Pleural effusions present a diagnostic challenge. Approximately 20% are associated with cancer and some 50% require invasive procedures to perform diagnosis. Determination of tumour markers may help to identify patients with malignant effusions. Two strategies are used to obtain high specificity in the differential diagnosis of malignant pleural effusions: a) high cut-off, and b) fluid/serum (F/S) ratio and low cut-off. The aim of this study is to compare these two strategies and to establish whether the identification of possible false positives using benign biomarkers – ADA, CRP and % of polymorphonuclear cells – improves diagnostic accuracy.We studied 402 pleural effusions, 122 of them malignant. Benign biomarkers were determined in pleural fluid, and CEA, CA72-4, CA19-9 and CA15-3 in pleural fluid and serum.Establishing a cut-off value for each TM for a specificity of 100%, a joint sensitivity of 66.5% was obtained. With the F/S strategy and low cut-off points, sensitivity was 77% and specificity 98.2%, Subclassifying cases with negative benign biomarkers, both strategies achieved a specificity of 100%; sensitivity was 69.9% for single determination and 80.6% for F/S ratio.The best interpretation of TM in the differential diagnosis of malignant pleural effusions is obtained using the F/S ratio in the group with negative benign biomarkers. Pleural effusions present a diagnostic challenge. Between 15 and 30% are associated with cancer and between 40 and 60% require invasive procedures to perform diagnosis .Equally, the cut-off points may differ according to the aim of the study –10.Another cause of discrepancy between studies is found in the types of benign diseases recorded. It has been reported that tuberculosis, complicated parapneumonic effusions and empyemas may have high concentrations of tumour markers in pleural fluid, related to the inflammation of the mesothelial cells or nearby tissues –14.. AdeParamalignant effusions represent another source of discrepancy. Patients with these effusions have cancer, but no neoplastic cells are present in the pleural mesothelium; however, patients may have high concentrations of tumour markers in serum. So, tumour markers may be found in effusion fluids alongside other macromolecules such as albumin, and they may be present in high concentrations in the pleura. Other benign diseases with high serum concentrations of tumour markers may present similar behaviour .Therefore, in order to obtain high specificity using only values of TM in effusion, high cut-off points have usually been used. However,, our group described a strategy based on two criteria including a low cut-off and the fluid/serum (F/S) ratio . AnalysiThe aim of the present study was to compare the diagnostic accuracy of these two strategies: a cut-off point for each tumour marker in fluid effusion to obtain maximum specificity, and the F/S ratio in pleural effusions in order to validate previous results. We also intended to establish whether the classification in groups according ADA, CRP and % of polymorphonuclear cells, might help to improve diagnostic accuracy.From January 2008 to December 2012, fluid and serum samples were collected from consecutive patients of all medical specialties at our center who presented pleural effusions. Diagnostic procedures were performed by assessors who were blind to the study data.The reference method used was pathological confirmation of cancer in serous effusions or definitive diagnosis assessed during the three months following the determination of TM. Serous effusions were defined as malignant when the presence of neoplastic cells was detected by cytology, biopsy or autopsy. Paramalignant effusions were defined as effusions in which no neoplastic cells were detected by any of the methods described above in patients diagnosed with cancer.In order to identify benign effusions we determined the following test in fluid and/or serum: protein, albumin, Nt-ProBNP, LDH, microbiological cultures, and if necessary antinuclear antibodies, anti-cyclic citrullinated peptide, rheumatoid factor, thyrotropin, and serological tests for viruses, bacteria and fungi.Effusion fluid and serum samples were collected and analysed on the same day. CEA, CA15-3, CA 72–4 and CA19-9 were determined using an electrochemiluminescence method on a Cobas 601 analyser . The analytical variation expressed as the between-assay coefficient of variation was 5.0, 3.0, 4.2 and 4.5% for CEA, CA15-3, CA72-4 and CA 19–9 at concentrations of 5 μg/L, 32 KU/L, 8.3 KU/L and 29 KU/L respectively. In the first approach, using a single determination in pleural fluid, we established the cut-off for each TM at a specificity of 100% with the ROC curve. For the second approach, simultaneous determinations were performed in fluid and serum; effusions were considered malignant when at least one of these TM CEA, CA15-3, CA72-4 or CA19-9 in fluids were above the URL and the F/S ratio was above 1.2. TM in serum were determined only in patients presenting TM values in pleural fluid above the upper reference limit (URL) in serum .The criteria used to suggest that an effusion might be a false positive  were %PN > 90, CRP > 50 mg/L or ADA >45U/L . The useROC analysis was used to establish cut-off points for each TM at a specificity of 100%.Sensitivity, specificity, negative predictive values (NPV) and positive predictive values (PPV) were calculated for each TM and for the combination of TM. All statistical analyses were performed using IBM® SPSS® Statistics for Windows v.20  and Stata® v.10 .A total of 402 consecutive pleural effusions were included, from 148 women and 254 men with ages ranging from 15 to 93 years . Out of the effusions assessed, 280 (69.7%) had a benign aetiology and 122 (30.3%) were malignant . Other studies have shown discriminant values ranging from 40 to 50 μg/L for CEA, 53 to 75 KU/L for CA15-3 and 8.9 to 16 KU/L for CA 72–4 , 13, 24,The main problem with the use high cut-offs for TM in the differential diagnosis of pleural effusion is the wide range of cut-offs, sensitivities and specificities that we find in the literature, indicating that the discriminant values may depend on the cohort. On this point, considerable differences may be found depending on the type of immunoassay used. Assessing CA15-3, Slev et al. . found cComparing the two strategies, the simultaneous determination of TM in serum and effusions and the F/S ratio > 1.2 achieves a 10% higher sensitivity than the single determination, but a lower specificity (98%). In order to improve these data, we assessed the two strategies in group A alone . In this group, simultaneous determination reached 100% specificity and maintained ranges of sensitivity. Thus, in patients with negative cytology , sensitivity rose from 54.5 to 72.7%.Using the F/S ratio and classifying according to ADA, CRP or %PN, we obtained the same specificity in two different studies, and so it seems that this strategy does not vary over time or between series in effusions with negative ADA, CRP and %PN. This strategy uses as a reference the serum concentration measured with the same immunoassay. It avoids some of the problems associated with the single determination of TM, for example the increase in TM concentrations in fluid in patients with high TM concentrations in serum.Classifying patients on the basis of biomarkers such as ADA, CRP and %PN also allows identification of some benign effusions with high concentrations of TM, thus increasing diagnostic accuracy. The group without suspicion of false positives has higher sensitivity and specificity using either strategy, while in the group of potential false positives sensitivity is much lower with both. In the patients in group B  included all false positives due to benign release of TM in fluid in both groups, we also found a decrease in sensitivity because this group included fewer malignant effusions and because more than 40% of tumours were non-epithelial, compared with a rate of 18% in group A.The data suggest that the best diagnostic accuracy (with very high likelihood ratios) is achieved by using the F/S ratio in group A, that is, patients without suspicion of false positives due to benign diseases. We propose an algorithm for the use of tumour markers in pleural effusions Fig. . The strPatients with a previous diagnosis of neoplasia and the appearance of pleural effusion with negative cytology and positivity for tumour markers can be taken as presenting disease progression; similarly, the presence of pleural and effusion and positive tumour markers but negative cytology in newly diagnosed localized tumours may indicate pleural metastasis. These and other practical considerations raised by these data should be addressed in a case by case team discussion.The main limitation of this study is the fact that it was performed at a single centre. Multicentre studies are needed to validate the results and to determine whether they are also applicable to other measurement systems.To obtain the maximum diagnostic yield from the measurement of tumour markers in pleural effusions, we support simultaneous determination of markers in fluid and serum with a low cut-off point in patients in whom no increases in TM due to benign disease are suspected . In patients in whom increases in TM due to benign disease are suspected, the strategy of a single determination in fluid may offer better diagnostic accuracy.


