift experienced by each window, we quantified the amount of lateral and axial drift present in movies taken through the AIW, the PIW, and the SWIP by tracking an anatomical feature  over time in different mice. As shown in figure 2To demonstrate the utility of the SWIP, we next used it for several applications. First, we visualized single cell dynamics in both the normal and diseased pancreas, as well as observing the morphologic changes that occur during the onset of chemically induced pancreatitis.. 3.3a; electronic supplementary material, movie S7). Appearance and disappearance of cells could not be accounted for by a shift in imaging plane as other structures such as blood vessels remained stationary in the images. In addition, many fields of view showed cells containing subcellular structures (figure 3b) such as nuclei and organelles (structures that exclude the CFP) that would also continually appear, disappear and translocate over time .Given that the healthy pancreas has never before been visualized using single-cell resolution intravital imaging, we first implanted the SWIP into mice whose pancreatic epithelia were labelled by cyan fluorescent protein . Our expectation was that the cells of this mature and fully differentiated tissue would be static, as can be seen when imaging the ducts and lobules of the breast . Surprisingly, we found unexpected cell movement in the healthy pancreas with cells continually appearing, disappearing, and translocating during imaging . The histological appearance of PBS treated mice was similar to untreated mice when viewed by IVI or H&E and morphologically similar to the healthy human pancreas (figure 4b).Given the permanent nature of the SWIP, we were able to use microcartography, our previously published field re-localization technique ,24 to refigure 4c). Similar histologic changes were seen by H&E staining (figure 4d). In addition to these changes, several other morphological changes indicative of cell death were able to be observed, including fluorescent fragments (clustered or in interlobular spaces) and intracellular vacuoles (figure 4e). Cell death due to photo-toxicity of the IVI can be ruled out, as this was observed without cerulein treatment, even in our 12 h imaging . Comparing these images with H&E stained slides of acute pancreatitis tissue in mice (figure 4f), the structures observed by IVI show the same morphology as histological changes previously identified as apoptotic bodies, autolysosomes and autophagic vacuoles [figure 4g). In addition to these snapshot images, IVI allows observation of the formation of these events in real time. As an example, figure 4h and electronic supplementary material, movie S7 show the formation of cellular fragments as an acinar cell apparently undergoes apoptosis. CFP signal intensity changes observed in the cytoplasm of these cells which points to cytoplasmic condensation and fragmentation into apoptotic bodies. This is consistent with what we observed when staining these tissues with cleaved caspase-3 (CC3), a marker of cell apoptosis. Pancreata taken from PBS-treated mice showed no appreciable level of CC3 staining , while rounded CC3 positive cells were evident in the cerulein-treated mice .However, in mice treated with cerulein, we observed a large number of histological changes commonly observed in samples of pancreatitis taken from rodents ,29 and ffigure 4vacuoles –30. Simi. 3.5Given the high level of image stability provided by the SWIP, we were able to use our previously published technique for mosaicked acquisition of IVI movies, large-volume high-resolution intravital imaging (LVHR-IVI) . In thisb; electronic supplementary material, movie S11) and single cell migration .Using LVHR-IVI, we captured a 4 × 4 mosaic covering 1.1 × 1.1 mm of tumour tissue over a period of more than 1 h  .a–c). As can be seen in figure 6a,b and electronic supplementary material, movies S13 and S14, we were able to observe the extravasation of serum contents through localized transient vascular openings followed by its rapid clearance. We observed this extravasation of dextran in the KPCY tumour bearing model (2 of 6 mice imaged) but not in non-tumor bearing mice . The temporal dynamics of bursting (figure 6c) followed similar kinetics to those observed in mammary tumours [We IV injected KPCY animals with 155 kD TMR to label tumour vasculature and imaged their pancreata through the SWIP a–c. As c. 4ex vivo [Intravital imaging has elucidated many physiological and pathological processes in various organs –36. A prex vivo , with thex vivo , or usinex vivo , kidney ex vivo  or subcuex vivo ,41. Whilex vivo ,14,42.Here, we present a new SWIP process, which improves long-term intravital imaging of both the healthy and tumour-bearing pancreata. In addition to comparing the performance of the SWIP to two other windows reported in the literature, we used the SWIP to image (1) single cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) physiology of PDAC in both orthotopically injected and autochthonous models. While the mechanical design of window frame used for the SWIP, and the use of a cross-stitch have been published before , neithera,c) during the initial stages of pancreatitis in cerulein-treated mice, we could clearly observe the onset and progression of morphological changes that were previously reported using fixed tissues, including acinar cell autophagy (characterized by structures similar to cytoplasmic vacuoles and autolysosomes), cell apoptosis , interstitial edema and cell shrinkage [figure 4h; electronic supplementary material, movie S9).Indeed, within the healthy pancreas, we observed a surprising and previously unreported level of cellular and subcellular dynamics. Why these cells are motile, and why the pancreas, in particular, would show this motion is an intriguing question worthy of further investigation. Moreover, using serial imaging  can be et al. [The flexibility of the SWIP also allowed imaging of pancreata in tumour-bearing mice, both orthotopically injected, and those spontaneously forming within genetically manipulated mice. Consistent with the report by Beerling et al. , we did During our IVI of the murine pancreas in the KPCY mouse, we used high-molecular weight dextran to label the vasculature and incidentally observed extravasation of this agent into the interstitium of the pancreas. These bursting phenomena display the same temporal dynamics as was previously observed in primary breast tumours ,44 and tOur work presents the first ever view of the dynamics of the pancreas at the minutes to hours timescale and at single-cell resolution, making all of the data presented novel biology and necessarily broad. This work is only a first foray into the unique, unanticipated biology of this organ. As with all intravital imaging techniques, the SWIP is limited in its ability to reveal information about cells and structures that are not explicitly labelled , and to functionally manipulate proteins and cells while simultaneously visualizing them. Combination of windows such as the SWIP with fluorescent reporters and control of protein or cell states with pharmacologic and/or optogenetic tools should remove these limitations.In summary, SWIP can be widely used in benign and malignant pancreatic diseases. With the improvement in the quality and stability of IVI, it can be a useful and promising tool to offer more information about the pathophysiology and cell biology of the pancreas.