Enthusiasm for research on the brain and its application in education is growing among teachers. However, a lack of sufficient knowledge, poor communication between educators and scientists, and the effective marketing of dubious educational products has led to the proliferation of numerous ‘neuromyths.’ As a first step toward designing effective interventions to correct these misconceptions, previous studies have explored the prevalence of neuromyths in different countries. In the present study we extend this applied research by gathering data from a new sample of Spanish teachers and by meta-analyzing all the evidence available so far. Our results show that some of the most popular neuromyths identified in previous studies are also endorsed by Spanish teachers. The meta-analytic synthesis of these data and previous research confirms that the popularity of some neuromyths is remarkably consistent across countries, although we also note peculiarities and exceptions with important implications for the development of effective interventions. In light of the increasing popularity of pseudoscientific practices in schools worldwide, we suggest a set of interventions to address misconceptions about the brain and education. Over the last few decades, the scientific community has shown an increasing interest in building bridges between brain science and education. This interest has promoted the creation of research teams, specialized journals, and meetings aimed at connecting neuroscience and educational practice . The entThe diverging views on the potential contributions of neuroscience to education among scientists and educational practitioners are due to several reasons . For ins® is offered in more than 80 countries , with similar characteristics to Brain Gyme brain” . This ace brain” . As mente brain” . HoweverThe purpose of the present study was to determine the prevalence of neuromyths among teachers from all levels of school education. As in previous studies, we were also interested in investigating which myths are more or less popular in this population. A better knowledge on the subject would be helpful to design more effective interventions to address neuromyths among Spanish teachers. In addition, we were interested in determining which factors predict belief in neuromyths. Specifically, we aimed to determine whether reading popular or scientific neuroscience literature prevents teachers from believing in neuromyths or, by contrast, if greater knowledge about the brain is associated with higher acceptance of misconceptions, as has been shown in previous studies . To provA second goal of the present study was to combine data collected in our Spanish sample and the evidence gathered in previous studies, to explore similarities and differences in the popularity of neuromyths across countries. As explained above, some of the previous studies on this topic have addressed the issue of cross-cultural variability in neuromyths by collecting data simultaneously from different countries e.g., . HoweverSD = 9.28). Participants were kindergarten teachers (22.8%), primary school teachers (32.9%), secondary school teachers (33.6%), vocational education teachers (3.5%), and teachers who worked in more than one level of education (6.3%). The sample was recruited from public (27.7%), private (6.3%), and state schools (64.9%). The average teaching experience of the participants was 16.9 years (SD = 9.69). Except for gender, age, and years of experience, these demographic and professional data were requested only for descriptive purposes and were not explored any further in subsequent analyses.The sample included 284 teachers from 15 independent Spanish regions . Eighty (28.07%) participants were males and 204 (71.57%) females. These percentages are proportional to the distribution of males and females in the total population of educators in Spain . The meaTeachers were contacted by email invitation to pseudo-randomly selected databases of schools available on the Internet, by personal invitation, or by two social networking sites. In the first case, after accepting the invitation, schools were asked to forward an email with information about the study to all their staff teachers. In the remaining two cases, information about the study along with an invitation to participate was sent by email, in the case of the direct invitation, or was published simultaneously on Twitter and Facebook, in the case of the social networking sites. In all the modalities, teachers who were interested in participating followed a link to the on-line survey. The research project was presented to the participants as a study about the role of neuroscience in education. Average completion time for each survey was approximately 15 min.The survey consisted of two parts. In the first part, participants gave their informed consent and provided background information about their age, sex, professional qualification , years of teaching, level at which they teach , position within the school , type of school they attended  and the region where their school was located. Teachers indicated whether they were interested in neuroscience applied to education and whether they thought this knowledge was important for their job. In addition, they were asked whether they had received in-service training in educational neuroscience  and, if so, how they got to known of its existence and the institution that had organized it. Additionally, they were asked whether any of the so-called ‘brain-based’ programs was being implemented in their schools. Furthermore, they indicated whether they read magazines about general or educational science and/or peer-review journal articles. To avoid misunderstandings, well-known examples of each type of publication were given. Finally, teachers were asked to provide information about any book, blog, or website about neuroscience that they consulted regularly.1 (see Tables 1 and 2), presented in random order. Participants were instructed to respond by marking one of three options: correct. incorrect, or do not know. All the neuromyths were false statements about the brain, while the general knowledge questions comprised true and false statements (see Table 2). The original survey was initially translated into Spanish by one researcher (MF). To guarantee the fidelity of the translation, the resultant version was back-translated into English by a second researcher (MAV) and both English versions were compared by a third person.In the second part, participants completed the survey developed by The α value for all statistical tests was set to 0.05. To examine which factors predicted belief in neuromyths, we conducted a multiple regression analysis with the number of myths as the dependent variable. The predictors were sex, age, years of experience, in-service training in educational neuroscience, reading popular magazines of science or education, reading scientific journals, consulting of blogs or web sites, and number of correct answers on general assertions about the brain. To examine the predictors of general knowledge about the brain, a second analysis was conducted with the percentage of correct answers to the general assertions as the dependent variable. In this case, the predictors were sex, age, years of experience, in-service training, reading popular magazines of science or education, reading scientific journals, and consulting of blogs and web sites.Table S2 in the Supplementary Material). Given that proportions do not follow a normal distribution, we conducted the (random effects) meta-analyses on double arcsine transformed proportions , books (27.8%), or in-training courses (16.9%). Among the latter, 52% knew about the existence of the courses from their own schools, 25% from the internet, and the remaining 22.9% from different sources such as the press, university, or friends. The most prevalent in-service training course topic was the multiple intelligences theory, which is not based on solid scientific evidence . Of all Table 1 summarizes the proportion of correct and incorrect responses for each neuromyth. For the overall sample, teachers failed to recognize 49.1% (SD = 17%) of neuromyths on average. In addition, 19.6% neuromyths (SD = 16.8%) were labeled as do not know. The most prevalent neuromyths were (1) “environments that are rich in stimulus improve the brains of pre-school children,” believed by 94% teachers; (2) “individuals learn better when they receive information in their preferred learning style,” believed by 91.1% teachers; and (3) “exercises that rehearse coordination of motor-perception skills can improve literacy skills,” believed by 82% teachers. In contrast, the most successfully identified neuromyths were (1) “children must acquire their native language before a second language is learned,” marked as false by 80.2% teachers; (2) “learning problems associated with developmental differences in brain function cannot be remediated by education,” marked as false by 78.5% teachers; and (3) “if students do not drink sufficient amounts of water their brains shrink,” marked as false by 64.7% teachers. Due to the important role bilingualism plays in some regions of Spain, the responses to the myth about the importance of acquiring a native language before learning a second language were analyzed separately for regions with several official languages. The results showed that among teachers who belonged to a region with just one official language, 52.3% believed the myth, while among the teachers who belonged to a region with two official languages, 76.9% believed the myth.B = 0.99). In addition, belief in neuromyths was predicted by general knowledge of the brain: Teachers who responded correctly to general knowledge questions about the brain were also more likely to believe in neuromyths (B = 0.31). Importantly, while having read scientific journals reduced belief in neuromyths (B = -0.92), having read educational magazines actually increased this belief (B = 0.52). The remaining factors did not predict belief in neuromyths (see Table 3).The multiple regression analysis revealed that women were more likely to believe in neuromyths that men (unstandardised SD = 16.04%) of the general statements about the brain and labeled 20.72% (SD = 16.8%) as do not know (see Table 2). Knowledge about the brain was predicted by reading primary scientific journals (B = 1.56), by in-service training (B = 1.38), and by reading books about neuroscience (B = 1.06). The remaining factors did not predict general knowledge of the brain (see Table 4).Teachers responded correctly to 62.29%  and the belief that it has been scientifically proven that fatty acid supplements can improve academic performance (I2 = 98.80%) are the items with the largest level of cross-cultural variation. The forest plots in Figure 1 show that these myths are very popular in some countries , but not in others .Beyond these similarities, the Over the last years, cognitive neuroscience has gradually taken on the challenge of understanding the neural mechanisms that enable human learning . HoweverThe results obtained in this study showed that Spanish teachers believed a considerable number of the neuromyths. Specifically, from a total of 12 neuromyths presented, five were believed by more than 50% of the educators. This result is very similar to the patterns observed in British and Dutch teachers and only slightly better than the results obtained in Latin America and Turkey. Conversely, the mean score on general knowledge questions about the brain was almost 60% amongst Spanish educators. This result is worse than the one obtained in the UK, Netherlands, and Latin America and could be due to the quality and quantity of the educational materials available in Spain. In this regard, as noted by As in previous studies , teacherAlong with the sources of information explored by the preceding studies, we were interested in analyzing other means Spanish educators employed to learn about the brain, such as books or web sites. Overall, the favorite sources of information reported by teachers contrasted with the ones that predicted knowledge about the brain. In other words, the resources that seem to promote general knowledge are the least popular among the teachers tested in the present study. Contrary to the results obtained by Supplementary Table S2). It should be stressed that both myths are present in several commercial educational packages, which may have contributed to their dissemination. For instance, in the case of learning styles, there is a huge industry devoted to publishing measurement instruments and guidebooks, and to organizing workshops and conferences targeted mainly at teachers (®, a program which has found widespread acceptance only in some countries, such as the UK (The eager interest in neuroscientific claims and their potential applications to education, together with the high prevalence of neuromyths amongst Spanish teachers, echoes the findings obtained in previous studies conducted in Asia, Europe, and Latin America . Neverthteachers . Similarteachers . Converss the UK . SimilarIn light of the present study and the preceding ones, the prevalence of neuromyths among educators is not an isolated phenomenon but, on the contrary, affects many different countries around the world. Given the gap that exists between scientists and practitioners, many experts agree that it is essential to establish interdisciplinary collaboration between neuroscientists and educators to inform each other and to create useful connections in both fields . In thisIn Spain, several researchers have expressed concerns about neuromyths in educational contexts . HoweverTo this day, neuroscientific findings are valuable for describing the mechanisms of learning. However, they cannot yet inform educational practice directly . While sAll authors developed the study concept. MF developed the study materials and conducted the analysis of the Spanish data. MV conducted the meta-analytic synthesis of studies. MF drafted the manuscript, and PG and MV provided critical revisions. All authors approved the final version of the manuscript for submission.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.


Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patient's tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies.Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection  Summary: We present a novel retinoblastoma nude xenograft mouse model which closely resembles the human disease and allows broad application possibilities and a comparison of in vivo and histological analysis. Retinoblastoma (Rb) is the most common primary intraocular malignancy of infancy with an incidence of 1/15,000 to 1/20,000 births. It is estimated that annually Rb affects 7000-8000 new patients worldwide , and surKnowledge on tumour genesis was increased enormously with the development of mouse models for retinoblastoma . For exaOphthalmic imaging, like wide-field photography and echography, are reliable tools, not only in diagnosis but also for detecting regression or progression patterns of Rb . Intraocin vivo and ex vivo follow ups were compared. In addition, ultrastructural analysis of the xenograft Rb tumours was performed in order to assess the relevance of our Rb mouse model.The aim of this study was to generate an animal model for Rb that closely resembles the human disease for the purpose of developing new therapeutic options or comparing the efficacy and side-effects of existing treatments. To this aim, retinoblastoma cells of the human Rb−/− cell line Y79 were intravitreally injected into the eyes of immune-deficient nude mice to induce tumour growth. Development and spread of the tumours were characterized by scanning laser ophthalmoscopy (SLO), fluorescein angiography (FA) and OCT, as well as by histology including an analysis at the ultrastructural level, and the Starting at week five after the injection, the eyes began to swell in individual animals. We determined four stages depending on the tumour progression as shown in As shown in The SLO/OCT analysis could only be performed in stage 0 eyes with tumours at very early stages or in eyes without a tumour. In eyes with tumours at later stages of growth (SI-SIII) no analysis was possible, because the tumour covered the fundus.In all cases where SLO/OCT analysis was possible, the results showed a good correlation with the results of the histological analysis. Using OCT, not only could we detect the tumour itself, but could also get information about its growth characteristics. The tumour shown in In the tumour-bearing eyes tumour cells could be observed in the vitreous, retina and subretinal space. An overview of the exact number of eyes and areas of tumour growth up to the time points of analysis is presented in For the initial phase of tumour growth (week 3), eight eyes were analysed. Tumour cells could be seen in the vitreous and on the retina in four of them. In two eyes the tumour grew through the retina, in one of these eyes even subretinal tumour growth could be observed.In all tumour-bearing eyes which were analysed 6 weeks after injection, the tumour completely replaced the vitreous and grew into the anterior chamber, in seven eyes the tumour invaded or damaged the lens, and in two eyes the tumour did not penetrate the retina A. In theIn advanced tumours (week 9 and 12) the tumour replaced