This secondary analysis of randomized clinical trials estimates the time needed to potentially derive clinical benefit from intensive blood pressure treatment in patients 60 years and older. Recent guidelines recommend a systolic blood pressure (BP) goal of less than 150 mm Hg or even 130 mm Hg for adults aged 60 years or older. However, harms from intensive BP treatments occur immediately , and benefits for cardiovascular event reduction emerge over time. Therefore, harms with low chance of benefit need to be clearer, particularly for those with limited life expectancy.To estimate the time needed to potentially derive clinical benefit from intensive BP treatment in patients 60 years and older.This secondary analysis included individual patient data from published randomized clinical trials with 27 414 patients 60 years or older with hypertension. Patient-level survival data were reconstructed when the original data were not available. Published trials were identified by searching PubMed until October 15, 2021.Intensive BP lowering vs standard BP lowering with the treat-to-target design.Major adverse cardiovascular event (MACE) defined by each trial, which was broadly similar with all trials including myocardial infarction, stroke, and cardiovascular mortality.P < .001). On average, 9.1  months were needed to prevent 1 MACE per 500 patients with the intensive BP treatment . Likewise, 19.1  and 34.4  months were estimated to avoid 1 MACE per 200  and 100  patients, respectively.Six trials  with 27 414 participants  were included in the analysis. Intensive BP treatment with a systolic BP target below 140 mm Hg was significantly associated with a 21% reduction in MACE (hazard ratio, 0.79; 95% CI, 0.71-0.88; In this analysis, findings suggest that for patients 60 years and older with hypertension, intensive BP treatment may be appropriate for some adults with a life expectancy of greater than 3 years but may not be suitable for those with less than 1 year. What is the time to benefit of intensive blood pressure treatment in adults 60 years and older?In this analysis of 6 randomized clinical trials consisting of 27 414 individuals with hypertension aged 60 years and older, 19.1 months and 34.4 months were needed to avoid 1 cardiovascular event for 100 and 200 patients, respectively.These findings suggest that intensive blood pressure treatment may be appropriate for some adults with hypertension aged 60 years and older with a life expectancy of greater than 3 years but may not be suitable for those with less than 1 year. As such, to help clinicians individualize BP control therapy among older patients with hypertension, we conducted an analysis of individual participant data on the basis of the available evidence from randomized clinical trials to determine the TTB of more vs less intensive BP control.Patients aged 60 years and older usually have tremendous heterogeneity in cardiovascular risk, such as diabetes, poor kidney function, left ventricular hypertrophy, and hyperlipidemia. Thus, clinicians need to individually weigh benefits against potential risks  when considering intensive BP control.The Xi’an Jiaotong University Health Science Center institutional review board approved this study. Patient consent was not required for this secondary data analysis.PRISMA) reporting guidelines. Two independent reviewers (Yongjuan W. and Y.G.) searched previous systematic reviews and meta-analyses in PubMed to identify published clinical trials of intensive BP in the older adult patients with hypertension. We also searched subsequently published relevant trials until October 15, 2021. We adopted the search terms used in the systematic review performed by Bavishi et al,1 which included the following Medical Subject Headings  terms: randomized controlled trial, target BP, goal BP, intensive BP, tight BP, elderly, and older patients.We performed this study on the basis of up-to-date published research. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses  compared intensive BP lowering vs standard BP lowering with treat-to-target designs , but not those with placebo as a control arm because these trials were structured to answer separate and largely incompatible clinical questions; (2) enrolled patients 60 years and older with hypertension because these patients may experience the up-front harms from the intensive BP treatment with little chance that they survive to receive the benefit; (3) provided follow-up data on cardiovascular events; and (4) had vector Kaplan-Meier (KM) curves to extract individual participant time-to-event data if original individual data could not be accessed.https://www.digitizeit.xyz/) following the instructions from Liu et al13 and Guyot et al.14 After extracting the raw data of time and survival probability, a STATA function developed by Wei and Royston15 was used to rebuild the individual data (stage 2). This validated algorithm has been used in various research.17 We presented the side-by-side comparisons of reconstructed and original curves  from KM curves by DigitizeIt software (The primary outcome is time to the first major adverse cardiovascular event (MACE), originally defined by individual trial as a composite of cardiovascular outcomes. Although the definitions of MACE differed across the included trials, they were broadly similar because all trials included myocardial infarction, stroke, and cardiovascular death. Detailed components of MACE in each trial are shown in eTable 2 in the The cumulative MACE rates at each time point in the standard and intensive BP control groups from the included trials were estimated using stratified KM curves. To address the between-study heterogeneity arising due to the clustering of participants at the study-level, the hazard ratio (HR) and its 95% CI were calculated using the stratified Cox proportional hazards model. The above analysis was repeated for different target systolic BPs .To estimate the time to specific absolute risk reduction (ARR) thresholds , we fitted Weibull survival curves using the individual participant data. We calculated TTB and its CI using the conventional frequentist method with Monte Carlo simulations. The details of these procedures are reported in eMethods in the 23 and an additional 601 studies after the latest previous electronic search included in the reviews. A total of 619 were excluded, and the remaining 67 underwent full-text assessment. Of them, 61 trials were excluded for the following reasons: not treat-to-target design (n = 51), participants were not older adults (n = 8), and no KM curves or patients-at-risk table for data reconstruction (n = 2). Finally, 6 trials were included in the analysis. Of them, original individual data from SPRINT 24 and ACCORD BP 25 were obtained through the National Heart, Lung, and Blood Institute on approval. We reconstructed the individual data for the remaining 4 trials: Cardio-Sis ,26 JATOS ,27 VALISH ,28 and STEP.4 The design and results of the included clinical trials have been reported previously.28 The search results are illustrated in We identified 85 trials from 7 systematic reviews and meta-analyses24 Cardio-Sis,26 and STEP4 trials. Other characteristics of the included trials are shown in The characteristics of the included trials are summarized in P < .001) (P = .57) (P < .001) (P = .005)  A. AlthouP = .57) B, we fou < .001) C and tar = .005) D.Analyses to determine the TTB at different clinical meaningful thresholds indicated that 9.1  months were needed to prevent 1 MACE per 500 patients with the intensive BP treatment (ARR = 0.002). Similarly, 19.1  and 34.4  months were estimated to avoid 1 MACE per 200 (ARR = 0.005) and 100 (ARR = 0.01) patients . The TTBTo our knowledge, this is the first study to use robust quantitative methods to determine the TTB for the prevention of cardiovascular events with intensive BP control in patients 60 years and older. It fills a critical gap for individually weighing benefits against potential harms while considering intensive BP control in this population, especially those with limited life expectancy. In this study, we found strong evidence for intensive BP treatment  to lower MACE events, and intensive BP treatment took 9, 19, and 34 months on average to prevent 1 MACE in 500, 200, and 100 patients, respectively, which suggested that intensive BP may be appropriate for older patients with a life expectancy greater than corresponding years after considering their adverse events  from more aggressive BP treatment.29 which contrasts with the American College of Physicians and American Academy of Family Physicians BP guideline where SBP less than 150 mm Hg is recommended in adults 60 years and older.30 These also differ from the 2018 European Society of Cardiology/European Society of Hypertension BP guideline, in which a BP of 130 to 139/70 to 79 mm Hg is considered.31 This is mainly due to the limited evidence and uncertain treatment effect from some trials28 in such population. Recently, the STEP trial4 showed that in patients aged 60 to 80 years with hypertension, targeting a reduction in SBP to 110 to 130 mm Hg resulted in a significantly lower incidence of MACE than a targeted reduction to 130 to 150 mm Hg. This was in line with the final report of SPRINT trial and its subgroup analysis report for patients 75 years and older.33 More recently, in a meta-analysis with more than 350 000 individual participants from 51 randomized clinical trials, pharmacological BP lowering to less than 120/70 mm Hg was effective into old age.2 This was consistent with our findings, which included more than 27 000 participants with a mean age of 70 years from 6 BP target trials.28The concept of intensive BP control has been extensively discussed. However, controversies still exist among the current guidelines for treating older patients. A BP target of less than 130/80 mm Hg is recommend for most adults 65 years and older in the 2017 American College of Cardiology/American Heart Association BP guideline,8 Previously, TTB was estimated through visually identifying the time point at which the curves separate.34 Clearly, this approach was subject to visual bias. Some other studies assessed the TTB by estimating the timing until the treatment effect reached statistical significance but heavily relied on an arbitrary P value.36 In the present study, we adopted the method proposed previously12 to calculate the time to reach the clinical meaningful ARR. Based on our analysis among populations with a mean age of 70 years, we found that it took 9.1 months on average to prevent 1 MACE in 500 people, suggesting that for most patients with a life