most of the eye's structures, like the vitreous, the lens and retina. Here in all six tumour-bearing eyes the sclera was the only part of the eye's tissue remaining C.Histologically, the tumours were composed of typical undifferentiated hyperchromatic cells with scanty cytoplasm having a rosette-like growth pattern, as described for the original tumour . All tumA tumour was found in 67.5% of the analysed eyes (29 of 42) 12 weeks after the injection of the Y79 cells.We screened tissues near the tumour, like the brain and skull, for metastases as well as the kidneys, lung, heart, liver, and spleen for the appearance of distant metastases by analysing HE-stained cross sections of these tissues. Only in one mouse could metastases in the brain be found. The metastases were found in the mouse, which after having reached stage III for one eye was kept for the longest period of time (35 days) before being killed and analysed  supplemented with 10% fetal bovine serum and 2 mM L-glutamine. The cells grew as a suspension culture and were cultured and passaged as recommended by the ATCC. For the injection, cells from passage 4 were used.The human retinoblastoma Y79 cell line was purchased at American Type Culture Collection . The cells were cultured in RPMI-1640 medium ] were used for the study. The animals were kept in individually ventilated cages (IVC) in our animal facility.The mice were handled at all times in accordance with the German Animal Welfare Act and were under the control of the Animal Protection Agency and under supervision of veterinarians of the University of Tuebingen. The experiments were approved by the local authorities (Regierungspräsidium Tuebingen AK 6/12).Each animal was first anaesthetized with an intraperitoneal injection of a three component narcosis .®, Darmstadt, Germany) containing 2×104 Y79 human retinoblastoma cells were injected into the vitreous of each eye through the sclera using a Hamilton syringe with a 26 gauge cannula. Special care was taken to prevent lens damage or posterior retinal punctures. After the injection, the eyes were treated with antibiotic eye drops . Finally the mice were subcutaneously injected with an antidote  which neutralized the anaesthetic.The pupils were dilated with 1 to 2 drops of Medriaticum drops  and a drop of topical anesthetic Novesine  was applied. Methocel  eye drops were used to avoid drying of the eyes. Injections were performed using a surgical microscope. Two microlitres of sterile phosphate buffered saline  directly to the outlet of the device, and an additional custom-made 100 dpt contact lens directly on the eyes of the mice. The mice were anaesthetized by a peritoneal injection of a three component narcosis (as described above), and the pupils were dilated with 1 to 2 drops of Medriaticum . Methocel  eye drops were used to avoid drying of the eyes and to ensure the adherence of 100 dpt- lenses on the mice eyes. The mice were put in front of the device on the XYZ-table and positioned for the analysis. The mice were covered with cloth to avoid hypothermia.Three, six, nine and twelve weeks after injection, groups of five mice were formed. Mice which showed tumour-caused phenotypical changes were primarily analysed. For the analysis a Spectralis™ HRA+OCT SLO/OCT device was used . The whole procedure was performed as described in ® 10%  was given subcutaneously to the mice to make it possible to visualize the retinal and tumour vessels using the FA (fluorescein angiography) mode of the SLO device. After that the OCT-imaging was performed. A detailed protocol for anaesthesia and imaging is described elsewhere . The tissues were processed and embedded in paraffin using conventional automated systems. The blocks were cut to obtain serial 4 µm thick sections and stained with conventional hematoxylin-eosin (HE). The slides were examined by the means of a light microscope.Directly after the en bloc stained with uranyl acetate and lead citrate, and embedded in Epon after dehydration in a graded series of acetones. Semi-thin sections (0.2 µm) were stained with Toluidine Blue and examined by light microscopy . For electron microscopy, the sections were cut ultrathin (0.07 µm) and analysed with a Zeiss 902 A electron microscope .The other eye of each mouse was fixed in 5% glutaraldehyde for electron microscopic analysis. After the fixation (min. 3 days) the eyes were screened under a binocular for areas of interest (aoi), especially tumour-containing areas. The samples containing these aoi were cut (1 mm×1 mm). These specimens were post-fixed with 1% OsO4 at room temperature in 0.1 M cacodylate buffer (pH 7.4),


Quantitative ultrasound (QUS) can probe tissue structure and analyze tumour characteristics. Using a 6-MHz ultrasound system, radiofrequency data were acquired from 56 locally advanced breast cancer patients prior to their neoadjuvant chemotherapy (NAC) and QUS texture features were computed from regions of interest in tumour cores and their margins as potential predictive and prognostic indicators. Breast tumour molecular features were also collected and used for analysis. A multiparametric QUS model was constructed, which demonstrated a response prediction accuracy of 88% and ability to predict patient 5-year survival rates (p = 0.01). QUS features demonstrated superior performance in comparison to molecular markers and the combination of QUS and molecular markers did not improve response prediction. This study demonstrates, for the first time, that non-invasive QUS features in the core and margin of breast tumours can indicate breast cancer response to neoadjuvant chemotherapy (NAC) and predict five-year recurrence-free survival. Locally advanced breast cancer (LABC) is an aggressive subtype of breast cancer which is clinically defined as a tumour that is greater than 5 cm or involves the skin or chest wall. LABC also includes inflammatory breast cancers and patients with fixed axillary lymph nodes or ipsilateral supraclavicular, infraclavicular, or internal mammary nodal involvement234in vivo in a variety of applications to reveal information about tissue microstructure, enabling the differentiation of disease from normal tissue and the characterization of disease into its subtypes. For instance, parameters derived from linear regression analyses of RF power spectra, including midband fit (MBF), spectral slope (SS), and spectral 0-MHz intercept (SI), have been used to characterize intraocular tumours and to detect prostate cancer, cardiovascular disease, and cancerous lymph nodes9101115in vivo in response to various treatments including photodynamic therapy, radiation therapy, chemotherapy, and anti-vascular therapy171819et al.et al.et al.Clinical ultrasound is widely used in medicine due to its cost-effectiveness, large penetration depth (~ 7 cm) and real-time imaging capability. Furthermore, the radiofrequency (RF) backscatter signal derived from ultrasound provides information about the tissue microstructure otherwise not resolvable by conventional ultrasound images (B-mode images). Quantitative ultrasound (QUS) techniques examine the frequency dependence of the RF signal backscattered from tissues and have been applied et al.et al.et al.Baseline QUS imaging features were evaluated in terms of their ability to predict breast tumour response in advance of any treatment.In addition to examining textural features within the tumor core as done previously, QUS features in the tumor’s surrounding tissue, referred to here as the margin, extending 3–10 mm from the core were analyzed. Quantitative features such as core-to-margin ratio (CMR) and core-to-margin contrast ratio (CMCR) QUS features were examined here, for the first time, and were considered as potential ultrasound-based biomarkers of tumour aggressiveness that determine the likelihood of tumour response.Molecular biomarkers including estrogen receptor (ER), progesterone receptor (PR), and HER2 were investigated as stand-alone and as additional biomarkers for response prediction.In addition to good versus poor response classification done previously in a different therapy monitoring studyUsing the same database of patients and their breast ultrasound images, the study here builds upon previous studies by Sadeghi-Naini Linear regression spectral parameters including MBF, SS, and SI, and more complex backscatter model parameters including ASD and AAC were computed from a region of interest (ROI) within the tumour and its margin. Additionally, an attenuation coefficient estimate (ACE) was obtained from the core ROI as a spectral correction factor and as a potentially predictive parameter. Higher-order statistical  and image quality features were extracted from parametric images based on the aforementioned parameters and were subsequently submitted to a classifier to predict the response of the patients. Furthermore, molecular markers including ER, PR, and HER2 were investigated as separate and additional predictors of response. Three types of classifiers were investigated for response prediction: Fisher’s linear discriminant (FLD), k-NN, and support vector machine (SVM). The clinical and pathological response (good versus poor response and complete versus incomplete response) of each patient was determined at the end of the treatment according to response evaluation criteria in solid tumours (RECIST)The prospective study was conducted under the regulations and guidelines in accordance with the research ethics board at Sunnybrook Health Sciences Centre (SHSC), Toronto Canada. All experimental protocols were reviewed and approved by the research ethics board at SHSC prior to commencing the study. After obtaining informed consent, ultrasound radiofrequency (RF) data and corresponding anatomical images (B-mode) were acquired from the affected breast of 56 LABC patients using a clinical ultrasound system equipped with a 6 MHz center frequency linear array transducer . All patients enrolled in this study were diagnosed with LABC with the exception of four early stage breast cancer patients who received NAC due to presenting with lymph node disease, and long surgery wait time. There were no cases of inflammatory breast cancer in this study. All patients completed a full course of NAC, which lasted 4–6 months, after which they underwent mastectomy or lumpectomy. The imaging session occurred within one week of diagnosis and prior to NAC initiation. Ultrasound RF data were acquired at a sampling rate of 40 MHz. The focus was set at the midline of the tumour using electronic beam focusing, and the imaging depths varied from 4 to 6 cm, depending on tumour size and location. Approximately 4–7 image planes were acquired, covering the full extent of the tumour volume, spaced at 5 mm intervals. In order to capture the heterogeneity of the tumour, ROIs were selected from central and peripheral zones of the tumour symmetrically.Patient clinical features including age, tumour size, hormone receptor statuses, and RECIST-based tumour size change were obtained from clinical evaluations and reports, biopsy results, and imaging reports. All patients in this study completed full courses of chemotherapy. Over 90% of patients received anthracycline-taxane based regimens. Enrolment of patients in terms of chemotherapy was not limited to one tumour type or particular regimen in order to facilitate maximal enrolment. Patients had clinical MRI scans before treatment and after as part of standard of care practices for tumour size measurements and assessment of chest wall involvement. The RECIST-based change was defined as the percent change in tumour size (longest diameter) between pre-treatment and post-treatment times . Pre-/post-treatment tumour sizes were obtained from pre-/post-treatment magnetic resonance imaging and post-surgical pathology reports. After mastectomy/lumpectomy, standard clinical pathology was carried out as part of patients’ standard of care. The method of clinical and pathological response determination (good/poor) in this study was based on the RECIST criteria with the addition of accounting for residual tumour cellularity upon completion of chemotherapy. A recent study demonstrated that residual tumour cellularity is also an important prognostic factor in breast cancer neoadjuvant treatment, which should be taken into account in conjunction with the RECIST metricROIcore. In order to compare the QUS features inside the tumour with those immediately surrounding the tumour where there may be microscopic tumour extensions, another ROI was generated automatically, which was referred to as ROImargin. This ROI was prescribed with a pre-defined thickness of 3 mm, 5 mm, or 10 mm. An example tumour segmentation with a 5 mm margin is presented in et al.et al.et al.ROIcore, and thus, no parametric image was available for this parameter. In order to characterize structural patterns in ROIcore, a gray-level co-occurrence matrix (GLCM)–based texture analysis was performed on the newly obtained parametric images as described in Tadayyonbet al.et al.et al.For QUS analysis purposes, an ROI was generated by manually outlining the core of the tumour . This was referred to as C and γ were optimized using a grid search, where C is the soft margin parameter and γ is the free parameter of the RBF kernel.Prior to performing patient response classification based on their tumour QUS and/or molecular features, the features were sorted by their statistical significance, from lowest to highest p-value . Starting with the lowest p value feature as the initial model, features were sequentially added to or discarded from the model until there was no improvement in classification accuracy based on RECIST and pathological response. This process is referred to as sequential forward feature selectionP(Cj|x), was defined as the estimated probability that the correct label, C, for x, is Cj and follows Bayes’ theoremk-NN classifier, was computed using the ratio of sum of the weights of the neighbors that belong to class j, i.e., Wi∈Cj, to the sum of the weights of all neighbors, i.e., Wi. The formulation for P(Cj|x) is ref. 34In a leave-one-out cross-validation scheme, the k-NN classifier predicts the class association of a test point in the feature space based on the class which forms the majority of the points neighboring the point of interest, and based on the distance between those points and the point of interest. The latter two non-linear classifiers are favourable when the classes cannot be separated by a line and a large number of features is available. All classifier predictions were made using leave-one-patient-out cross-validation. In order to evaluate, statistically, how well a classifier could separate the two groups (good versus poor response patients), a Mann-Whitney test or a t-test  was performed on the estimated posterior probabilities of each group. The estimated posterior probability, nbd represents the neighborhood . The weight represents the distance between each data sample and its neighbor.Where A “patient-by-patient” clinical characteristics is presented in A representative conventional ultrasound image (B-mode image) of a patient’s breast tumour, its corresponding core and margin ROIs, and a resulting SI parametric image are presented in k-NN) and using the optimal feature set obtained through sequential forward feature selection. Reported values are sensitivity, specificity, accuracy, and AUC. For AUC, 95% confidence bounds are reported, which were obtained through bootstrapping (1000 samples). As evident, the k-NN classifier provided the most favourable classification, with a sensitivity of 90%, a specificity of 79%, an accuracy of 88%, and an AUC of 0.81, for predicting response to chemotherapy in advance of it being administered. ROImargin, including 3, 5, and 10 mm thicknesses. Results suggested that 5 mm is the optimal margin thickness for characterizing a patient’s tumour responsiveness.SICMR, and SICMCR Other parameters also appeared in this feature set, including MBF (one feature), SS (two features), and ASD (one feature). Results suggest that the statistical and image quality features of spectral parameters provided more discriminatory information about the response characteristics of a tumour than backscatter model parameters such as ASD. Removal of ASD from the feature set resulted in a 2% decrease in classification accuracy (from 88% to 86%). Above all, a Mann-Whitney test on the posterior probabilities of the good response and poor response groups demonstrated highly statistically significant results (p < 0.001), as presented in the last row of k-NN 5 mm-margin model). The RFS curve based on the QUS predictions was obtained in the same manner as the RFS curve based on clinical response, except that the classification of each patient as a survivor or non-survivor (recurrence or death) used for generating the curve was based on the QUS predictions. The QUS predictions were based on the k-NN classifier and optimal feature set reported in In this study, pre-treatment textural and image quality features of QUS parametric images of breast tumours and their margin zones were demonstrated to be predictive of chemotherapy responsiveness in LABC patients undergoing NAC treatment with an 88% classification accuracy. The classification results were verified with ultimate clinical responses of the patients determined based on MP criteria. Parameters of the predictive