expectancy of less than 1 year, the harms of intensive BP control may outweigh its benefits. Likewise, it took 34.4 months to prevent 1 MACE from intensive BP control for 100 patients, suggesting that for most patients with a life expectancy greater than 3 years, the benefits may likely outweigh the harms. Of note, a longer TTB was observed for trials with target SBP less than 120 mm Hg, which may reflect a higher cardiovascular disease (CVD) risk in the early period or immediate harms such as injurious falls, kidney disease, and other complications of hypotension following more aggressive BP treatment in comparison with target SBP less than 130 mm Hg, even though the treatment benefit was found at the end of the study.Even though the treatment thresholds differ between guidelines, clinicians are advised to determine BP targets based on a thorough review of comorbidities and patients’ life expectancy. A framework for individualizing prevention decisions incorporating TTB is being increasingly discussed. Analyzing and reporting this measurement would add more information about treatment effectiveness to clinicians’ evidence base.37 recommend that clinical judgment, patient preference, and a team-based approach to assess risk–benefit is reasonable for decisions regarding intensity of BP lowering and choice of antihypertensive drugs for older adults. The TTB as part of this information may help clinicians by providing a framework for therapeutic decisions to prioritize and individualize therapies to reduce CVD risk expeditiously. Meanwhile, it may also help patients with hypertension to recognize the potential for rapid benefit from the prescribed therapy.The summary TTB results of the present study provided a global estimate for prevention with intensive BP control; individual patients may be best served by focusing on TTB results from studies with similar intensive BP control interventions or patient characteristics. The degree to which an individual patient will benefit from intensive BP control will likely depend on their risk profile and potential harm. The BP guidelines33 it was still unclear whether this benefit could be replicated in patients older than 80 years because this specific age group has been traditionally excluded or underrepresented in clinical trials. Thus, uncertainties regarding the TTB estimates may exist among this specific population. Second, although we performed several sensitivity analyses among these included trials, such as the exclusions of ACCORD BP trial25 with a diabetic population only or trials with kidney outcomes included in the definition of MACE, we may still not extensively assess the association with TTB estimates from the heterogeneities across trials. Third, the algorithm could not provide us with additional patient-level covariates for further detailed analyses by different characteristics  even though it enabled us to closely approximate the original patient-level survival data within each individual study. Therefore, we were not sure whether patients at greater CVD risk might have a shorter TTB. Fourth, the concept of intensive BP interventions as well as its health complications among patients with hypertension has evolved tremendously in recent years. It is important to recognize the limitations of different comparators across different trials, which may affect our estimations. Finally, harms, such as injurious fall, hypotension, syncope, electrolyte abnormalities, and acute kidney injury or failure, have been reported to be associated with intensive BP treatment.39 A study from the SPRINT trial found that serious adverse events from intensive BP treatment (<120 mm Hg) were seen 9 months earlier than the decrease in MACE.34 However, the present study could not access these safety data to detail the time to harm. Therefore, further research is needed in this area.First, although results from the SPRINT trial confirmed the benefit from intensive BP treatment among patients older than 75 years and did not show evidence of an interaction between treatment and age group,In this analysis, we found that treating 100 older patients (≥60 years) with hypertension for approximately 3 years would prevent 1 MACE. These findings suggests that intensive SBP control may be most appropriate for older adults with a life expectancy of greater than 3 years. For those with a life expectancy of less than 1 year, the harms of intensive BP control may outweigh the benefits. These results reinforce the importance of individualizing intensive BP control decisions by incorporating each patient’s values and preferences.


The quality of foods taken during breakfast could contribute in shaping diet quality. This study determined the regularity of breakfast consumption and breakfast quality based on the food, energy and nutrient intakes of Filipinos.Data from the 2018 Expanded National Nutrition Survey (ENNS) was extracted for analysis. There were 63,655 individuals comprising about 14,013 school-aged children (6–12 years old), 9,082 adolescents (13–18 years old), 32,255 adults (19–59 years old), and 8,305 elderly (60 years old and above). Two-day non-consecutive 24-h food recalls were used to measure food and nutrient intakes. Diet quality was measured using Nutrient-Rich Food Index (NRF) 9.3. The sample was stratified by age group and NRF9.3 tertiles.Results showed that 96 – 98% Filipinos across age groups were consuming breakfast. Children age 6–12 years have the highest NRF9.3 average score (417), followed by the elderly (347), adolescents (340), and adults (330). These scores were very low in comparison with the maximum possible NRF score which is 900. The essential nutrient intakes of respondents were significantly higher among those with the healthiest breakfast diet (Tertile 3) compared to those with the poorest breakfast diet (Tertile 1). However, participants in the healthiest breakfast diet did not meet 20% of the recommendations for calcium, fiber, vitamin C, and potassium.This study revealed that majority of the population are regular breakfast consumers. However, the breakfast consumed regularly by Filipinos were found to be nutritionally inadequate. And even those classified under Tertile 3 which were assumed as having a better quality of breakfast were still found to have nutrient inadequacies. Thus, the study suggests that Filipinos must consume a healthy breakfast by including nutrient-dense foods such as fruits, vegetables, whole grains, fresh meat, and milk to provide at least 20–25% of the daily energy and nutrient intakes.The online version contains supplementary material available at 10.1186/s12937-022-00804-x. Breakfast is often regarded as the most important meal of the day. The American Heart Association defines breakfast as the first meal of the day eaten within 2 h after waking up . Latest Throughout the years, a significant amount of literature has supported the beneficial effects of breakfast consumption. Several studies have consistently recorded a broad variance in breakfast’s contribution to nutrient intake and diet quality in different parts of the world –9. Daily12In the Philippines, there is limited information about breakfast consumption.A typical Filipino diet in a day consists of about three and a half (3 ½) cups of cooked rice, one (1) matchbox of fried fish, and half (1/2) cup of boiled vegetables per day and these are usually consumed during the three (3) major meals of the day: breakfast, lunch and supper . Previous studies found that breakfast food pattern in the Philippines and specific provinces was rice, bread, fish, egg, and coffee which were mainly composed of foods rich in carbohydrates, protein, and fat 17. It wan = 63,655. The breakdown per age of the total sample size are as follows: 14,013 school-aged children (6–12 years old), 9,082 Adolescents (13–18 years old), 32,255 Adults (19–59 years old), and 8,305 Elderly (60 years old and above) Fig. For this study, data was derived from the 2018 Expanded National Nutrition Survey (ENNS) dietary component survey. The coverage population of the ENNS is about 40 provinces with over 80,540 surveyed individuals with a response rate of 81.5%. The 2018 ENNS utilized the 2013 Master Sample List developed by the Philippine Statistics Authority (PSA). This sampling design followed a two-stage cluster sampling technique. The first stage involved the selection of Primary Sampling Units (PSUs), which involved sampling domains from 81 provinces, wherein 16 sample replicates was drawn from each domain. The second stage involved the selection of households from the 16 sample replicates. During this stage, the selected households served as the final sampling unit [Breakfast was defined based on the meal code number 1 indicated in the questionnaire which was self-reported as “Breakfast” by the interviewee. On the other hand, breakfast skipping was defined in this study as no breakfast consumption or less than 50 kcals of energy consumed at breakfast otherwise regular consumer if more than or had 50 kcal . Two dayDiet quality was measured using the Nutrient Rich Food Index (NRF) 9.3. The NRF9.3 is a validated measurement tool to determine the nutrient density of the total diet. The determination of the nutrient density score for the NRF 9.3 is calculated by the sum of the percentage daily reference values (DRVs) of the nine nutrients that are recommended  minus the sum of the percentage of DRVs for the three nutrients to limit . Total sugar intake was used since added sugar was not included in the survey. The closer the value is to the maximum score of 900 would indicate better quality of the overall diet. The basis for the NRF9.3 algorithm was from a nutrient profiling model on linking food items that have the highest nutrient density at the same time, affordable and engaging , 22, 23.The Philippine Dietary Reference Intake (PDRI) and other standard recommendations were used to calculate the reference daily values (DVs) . The following were the qualifying nutrients and standard reference amounts: protein (50 g), fiber (25 g), vitamin A (1500 RE), vitamin C (60 mg), vitamin D (10 mcg), calcium (1000 mg), iron (18 mg), potassium (3500 mg) and magnesium (400 mg). The 3 disqualifying nutrients and maximum recommended values (MRVs) were: added sugar (50 g), saturated fat (20 g) and sodium (2400 mg).The following is the NRF 9.3 calculation formula:withandintakei is the intake of each nutrient, ED is the energy density, and DVi is the reference daily value for that nutrient. In NR calculation, the intake of each nutrient for each subject was normalized for 2000 kcal and expressed as a percentage of the reference