model including classifier type and tumour margin thickness were optimized for best response classification, yielding the k-NN classifier incorporating 5 mm- margin thickness. Furthermore, 5-year RFS curves of the LABC patients obtained using the predictive model followed closely with that obtained using clinical information comprised of RECIST-based response and post-surgical histopathology. MP scoring demonstrated equivalence with clinical-response criteria (RECIST) but provided additional information about microscopic pathological details. It was important to use MP scoring as tumour cellularity is taken into account in this methodology whereas RECIST-only criteria rely on anatomical measures which do not always accurately reflect biological tumour response.et al.ex vivo samples of breast tissues including fat, parenchyma, and infiltrating ductal carcinoma demonstrated that frequency-dependent attenuation was discriminative among those tissues. Similarly, Landini et al.et al.According to findings of this study, the ACE parameter demonstrated the most statistically significant discrimination between tumours of good response and poor response patients. Other studies involving breast tissue characterization using QUS obtained similar results for differentiating benign from malignant breast features35The high accuracy of our QUS-based model in predicting tumour responsiveness (88%) can be attributed to the fact that the model includes multiple parameters, accounting for breast tissue composition and stiffness (ACE), microstructural size (i.e. SS and ASD), and microstructural and cellular organization of tissue (MBF and SI). The fact that the acoustic features of both the tumour core and its margin contributed to the classification suggests that the margin may account for the presence of microscopic “limbs” infiltrating from the primary tumour mass into surrounding normal tissue.k), whereas the SVM requires tuning of the cost (C) and kernel parameters (γ).The poor performance of the FLD (75% accuracy) was not unexpected, as the task at hand is too complex for a linear algorithm such as FLD. The task at hand was differentiating breast tumours of mostly the same histological type (IDC) which are clinically and ultrasonically (B-mode) indistinguishable, but differing in their response characteristics to chemotherapy. The reason for the superior performance of the k-NN classifier over the SVM (4% higher accuracy) could be attributed to the fact that the k-NN classifier is easier to optimize since it generally requires only one parameter to be tuned, i.e. the number of neighbors parameter .A recent study by our groupThere is now mounting evidence suggesting that molecular subtypes of tumours play an important role in developing or inherently having drug resistanceA more promising marker is Ki-67, which has been demonstrated to be predictive of breast tumour response to chemotherapy at baseline (p = 0.0001)The method developed here may however be combined with monitoring of cell-death responses using quantitative ultrasoundIn this study we propose that ultrasound-based microstructural tumour characteristics can also be used to predict tumour response to therapy. Here QUS and textural features, in principle, reflect tumour structural changes at a cellular level and also at a tissue organizational level. Such features are recognized to become more disorganized with aggressive tumours as reflected in the ultrasound features studied here. The method here provides a manner to non-invasively quantify tumour structural-based changes in tissue with links to patient clinical outcomes. The particular features which worked best appeared to be tumour core based parameters and also core-to-margin contrast ratio-based parameters. Higher-order model-based parameters such as the effective scatterer size and the acoustic-scatter concentration estimate also appeared in the feature set which best predicted for response. The use of texture parameters was critical in order to obtain the sensitivity and specificity reported in this study and it is posited that such texture-based features better reflect the structural heterogeneity that can develop in tumours.The features in the optimal set here therefore likely span tumour structure in its core but also through the use of margin-to-tumour ratio features include its microscopic effects on surrounding tissue. In addition, the ultrasound-based predictions of response here were reflected in terms of 5-year patient survivals signifying real-life implications for such ultrasound-based predictions.Other modalities have relied on similar mean-value and texture analyses including ultrasound spectroscopy, diffuse optical spectroscopy and MRI methods for therapy response monitoring43444545a priori may aid in stratifying patients for adaptive chemotherapy whereby the treatment offered is customized based on tumour aggressiveness. For instance, if a patient is deemed to be responsive based on the QUS prediction, then standard first-line chemotherapy can be administered according to the patient’s tumour characteristics. Conversely, if a patient is deemed to be non-responsive, a more aggressive first-line chemotherapy regimen can be administered in order to maximize chances of positive response. This is already carried out in the adjuvant setting with testing through molecular assays such as Oncotype Dx. The research here offers the possibility to do so in a neoadjuvant setting using tumour structure characterization non-invasively through the use of ultrasound.The work in the study here had link to patient responses of chemotherapy in terms of tumour responsiveness and thus naturally has links to patients’ outcome in terms of survival. This type of testing In this study, a QUS-based multiparametric classifier incorporating texture and image quality features that account for tumour core and a 5 mm thick surrounding margin was demonstrated to be a sensitive (90%) and specific (79%) pre-treatment predictor of tumour response to therapy and 5-year RFS (p < 0.05). Pre-treatment image-based surrogates of response stand to personalize health care by minimizing drug toxicity and maximizing chances of long-term survival.How to cite this article: Tadayyon, H. et al. A priori Prediction of Neoadjuvant Chemotherapy Response and Survival in Breast Cancer Patients using Quantitative Ultrasound. Sci. Rep.7, 45733; doi: 10.1038/srep45733 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Sol-gel transformations in HPMC (hydroxypropyl methylcellulose) are being increasingly studied because of their role in bio-related applications. The thermo-reversible behavior of HPMC is particularly affected by its properties and concentration in solvent media, nature of additives, and the thermal environment it is exposed to. This article contains investigations on the effects of salt additives in Hofmeister series on the HPMC gelation. Various findings regarding gelation with salt ions as well as with the ionic and non-ionic surfactants are presented. The gel formation in physiological salt fluids such as simulated gastric and intestine fluids is also examined with the interest in oral drug delivery systems. The processes of swelling, dissolution and dispersion of HPMC tablets in simulated bio-fluids are explored and the release of a drug from the tablet affected by such processes is studied. Explanations are provided based on the chemical structure and the molecular binding/association of HPMC in a media. The test results at the body or near-body temperature conditions helped in understanding the progress of the gelation process within the human body environment. The detailed interpretation of various molecule level interactions unfolded the sol-gel mechanisms and the influence of a few other factors. The obtained test data and the established mathematical models are expected to serve as a guide in customizing applications of HPMC hydrogels. Due to 2(OH)xOCHyOC3H7)zperature . Celluloperature ,6. They zperatureMany techniques are available for studying the sol-gel transitions in HPMC hydrogels. These processes primarily include dynamic light scattering , differeThis article presents studies on the gelation processes for HPMC in various ionic media. The ability of typical salting-out and salting-in salts in affecting the thermogelation of HPMC is systematically studied. The enthalpy and entropy changes (ΔH and ΔS respectively) determined from the DSC curves are discussed along with the contributions from the salt ions in the Hofmeister series. The findings for the HPMC solutions with the salt as well as with ionic and non-ionic surfactant additives are presented. Gel formation in physiological salt fluids