DVs. Previously, nutritional % DVs were capped at 100, so that an extremely high intake of one nutrient could not compensate for a dietary deficiency of another. Only the portion in excess of the recommended quantity was taken into account for the LIM.where Food group consumption was express as actual intake (in grams) and percentage of consumer. Consumers of each food group were scored 1 if they consumed at least 10 g otherwise 0 (< 10 g) . Only thThe percentage contribution of breakfast to daily nutrient intake was calculated by calculating the total intake of each nutrient at breakfast divided by the total intake of each nutrient at daily intake multiplied by 100. The percentage contribution of breakfast among people with the healthiest breakfast quality to the daily recommendation was calculated by dividing their intake to the recommendation for each nutrient and their respective recommendations and then multiplied by 100. Same calculation was done for the percentage contribution of nutrient intake from all meals to daily recommendation.Quality control of the dietary intake data was conducted in two steps. In the first step, the foods reported by a participant like coding information, and quantity were reviewed. In the second step, scatterplots and histograms was used to determine the outliers of the datasets. For implausible micronutrient intake, excessive intakes were defined as those that exceeded 1.5 times of the 99th percentile of the observed intake distribution of the nutrient in the corresponding sex, and age group. Intakes above the upper limit were substituted by a random value generated from a uniform distribution in the intervals with lower bound equal to the 95th percentile of the observed intake and an upper bound equal to 1.5 times the 99th percentile . After vp < 0.05.PCSIDE version 1.02  was utilized in estimating the usual nutrient intakes at breakfast. This program estimates distributions of usual nutrient intake by removing the effect of day-to-day (intra-person) variability in intake from daily intakes , 30. AllTable Tables In Table Table Tables Figure Figure Adolescents with the healthiest breakfast diets met the 20% recommended intake of protein (26%), vitamin D (29%), vitamin A (22%) and magnesium 24%). Only 7% of fiber at breakfast was consumed. Poor intakes of calcium (12%), iron (15%), and vitamin C (12%) were observed at breakfast for adolescents. There is a slightly low contribution (11%) of total sugar, saturated fat (17%), and sodium (13%) for this group. Overall, it is noticeable that most micronutrients were inadequate especially calcium, fiber and vitamin C  were adequate in energy (23%), protein (27%), iron (20%), vitamin D (28%), vitamin A (20%), potassium (23%) and magnesium (28%) at breakfast. However, contribution of calcium (17%), vitamin C (12%), and fiber (9%) at breakfast is low, which is an unceasing problem up until this age group Fig. .Fig. 5CoAmong the elderly with the healthiest breakfast diets, their breakfast consumption reached more than the 20% recommended intake of energy (21%), protein (21%), iron (23%), and magnesium (23%). However, the composition of their breakfast was inadequate in fiber (10%), vitamin D (9%), vitamin C (15%), and calcium (17%). There was only 1% excessiveness of total sugar (21%), and no excessiveness of saturated fat and sodium. On a daily basis, they were inadequate of calcium, fiber, vitamin D, and vitamin C Fig. .Fig. 6CoThe present study determined the regularity of breakfast consumption and its contribution to the daily energy and nutrient intakes of Filipinos in order to serve as basis for breakfast recommendations in the Philippines. To our knowledge, the present study was probably the first attempting to describe the breakfast consumption of Filipinos. With the use of the secondary data from the 2018 ENNS gathered by the DOST-FNRI, this study was able to analyze the breakfast intakes of Filipinos . AlthougResults from the analysis reported that majority of the Filipino population (96-98%) regularly consume breakfast. This strongly identifies that breakfast is an important meal for Filipinos. As stated in a previous literature, the regularity of breakfast consumption and meal times were closely related to healthy lifestyle habits and could play an important role in providing adequate nutrients –33.In terms of breakfast energy and nutrient intakes, the present analyses also allowed this study to identify food choices and breakfast patterns that were associated with the highest quality of breakfast diets, as captured by NRF 9.3 scores. Participants were divided into tertiles based on their NRF 9.3 scores for each age group to investigate the associations between breakfast nutrient intake and overall diet quality. The age groups with the highest NRF 9.3 score or Tertile 3  were characterized in this study to consume higher amounts of essential nutrients and more diversified diets as per food groups consumption, compared to lower tertiles. Moreover, a higher NRF score was associated with a higher consumption of desirable food groups making the index an ideal measurement of overall diet quality.Upon analysis, the current study reported that energy intakes at breakfast does differ across NRF 9.3 tertiles. More so, it was revealed that Filipino children aged 6–12 years had the highest NRF average scores 417), followed by elderly (347), adolescents (340), and adults (330). Yet, all these scores were still considered to be very low in comparison with the maximum possible NRF score which is 900; and if compared with the findings from previous IBRI studies conducted in Western countries , followe. These rFilipino children and adolescents with the healthiest breakfast diets (Tertile 3) were associated with higher intakes of energy, total protein, total carbohydrates, total fiber, and micronutrients  compared to counterparts (Tertile 1 and Tertile 2). While for Filipino adults and elderly, those consuming the healthiest quality of breakfast diet exhibited higher intakes of energy, total protein, total carbohydrates, total fat, MUFA, PUFA, total fiber, micronutrients , and cholesterol. Even so, participants classified under the group with the healthiest diet (Tertile 3) still did not meet 20% of the dietary recommendations for calcium, fiber, vitamin C, and potassium, which clearly represents an opportunity for needed improvement.These results were reflected in the breakfast consumption of food groups as higher intakes of fresh fish, chicken egg, fresh meat, green leafy vegetables and other vegetables, and lower intakes of rice, coffee, sugars, and other cereal products were found among groups with the healthiest breakfast diet. These food groups are included in the 2012 Nutritional Guidelines for Filipinos (NGF) which reflects the impact of its consumption towards a healthier diet . The curThe investigation for the main sources of these aforementioned nutrients that Filipinos were found to be inadequate had revealed that the food groups that were associated as the richest sources of these nutrients were not consumed at breakfast. According to the food consumption survey, vitamin C intakes in the Philippines had an 83%-95% inadequacy rate which is very alarming . This is39Among Filipino breakfast consumers, more than 20% of the total daily energy intake was from breakfast. This finding is in line with a previous breakfast study conducted in Denmark , which iIndeed, results in this study had revealed that emphasis should be given on the consumption of fruits, vegetables, milk, and fresh meat which were among the least consumed food groups during breakfast among the Filipino population. Yet, it is also alarming that fruits as well as milk and milk products were found further down the list of mostly consumed food groups among Filipinos. When considered together, the poor food choices at breakfast are likely contributing to the suboptimal intake of some nutrients at this meal  , which could be improved by the higher consumption of fruits, vegetables, fresh fish, fresh meat, and milk, and lower consumption of refined grains, coffee and sugary products.Datasets for the food recalls did not record whether the food recalls were on a weekday or weekend. With the large sample size used in this study, this consideration could already be factored-in. The usage of dietary supplements was not included in this study since the goal was to estimate nutrient intakes through food and beverages only. This might result in an underestimate of total daily micronutrient consumption.This study revealed that majority of the population are regular breakfast consumers. However, the breakfast consumed by Filipinos were found to be of poor quality. And even those classified under tertile 3 which were assumed as having a better quality of breakfast were still found to have nutrient inadequacies. Based on the findings obtained, it could be suggested that Filipinos must consume a healthy breakfast by including nutrient-dense foods such as fruits, vegetables, whole grains, fresh meat, and milk to provide at least 20–25% of the daily energy and nutrient intakes. The results from this study also provide strong evidence for a positive impact of breakfast consumption on overall diet quality. These findings could help to inform the development of nutrient-based recommendations for a balanced breakfast for the first time in the Philippines.The findings of this study opened a window of opportunity in improving the diet quality of breakfast in the Philippines by re-emphasizing recommendations in the Pinggang Pinoy and on the consumption of nutrient-dense foods. A future favorable turnout of the study would be the expansion of the national feeding program with the inclusion of breakfast and the possibility of extending the feeding until the adolescent age. As per Republic Act 11,037, which is the signed law that enacts free meals at public schools for undernourished kids, the current national nutrition program partnered by the Department of Education (DepEd) and Department of Social Welfare and Development (DSWD) only provides lunch meals to preschool until elementary children. Expansion from breakfast to lunch and targeting up until the adolescent age may help in mitigating micronutrient deficiencies through age groups, since breakfast is consumed by majority of the population.Additional file 1:Table S1. Age- and sex- specific reference daily values for young age group used for the calculation of NRF 9.3. Table S2. Age- and sex- specific reference daily values for adult age group used for the calculation of NRF 9.3.Additional file 2: Table S3. Food Group Classification.