such as simulated gastric and intestinal fluids (SGF and SIF respectively) is examined to have a better insight into the oral drug delivery systems.Three different grades of HPMC, as listed in 2HPO4, K2HPO4, and Na2SO4) and a trivalent salt (Na3PO4), which were used as received. In addition, sodium hydroxide (NaOH) and monobasic potassium phosphate (KH2PO4) required for preparing the buffer solutions and the hydrochloric (HCl) acid (37%) were purchased from Fluka Chemical Corp., WI, USA. Surfactants, sodium n-dodecyl sulfate (SDS), sodium n-decyl sulfate (SDeS) and Triton X-100 were purchased from Sigma-Aldrich Inc., USA. Sodium n-hexadecyl sulfate (SHS) was ordered from Alfa Aesar-A Johnson Matthey Company, MA, USA. Indomethacin, the drug that was loaded into HPMC tablets where needed, was procured from Aldrich, USA. To prepare the aqueous solutions, de-ionized (DI) water from a Millipore  Alpha-Q water-purifying system was used as obtained at room temperature and at different thermal conditions.Various salts in Hofmeister series were procured for studying their salting-in and salting-out effects on the gel formation. Analytical grade salts, purchased from Sino Chemical Co. Ltd., Singapore, included monovalent salts , divalent salts  for 24 hours so as to obtain homogeneous and transparent mixtures before their use for testing.Various calorimetric measurements were conducted using a micro-differential scanning calorimeter . A 0.5158 mL of sample solution and an equal amount of reference fluid (deionized water or other solution as required) were hermetically sealed into the sample cell and the reference cell, respectively. DSC curves for cooling and heating (at a rate of 1.0 °C /min) were recorded in the temperature range from 20 to 90 °C.A control-strain rheometer  was used to measure the flow properties and dynamic viscoelasticity of gel solutions. The rheometer was equipped with two sensitive force transducers for torque ranging from 0.004 to 100 g cm. The sample solution was poured onto the parallel-plate geometry (50 mm in diameter) and a small amount of silicone oil was applied at the periphery of the solution to prevent evaporation. The dynamic storage modulus (G′) and loss modulus (G″) were examined as the functions of temperature.Micro-structural changes during and after the gel formation were examined using a confocal microscope  coupled with a hot plate . A UV-vis spectrometer  was used for light transmittance measurement through the sol-gel media at various temperatures, where the heating rate was controlled by a Peltier-type temperature controller.The number average molecular weights (Mn) of the polymers were determined by gel permeation chromatography (GPC)  with a differential refractometer detector . The mobile phase used was DI water with the flow rate of 1 mL/min. The weight and number average molecular weights were calculated from a calibration curve using a series of Dextran standards .The HPMC tablets for studying their behaviour in bio-environment were prepared using the direct-compression method without any binder in Carver Laboratory Press .D,L-lactide-co-glycolide) (PLGA), and copolymers of N-isopropylacrylamide [et al. [− < ClO4− < I– < NO3− < Br− < Cl– < F– < H2PO4− < S2O32– < SO42−, whereas the influence of cations increases in the order Li+ > Na+ > K+ > Mg2+ > Ca2+ > Ba2+. However, the effect of cations is less significant than that of anions [Salts, including the common salt as discussed earlier, are known to influence the temperature-induced phase transitions in aqueous solutions of thermosensitive polymers, such as MC (methyl cellulose), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers, triblock copolymers of poly(ethylene glycol) (PEG) and poly were −72.6, −20.6, and −9.2 °C/M, respectively. The negative slope for the kosmotropic anions indicated that they promoted the sol–gel transition of the HPMC solutions. In contrast to kosmotropic anions, the slopes of chaotropic anions such as I− and SCN− were positive, and they were 9.4 and 14.3 °C/M, respectively, which indicated that they retarded the thermal gelation of the HPMC solutions. The results showed that the effects of anions onThe magnitude ofline see . The morAs most studies have pointed out and demonstrated that the ions exert their salting-out/salting-in effects via their water structuring capability and not via the direct interactions between the ions and the polymer chains ,31,32; aThe effects of ions on water structure are attributed to the competitions between ion-water interactions and water-water interactions . The for– provides the weakest competition for water molecules, which thus leads to salting-in effects and increased the solubility of the polymer.Because the chloride ion has the smallest radius and largest charge density among the halides investigated in this study, it has the strongest ability to compete for water molecules and form hydrogen bonds with water molecules, which minimizes or weakens water-polymer hydrogen bonding. On the other hand, the low charge density of ions such as Iet al. [hydr of the various salts anions yielded a straight line with a regression coefficient of 0.84, indicating that these two parameters were not well correlated linearly. On the other hand, viscosity B coefficient was used to quantify the degree of water structuring because it was related to the viscosity of an aqueous salt solution. Anions with a large viscosity B coefficient value exhibited strong water structure making capabilities. The regression coefficient for theHribar et al.  argued thydr and viscosity B coefficient was not well-defined, most anions followed a sequence. These seem to demonstrate that different mechanisms were involved in thermogelation of HPMC in presence of salts [Even though the linear correlation between slopesof salts .et al. [− and Cl− cause a strong electrostatic orientation of water molecules with respect to the anion, which makes water structure more ordered.An alternative explanation proposed by Hribar et al.  is that 2SO4 and 0.2 M NaI solutions. The figure legends are as follows: HPMC main chain ; water molecules ; hydrophobic substitution ; hydroxyl group ; ions ; hydrophobic interaction ; interchain hydrogen bonding .The figure below illustrated the physical structures of HPMC hydrogels in respectively 0.2 M Na− and SCN− have low charge densities, and water structures around them are less organized. Having no restraint on their movement, these water molecules tend to increase hydrogen bonding between them and the polymer, which results in an increase inAs a result of more ordered water structure, a less number of water molecules are freely available to solvate the polymer chains. This facilitates and accelerates the hydrophobic association of methyl substitutions causing reduction in43−) was more effective in the salting-out effects than the divalent anion (SO42−), followed by the monovalent anions such as Cl−. However, the trends did not relate well to the radius of the ions  of the polymer. The strong surfactant/polyelectrolyte interaction may lower the CAC value and counteract polymer solubility, resulting in gel formation ,48. In tIn the sections below, the investigations on the effects of three anionic surfactants, namely; SDS, SDeS, SHS, and one non-ionic surfactant, Triton X-100, on thermodynamic behavior of HPMC hydrogels are discussed.The relative heat capacity profiles depicting thermal behavior of HPMC/SDS solutions with different SDS concentrations determined using micro-DSC with increasing solution temperature are shown in In the absence of SDS, the DSC measurement for HPMC solution showed a small peak at 61 °C. However, with the addition of SDS, either the height or the position of the peak altered. As seen in Based on these DSC observations, a schematic diagram of the interaction between HPMC and SDS as well as the gelation of HPMC/SDS system was constructed and is shown in Cp values only increased quantitatively due to the presence of SDS. When the concentration of SDS was higher than 6 mM, the gelation phenomenon of HPMC was affected significantly. Thus, the concentration of 6 mM for SDS can be considered as the CAC value in the presence of HPMC. After reaching the CAC value, binding of SDS to HPMC occurred either through adsorption or through cluster formation Ti is the heat released during the gel formation process at temperature iT and is calculated asThe gelation kinetics of HPMC with and without SDS was studied using the DSC measurements. A concept of the degree of conversion,0H, is estimated asThe total heat of the gelation, ∆i(dH/dT) is the peak height of the thermogram at temperature iT.Correspondingly, the rate of gelation,i.e., 