Polymeric nanoparticles have attracted much attention as pharmaceutical delivery vehicles to prolong residence time and enhance the bioavailability of therapeutic molecules through the mucoadhesive phenomenon. In this study, chitosan:TPP nanoparticles were synthetized using the ionic gelation technique to analyze their mucoadhesive interaction with reconstituted porcine gastrointestinal mucus from a triborheological point of view under different pH conditions . The triborheological profile of the reconstituted mucus was evaluated at different pH environments through the oscillation frequency and the flow sweep tests, demonstrating that the reconstituted mucus exhibits shear thinning behavior regardless of pH, while its viscoelastic properties showed a change in behavior from a polymeric solution performance under neutral pH conditions to a viscoelastic gel under acidic conditions. Additionally, a rheological synergism analysis was performed to visualize the changes that occur in the viscoelastic properties, the viscosity and the coefficient of friction of the reconstituted mucus samples as a consequence of the interaction with the chitosan:TPP nanoparticles to determine or to discard the presence of the mucoadhesion phenomenon under the different pH values. Mucoadhesiveness evaluation revealed that chitosan:TPP exhibited strong mucoadhesion under highly acidic pH conditions, below its pKa value of 6.5. In contrast, at neutral conditions or close to its pKa value, the chitosan:TPP nanoparticles’ mucoadhesiveness was negligible. Mucus is a viscoelastic gel that protects the wet epithelial surfaces of gastrointestinal tissues  through On the other hand, gastrointestinal mucus consists of 95% of water, with the remaining 5% forming different molecules, including glycoproteins, antibodies, lipid complexes, inorganic ions, growth factors, DNA, cells and mucin ,7. MucinMucin consists of a protein core with regions rich in serine and threonine, enabling a high grade of O-glycosylation that makes it resistant to enzymatic degradation and capable of holding water, giving it its hydrogel properties . MoreoveIn the same way, the wide range of pH variation (between 1.0 and 8.0) throughout the gastrointestinal tract, considering from the oral cavity to the colon, represents a fundamental variable in the study of gastrointestinal mucus rheology. Since the environmental pH plays a key role in the structural and conformational modifications that mucus undergoes and, consequently, in the variations of its rheological properties . Mainly By considering the fluctuating pH conditions in the gastrointestinal tract, different polymer-based approaches have been studied for drug delivery purposes, including nanoparticles , hydrogeChitosan is a natural cationic polymer synthetized by the N-deacetylation of chitin. This biopolymer exhibits good biodegradability, high stability, low toxicity, biocompatibility, mucoadhesiveness, sustained release and tuneable physical properties ,24,25,26G′ and G′′) or in the viscosity (η) when the interpenetration of polymer and mucin chains occurs during the phenomenon of mucoadhesion [Mucoadhesion refers to the attachment of a natural or synthetic molecule to mucus for extended periods of time through different interfacial forces . The mucadhesion ,38.This work characterizes the viscoelastic, viscous and lubricating properties of reconstituted mucus through the fluctuating pH conditions to which mucus is subjected throughout the gastrointestinal tract with the aim of analyzing how mucus physicochemical changes affect its biological properties. Considering that mucus undergoes a pH-dependent transition when the gastrointestinal conditions change from acidic to neutral pH. However, this research focuses mainly on the application of the rheological synergism method to study the presence of the mucoadhesive phenomenon with the objective of determining the acidic conditions within the gastrointestinal tract that promote a greater degree of adhesion between mucus and chitosan:TPP nanoparticles. Additionally, this study analyzes the implications of the mucoadhesive phenomenon in the lubricating properties of mucus under different acidity environments.Mucin from porcine stomach type III, bound sialic acid 0.5–1.5%, partially purified powder and Chitosan, low molecular weight, were purchased from Sigma-Aldrich . Simulated Intestinal Fluid TS was obtained from RICCA Chemical. Sodium Tripolyphosphate (TPP) was acquired from Merck. All the other reagents were of analytical grade. w/v). The solutions were incubated at 37 °C under constant agitation at 200 rpm for 24 h. At the end of the incubation period, each sample was carefully checked to ensure that it maintained its original pale yellow color and a completely homogeneous consistency to ensure that there was no contamination. Subsequently, the pH of each solution was adjusted to the corresponding value  to perform all the different analyses. The pH adjustment was made by adding the appropriate volumes of 1 M HCl or 1 M NaOH until the expected pH was obtained.The preparation and manipulation of the reconstituted mucus samples were carried out under sterile conditions. Each sample of reconstituted mucus was prepared by diluting 500 mg of mucin in 10 mL of simulated intestinal fluid (SIF) to obtain solutions with a final mucin concentration of 5% . Simultaneously, a 1 mg/mL solution of TPP was prepared by adding 50 mg of TPP into 50 mL of 1% acetic acid solution. The pH of both solutions was adjusted to 5.0 using 1 M HCL. The chitosan solution was stirred at 800 rpm for 60 min, while the TPP solution was stirred for 30 min. Next, TPP solution was added dropwise to the chitosan solution, which was under magnetic stirring at 800 rpm. The mixture was stirred for 60 min at 800 rpm. Then, the mixture was sonicated for 30 min at 40 kHz. Finally, the nanoparticle solution was frozen at −20 °C and placed inside the freeze dryer for 48 h. It is emphasized that before some characterization methodologies  and triborheological tests , the lyophilized nanoparticles were diluted in distilled water at 3 mg/mL concentration and sonicated for 20 min at 40 kHz. Then, the pH of each solution of nanoparticles was adjusted to 2.0, 4.0, 6.0 or 7.0, respectively. Mean hydrodynamic diameter, polydispersity index (PDI) and zeta potential of chitosan:TPP nanoparticles were measured at 25 °C with 2 min of equilibrium using a Malvern Zetasizer Nano ZS. Dynamic light scattering (DLS) was used for the measurements of particle size and PDI with a laser wavelength of 633 nm and a 173° detection angle inside DTS0012 disposable cuvettes. Moreover, for zeta potential measurements, the electrophoretic light scattering (ELS) technique was utilized with DTS1070 disposable cuvettes. The analysis of chitosan:TPP nanoparticles morphology was performed by a JEOL JSM 6360 scanning electron microscope operated at an applied voltage of 15 kV using a high vacuum of the beam diameter and secondary electrons at magnifications of 5000×. Before the analysis, freeze-dried samples were placed on the sample holder and gold-sputtered on a JEOL JFC-1100E to enhance their conductivity properties.Additionally, lyophilized nanoparticles were diluted in distilled water at a concentration of 3 mg/mL and sonicated for 20 min at 40 kHz. A drop of the solution of nanoparticles was placed on the sample holder and dried at room temperature for 48 h under a vacuum. The dried sample was gold sputtered and analyzed in the SEM at an applied voltage of 20 kV using a high vacuum of the beam diameter and secondary electrons at magnifications of 1600×. G′) and loss (G′′) modulus. While the flow sweep test was carried out using shear rate values between 0.1 and 1000 s−1 to determine reconstituted mucus viscosity. Rheological profiling of reconstituted mucus was performed with a TA Instruments Hybrid Rheometer DHR-3 using a cone-and-plate configuration formed by a 60 mm diameter stainless steel cone geometry (0.9969°) and a Peltier system to control the sample temperature at 37 °C to mimic human body conditions. The analysis of the rheological properties of reconstituted mucus under different pH values included oscillation frequency and flow sweep tests. The oscillation frequency test was performed with an angular frequency range between 0.1 and 100 rad/s at a strain of 1% to identify reconstituted mucus storage ((a)G′) were compared to evaluate the mucoadhesive interactions among reconstituted mucus and chitosan:TPP nanoparticles. The evaluation was calculated as ΔG′ using Equation (1):G′ refers to the mucoadhesive interaction term (the elastic component is interpreted as the interaction between nanoparticles and mucus), G′mix refers to the elastic modulus of the mixture, G′muc refers to the elastic modulus of the reconstituted mucus and G′np refers to the elastic modulus of the chitosan:TPP nanoparticles;The values of the elastic modulus ((b)η’ refers to the viscosity due to mucoadhesive interaction, η’mix refers to the viscosity of the mixture, η’muc refers to the viscosity of the reconstituted mucus and η’np refers to the viscosity of the chitosan:TPP nanoparticles.The viscosities of the three samples  were contrasted to evaluate mucoadhesion through Equation (2):The rheological tests described in Triborheological profiles of reconstituted mucus under different pH conditions were evaluated in the DHR-3 Hybrid Rheometer (TA Instruments) using a plate-and-plate configuration conformed by a 40 mm diameter stainless steel plate and a Peltier system that acts as a temperature controller (37 °C) during the tests. The coefficient of friction was evaluated by performing a flow sweep test from 0.1 to 50 rad/s, applying a normal force of 1 N. Additionally, the triborheological performance of the mixture (reconstituted mucus–chitosan:TPP nanoparticles) was measured under the same conditions and compared with reconstituted mucus performance to evaluate the alterations in the coefficient of friction produced by the mucoadhesive interaction. p < 0.05 using Minitab 19 software .All experiments were performed in triplicate, and the data shown are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and post hoc Tukey tests was calculated with a significant level of Triborheology tests were carried out to determine the variations in the viscoelastic, viscous and lubricant profiles that reconstituted mucus undergoes through the different pH conditions presented in the gastrointestinal tract , considering a temperature of 37 °C to mimic the human body.Oscillation frequency tests showed that reconstituted mucus exhibited significant changes in its viscoelastic properties depending on the pH conditions, as G′) is predominant over the loss modulus (G′′) all the time. This behavior is explained by the protonation of the carboxylic groups and the breaking of disulfide groups of the mucin, which induces an unfolding and exposure of hydrophobic regions [At highly acidic conditions (pH = 2), the viscoelastic behavior exhibited a clearly gel-like response since the storage modulus  at very low frequencies followed by a modulus crossover to storage modulus (G′) dominance around an angular frequency of 1 rad/s. This performance is typical in solutions that act as weak gels [Instead, under neutral pH conditions (pH = 6 and 7), mucus behaves as a viscous polymeric solution with an initial dominance of the loss modulus  and loss (G′′) modulus is significantly reduced. Additionally, the order of the storage modulus is reduced by half, while the order of magnitude in the viscous modulus is lightly increased. The situation was expected, considering that the mucus isoelectric point is found around the pH range of 2.0–3.0, which represent the critical point in the charge shift and conformational structure of mucus [Moreover, under slightly acidic conditions (pH = 4), the transition from a weak gel behavior to a polymeric solution can be visualized because the gap between storage . The triborheological studies showed that, independently of the pH conditions, reconstituted mucus exhibited minimal variations in its lubricant properties under low velocities. However, when mucus reached the hydrodynamic lubrication regime, the sample with pH 2 exhibited a significant increase in its coefficient of friction (CoF) in comparison with the other mucus samples, as the Stribeck curves of The reconstituted mucus reached the boundary lubrication stage with CoF values around 0.16 ± 0.017 in the angular velocity range of 0.01 to 0.6 rad/s, followed by a rapid decrease in CoF values to 0.04 ± 0.008 during the mixed lubrication, which was found at the angular velocity range of 0.6 to 3.9 rad/s. Mucus tends to exhibit good lubricant performance in the boundary and mixed lubrication regimes with very low CoF values (around 0.01) because, during the application of shear forces, the friction is reduced because the energy is dissipated by the movements of the water molecules found in the mucus layer . HoweverFinally, reconstituted mucus reached the hydrodynamic lubrication, in which the pH represented an important variable. Mucus showed a dramatic increase up to 0.26 ± 0.027 in the CoF under highly acidic conditions (pH = 2). In contrast, the samples with slightly acidic (pH = 4 and 6) and neutral (pH = 7) pH exhibited more controlled behavior of their CoF values with an increase to 0.23 ± 0.036, 0.23 ± 0.038 and 0.19 ± 0.031, respectively. This performance is mainly attributed to the pH-dependent viscoelastic behavior of mucus because the higher viscosity of mucus under more neutral conditions allows the formation of fluid films at lower angular velocities, which, in conjunction with the shear thinning property, reduces friction as a result of a hydrodynamic drag at high angular speeds ,55.Chitosan:TPP nanoparticles were successfully synthetized by ionic gelation methodology. In the first instance, the formation of chitosan:TPP nanoparticles was determined qualitatively by observing a transition in the coloration of the chitosan solution, which changed from a clear solution to a light-blue one, producing the typical opalescence of colloidal particles in suspension due to the Tyndal effect, during the dropwise addition of TPP solution ,57. ThisMoreover, the sonication step after the ionic gelation method represented a critical stage during the synthesis since the ultrasonic waves in the range reduce particle size and polydispersity index (PDI) of the nanoparticles by crushing the aggregates formed during the synthesis ,60. In tOn the other hand, the wide variation in pH throughout the gastrointestinal tract represents a significant challenge for all the molecules that interact with this hostile environment that ranges from pH 1–3 in the stomach to pH 6–7.5 in the intestine  . ConsequThe synthesis of chitosan:TPP nanoparticles is achieved through chitosan’s ability to gel spontaneously on contact with polyanionic TPP solution as a result of inter- and intra-molecular cross-linking interaction among chitosan cationic amino groups and polyanionic phosphate groups . TherefoThe size variations exhibited in the results demonstrated that the pH of the medium represents a critical variable involved in the final particle size and PDI of the chitosan:TPP nanoparticles. Chitosan is a weak polyelectrolyte with a pKa value of around 6.5, so its degree of protonation is mainly affected by the pH of the solution; even chitosan is insoluble at neutral and alkaline pH . Under aOtherwise, zeta potential measures the electrostatic charges of nanoparticles, which represents a major factor in phenomena such as dispersion, flocculation or aggregation, and hence zeta potential is associated with the stability of nanoparticle solutions . Since cMorphology of the synthetized chitosan:TPP nanoparticles were examined with Scanning Electron Microscope, as Chitosan:TPP nanoparticles were visualized with a scanning electron microscope to obtain detailed information about their morphology, as Instead, Mucoadhesion describes the adhesive interaction between the mucus layer and the mucoadhesive polymer due to hydrogen bonding, Van der Waals forces, hydrophobic interactions, ionic interactions and physical entanglement ,78. One G′) under acidic conditions  because the elastic modulus (G′) is larger in the mixture of mucin–chitosan:TPP nanoparticles than in the individual and in the sum curves. These results showed a high affinity of chitosan:TPP nanoparticles for reconstituted mucus based on an interaction mainly mediated by the opposite charges of the two biopolymers that influence an entanglement of mucin and polymer chains [G′) in comparison with the sum of the elastic modulus (G′) values of the single components [G′) of the mucus and the nanoparticles are not significantly different (p < 0.05). Thus, a negligible mucoadhesion is established as a consequence of the deprotonation of the amino groups in chitosan:TPP nanoparticles at neutral pH, forming an insoluble polymer layer, making difficult the interpenetration among polymeric and mucin chains [The oscillation frequency tests displayed in r chains , causingmponents . Moreoven chains ,84. This mucoadhesive profile of chitosan:TPP nanoparticles is mainly attributed to the formation of secondary bonds and electrostatic interactions of positively charged amino groups of chitosan with negatively charged mucus moieties, such as sialic and sulfonic acid residues ,86. HoweAdditionally, a viscosity-based synergistic approach can be taken to determine rheological synergism (Δ η) based on the difference between the viscosity of the mixture and the sum of the individual components . The visp < 0.05) than the sum of the individual viscosities as p < 0.05), although it remains positive. These changes are produced by the influence of chitosan ionizable groups that tend to deprotonate near its pKa value (6.5), reducing the charge density in the surface of chitosan:TPP nanoparticles and, consequently, reducing the electrostatic interactions [Furthermore, the results demonstrated that at highly acidic values (pH = 2 and 4), there is a clear presence of a positive rheological synergism due to the viscosity values of the mixture being significantly higher  and highly positive zeta potential (23.55 ± 0.84 mV) in an extremely acidic environment. In contrast, the size of the nanoparticles increased up to 506.23 ± 49.98 nm with a zeta potential close to zero (3.87 ± 0.79 nm) at neutral conditions. This performance is related to the cationic nature of chitosan (pKa∼6.5) and the protonation/deprotonation of its amino groups. p-value > 0.05. The elastic modulus term (ΔG′) showed a positive synergism for all the samples under acidic conditions , while the sample at neutral conditions (pH = 7) exhibited a negligible synergism. Instead, the viscosity (η) synergism was only observed in highly acidic conditions (pH = 2 and 4), whereas slightly acidic (pH = 6) and neutral (pH = 7) samples showed imperceptible synergism. Considering that both rheological synergism studies are complementary, the mucoadherence phenomenon only occurs at acidic pH since the amino groups of chitosan are protonated and establish electrostatic interactions with negatively charged mucus. The mucoadhesive profile of the chitosan:TPP nanoparticles was evaluated by rheological synergism with a Finally, the Stribeck curves of the mixtures of reconstituted mucus–chitosan:TPP nanoparticles proved that mucoadhesion is related to enhanced lubricant properties because the physical chain entanglement and formation of electrostatic interactions among mucin and chitosan molecules are associated with better load-bearing and wear resistance properties.


Genomic DNA was extracted from young leaves and the PCR reactions were performed using ten primers for each SCoT, ISSR and RAPD marker. Only four out of ten primers showed amplicon profiles in all PCR markers. A total of 70 bands were generated by SCoT, ISSR and RAPD-PCR with 35 polymorphic bands and 35 monomorphic bands. The total number of bands of RAPD, ISSR and SCoT was 15, 17 and 38, respectively. The polymorphism percentages achieved by RAPD, ISSR and SCoT were 60.25%, 15% and 57%, respectively. The cluster analysis based on morphological data revealed two clusters. Cluster I consisted of violet and orange kalanchoe, and cluster II comprised red, yellow and purple kalanchoe. Whereas the cluster analysis based on molecular data revealed three clusters. Cluster I included only yellow kalanchoe, cluster II comprised orange and violet kalanchoe while cluster III comprised red, and purple kalanchoe. The study concluded that orange, violet and yellow kalanchoe are distinguished parents for breeding economically valued traits in kalanchoe. Also, the study concluded that SCoT and RAPD markers reproduced reliable banding patterns to assess the genetic polymorphism among kalanchoe genotypes that consider the basis stone for genetic improvements in ornamental plants.Determining the appropriate parents for breeding programs is the most important decision that plant breeders must make to maximize the genetic variability and produce excellent recombinant genotypes. Several methods are used to identify genotypes with desirable phenotypic features for breeding experiments. In this study, five kalanchoe genotypes were morphologically characterized by assessing plant height, number of inflorescences, number of flowers, flower length, flower diameter and number of petals. The analysis showed the distinction of yellow kalanchoe in the plant height trait, while the orange kalanchoe was distinguished in the number of inflorescences, the number of flowers and flower length traits, whereas the violet kalanchoe possessed the largest flower diameter and the highest number of petals. The molecular profiling was performed by random amplified polymorphism DNA (RAPD), inter-simple sequence repeats (ISSR) and start codon targeted (SCoT)- Kalanchoe is a medicinal plant largely used in folk medicine for the treatment of kidney stones, gastric ulcer, pulmonary infection and rheumatoid arthritis  and is gThe species of kalanchoe play an important role in scientific research of genetic diversity and evolutionary aspects of plants. Plant diversity has captivated humans throughout history, owing to the enormous variation in molecular and morphological features found in nature. The naturally occurring variations among plant species provide a familiar environment for evolution by natural selection, plant taxonomy and phylogeny are based on genetic diversity ,6,7.In the field of ornamental plant improvement, distant hybridizations are still a common method of promoting genetic diversity in plants. More than 100 plant species have derived from this variation in breeding and genetic improvement programs ,8,9 by aAllium species [Coffea Arabica plants [Punica granatum L.) [Dianthus [Several studies discussed the genetic diversity of various ornamental plants such as  species , ornamena plants , ornamenatum L.) , ornamenatum L.)  and DianDianthus .Conventional DNA markers have numerous applications in determining genetic diversity in plants. These markers include inter simple sequence repeat (ISSR) markers , sequencThis investigation was performed to assess the genetic diversity among five kalanchoe genotypes by combining morphological characterization with the RAPD, ISSR and SCoT molecular markers and phylogeny analyses.Five kalanchoe genotypes were maintained under greenhouse conditions and assessed for morphological features; number of petals, flower length, number of flowers, flower diameter, number of inflorescences and plant height  which ha−1), number of flowers (321 flowers plant−1) and flower length (2.1 cm). The violet genotype recorded the highest number of petals .The yellow kalanchoe genotype showed the highest plant height (40 cm), while the orange kalanchoe genotype got the lion’s share due to recording the highest number of inflorescences  values. A significant difference was noticed between the yellow kalanchoe genotype and the remaining genotypes, except for the orange kalanchoe in plant height character A. The anFlower length (cm) was significantly different among both orange and yellow genotypes and the rest genotypes, while violet, purple and red genotypes were insignificantly different from each other D. FlowerGenetic polymorphism analysis of kalanchoe genotypes was conducted by RAPD, ISSR and SCoT-PCR amplifications using 30 arbitrary primers (10 primers for each PCR reaction type)  to examine the molecular polymorphism among kalanchoe genotypes. Finally, only four primers for each reaction produced reliable polymorphic banding profiles within all the studied genotypes . The reaPhylogenetic relationships among the five kalanchoe genotypes were inferred based on the data recorded from morphological criteria. The clustering analysis grouped the five kalanchoe genotypes into two groups  . ClusterThe success of breeding programs is determined by the accurate selection of parents. At this point, breeders begin their search for a specific plant genotype that will meet market expectations. Even while recombination may play role in expanding the polymorphism among segregating populations, the ability of two parents to combine their progeny and their excellent performance in agronomic variables will identify whether the progeny will be successful elite lines ,19. GiveIn this investigation, five kalanchoe genotypes were characterized regarding their morphological and molecular features. The current results of morphological characterization revealed that some kalanchoe genotypes had advantages in some traits, and this can be considered as a cornerstone in breeding and genetic improvement of kalanchoe. The orange kalanchoe offered advantages in its number of inflorescences, number of flowers and flower length, which are important traits in the characterization of ornamental plants. Hence, orange kalanchoe genotypes may be used as parents in breeding programs for these parameters.Kalanchoe genotypes are morphologically diverse and hence are great candidates for intraspecific hybridization to produce new lines with favorable features.In the same context, the yellow kalanchoe presented advantages in both plant height and flower length, also the violet kalanchoe showed the highest score in flower diameter and number of petals which are considered the most important features in the ornamental field. For successful Kalanchoe breeding programs, it is important to completely characterize the morphological traits, especially those that have economic importance such as number of inflorescences, number of flowers and flower length. Achieving desired breeding results requires correct parent selection and understanding of the genetic distance either based on morphological features or molecular profiling and understanding the inheritance of the desired traits . This stThe current study emphasized the potential importance of DNA polymorphism detection for plant breeding programs and genetic improvement. Plant genetic diversity can be studied and detected with the help of molecular markers . For genThe molecular markers (RAPD and SCoT) produced reliable polymorphic banding patterns enabling the determination of genetic polymorphism among kalanchoe genotypes. The ISSR produced a low polymorphism ratio compared to both RAPD and SCoT markers. The RAPD and SCoT-PCR markers used in this study may be considered as more efficient in identifying the genetic polymorphism based on their polymorphic banding profiles than ISSR-PCR which produced a low polymorphism percentage. This may provide important clues in distinguishing the relationship among kalanchoe genotypes. These results are similar to those reported by Collard and Mackill , they foBased on the results obtained from morphological data and molecular profiles of the kalanchoe genotypes, we generated two phylogenetic trees. Phylogeny and alignment are important analyses to determine the genetic diversity among different species ,33,34,35Despite the current study may support the conventional breeding methods for genetic improvement of kalanchoe genotypes, however recent genetic approaches such as genetic engineering  or genomThe five genotypes of kalanchoe used in this study were obtained and classified by a plant taxonomist in the Faculty of Science. The plants were kept in a controlled greenhouse environment with natural light, at a temperature of 15 °C at night and 25 °C during the day.Morphological data; plant height, number of inflorescences, number of flowers, flower length, flower diameter and number of petals were collected. Plant height was recorded on the day when the first flower bloomed. Flower diameter, floral length, and the number of petals were recorded at the sticky stage of stigma . On the –1 and DNA was stored at –20 °C for the next amplification experiments.The genomic DNA was extracted from 5 g of sterilized (0.05% Clorox) young leaf samples using the cetyltrimethylammonium bromide (CTAB) method . A Nano 2 (25 mM), 1 μM of each primer (10 pmol) and 2 μL of genomic DNA (20 ng/μL). The final volumes were adjusted with sterile distilled water up to 25 μL. The RAPD-PCR-based amplification was performed using a 96 well plate thermal cycler (Applied Biosystem) as the following: 95 °C for 1 min for initial denaturation, followed by 40 cycles of 95 °C for 30 s, 1 min at the annealing of each primer and 72 °C for 1 min for extension and the final extensions were done at 72 °C for 5 min.PCR amplification was performed by ten primers as shown in 2 (25 mM), 2 μL dNTPs (200 μM), 2 μL Primer (10 pmol) and 1 Unit Taq DNA polymerase (Promega). The conditions of ISSR-PCR amplifications were used in an initial denaturation of 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, the annealing temperature for the various primers for 1 min, 72 °C for 1 min (extension) and final extension at 72 °C for 5 min.ISSR-PCR-based reaction was performed to detect the polymorphism among kalanchoe genotypes using the primers presented in 2, 2 μL 5× buffer and 1 Unit of Taq polymerase (Promega). The PCR amplification was set up for the initial denaturation at 94 °C for 5 min, followed by 35 cycles, each cycle comprised of 94 °C for 1 min, 53 °C for 1 min, then 72 °C for 120s, and the final extensions were done at 72 °C for 7 min.The SCoT primers  were cho1.5% agarose gel electrophoresis in TBE buffer was used to separate the results of the RAPD, ISSR, and SCoT reactions according to . The sizt-tests at p ≤ 0.05.Microsoft Excel was used to analyze the morphological data. For the number of petals, flower length (cm), flower diameter (mm), number of inflorescences, number of flowers, and plant height, significant differences were evaluated using Student The SCot, ISSR and RAPD-based PCR loci were scored as present 1 or absent 0, each of which was treated as independent. Genetic diversity was identified by comparing the banding patterns of all genotypes. Polymorphism levels were estimated by dividing the polymorphic loci by the total number of scored loci. Percentage fidelity of the RAPD/SCoT was calculated from the following equation . 100 bp ladder  was used to estimate band size. Genetic similarities among kalanchoe genotypes were calculated according to Dice coefficient measurement  using IBThis work discussed the genetic relationship among five kalanchoe genotypes through the analysis of morphological and molecular features. The information about molecular and phenotypic properties has great importance in the future selection of breeding populations, particularly for traits that possess commercial values. According to the findings, orange, violet, and yellow kalanchoe are distinct parents for breeding commercially valuable kalanchoe features. The study also concluded that SCoT and RAPD markers exhibited accurate banding pattern profiles to analyze the genetic diversity of kalanchoe genotypes as they gave a high polymorphism percentage among kalanchoe genotypes compared with ISSR marker that gave a very low polymorphism percentage.