2 mM, the curve for the degree of conversion shifted to the left hand side with a lower slope as compared to that of HPMC solution without SDS. The sol-gel transition for aqueous HPMC/SDS (2 mM) system was observed to occur at the temperature of 51 °C. At a higher SDS concentration of 6 mM, the curve for the degree of conversion shifted to the right hand side with its slope comparable to pure HPMC solution. The sol-gel transition of the aqueous HPMC/SDS system with 6 mM SDS occurred at a higher temperature of about 58 °C. Further increase in the SDS concentration resulted in continuous shifting of the degree of conversion curve with decreasing slope.The symbols in 1k and 2k are temperature-dependent rate constants and m and n are empirical constants. For non-isothermal gelation process, Equation (3) may be revised as:A popular kinetic model from Sourour and Kamal : as in E1k would have to be zero. Thus, a simplified expression for non-isothermal gelation taking place at a constant rate of heating is:k is the rate constant for the gelation process.Based on the fact that the gelation rate is zero at the beginning of the heating process, the value of Subsequently, kinetic parameters−1 to 0.50 min−1 with the increasing SDS concentration from 0 to 6 mM. As discussed above, SDS existed as dissociative ions in the aqueous HPMC/SDS hydrogels and no integrates came into being in this concentration range. Such free SDS molecules would attract the water molecules, resulting in an increasing hydrophobicity due to these water molecules available for HPMC chains [−1 with the increasing SDS concentration from 8 mM to 12 mM. With continuous heating, hydrophobic groups of the HPMC were exposed and intermolecular association occurred to form new junctions for the gel network. This process corresponded to the second peaks observed in −1.A reasonable mapping of the experimental data may be seen with Equation (5). Interestingly, the kinetic rate constant increased from 0.17 minC chains . The soli.e., 20, 30 and 40 mM, a strong “salt-in” effect in thermograms was observed and the gelation of HPMC occurred at higher temperatures. The occurrence of the highest relative pC on the thermogram shifted to a temperature of 20 °C higher. Although the shape of the thermograms had only one peak, the temperature range for the sol-gel transition became narrower with the increase in the concentration of SDeS. SDeS, being a similar surfactant to SDS with a polar head, has a propensity to bind to the hydrophobic segments of HPMC. Due to its higher concentration, the bound SDeS granted high-density electrostatic repulsive interactions, which allowed more hydrophilic surfactant shells around the HPMC resulting in a higher onset gelation temperature.pC was registered. It should be noted that SHS concentration of 0.2 mM is lower than its CMC, i.e., 0.45 mM of SHS in aqueous solutions [The thermograms of HPMC in the presence of SHS are presented in olutions ,58. Thusi.e., 0.1, 0.2 and 1.0 mM, minor “salt-out” effect (about 1.8 °C) was observed and the HPMC gelation occurred at lower temperature. The enthalpy change in the gelation process was much less. It has been reported that the CMC value for Triton X-100 in aqueous solution is approximately 0.2 mM [It is known that interactions between non-ionic surfactants and neutral polymers are very weak ,59. Hydry 0.2 mM . Any varAs seen in  representing the polar head of surfactant molecule and  representing the electro-static repulsory, With The different strengths of electrostatic repulsion for SDeS and SHS molecules, as shown schematically in Considering its non-ionic nature, the addition of Triton X-100 did not introduce any electro-statically repulsive interactions among the surfactant molecules, as shown schematically in With the addition of surfactant, be it either anionic or non-ionic, the bound surfactant around the hydrophobic parts of HPMC will require more energy to induce alignment of the polymer chains as well as to dismantle the micellar structure. This automatically raises the energy requirement for the system; the magnitude, however, is controlled by the electrostatic repulsion and its strength.In summary, anionic surfactants increase the energy barrier of the sol-gel transition due to their priority binding to the hydrophobic parts of HPMC, which induces polar outshells and thus hinders the free access to HPMC chains at elevated temperature. The non-ionic surfactant shows much less influence on the gelation of HPMC solution. The difference in the chemical structure of and the electrostatic interaction between the surfactant and HPMC molecules determines the thermal energy requirements for sol-gel transitions in the ternary mixtures of surfactant-HPMC-water.HPMC is the most commonly used hydrophilic carrier for oral-administered controlled drug release systems  as it di2PO4 and NaOH. SGF, without pepsin, was prepared using NaCl and HCl according to the specification given in the United States Pharmacopeia (25th edition). The compositions of the various buffer solutions are listed in 2PO4; NaOH was added to adjust pH values. For the SIF buffer solutions with different pH values but with the same buffer content (8.6 g/L), NaCl was added to keep the buffer content constant. Unless mentioned otherwise, the pH of SIF used was 7.4 and the solvent was H2O. The pH of SGF was 1.2, whereby the concentration of HCl was 70 mM and NaCl was added to tune the buffer content. The HPMC solutions were prepared by dissolving the polymer into respective buffer solutions.It was of interest to investigate the effects of physiological buffers on the thermogelation behavior of HPMC. Thermal properties of HPMC in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were examined. SIF, without pancreatin, was prepared using KHWhen the number average molecular weights (Mn) of the polymers were determined by GPC, polymer sample was diluted 6 times and the solution was then filtered. The LCST  values for the solutions were determined at the temperatures which showed an optical transmittance of 50% during UV-vis spectrometer measurements. During rheological studies, and G′ and G″ values were examined as a function of temperature from 20 °C to 75 °C at a frequency (×) of 1 rad/s and a heating rate of 1 °C/min. Isothermal frequency sweeps (x = 0.1–100 rad/s) on HPMC solutions were also performed at 5 wt% strain.2O and D2O) are presented in The thermogram of 10 wt% aqueous solution of HPMC as well as the thermograms of HPMC containing SIF (8.3 g/L) in two solvents . During the heating process, a broad endothermic peak was observed while two exothermic peaks  were present during the subsequent cooling process. The similarities between the patterns of the thermograms for HPMC in the presence of SIF and in aqueous solutions indicate that they share the same mechanisms as described previously [onset), the water cages start to deform and break to expose the hydrophobic substitutions to the aqueous environment. The exposed hydrophobic substitutions form hydrophobic aggregation domains via hydrophobic-hydrophobic interactions. These domains are connected through hydrophobic groups, yielding a three-dimensional physical network of HPMC chains.The curves for HPMC in SIF were found similar to those in aqueous solution. However, Tperature , shiftedeviously ,68. It heviously . The coneviously . The intIt is worth noting that the endothermic peak  and midp− and H2PO4− while those of SGF contain Cl−, which are water structure makers or salting-out anions according to Hofmeister series. In their presence, the adjacent water molecules were polarized and rearranged in the electric field of the ion [− and H2PO4− are 0.112 L mol−1 and 0.34 L mol−1, respectively [− and H2PO4−, causing depletion in the strength and number of water cages and resulting in enhanced hydrophobic association at low temperatures.As shown in  the ion . Viscosiectively . Ions wiectively . The strectively ,18,26,69et al. [The broad exothermic peak and the smaller shoulder observed during the cooling process indicated that gel-sol transition proceeded in two successive transitions. Similar trends in the thermograms of HPMC and MC in aqueous solutions have been reported by Hirrien et al. , yet the2O and H2O. As seen in max in D2O moved to a lower temperature. More interestingly, the Tmax has approximately a linear relationship with the mole fraction of D2O  (PNIPAAm) studied by Kujawa and Winnik , where tthe Tmax ,71. Addimax in D2O was assumed to be due to the interchain hydrogen bonding rather than the combined effects of the interchain hydrogen bonding and enhanced hydrophobic interactions. This is because a non-linear change in the Tmax with D2O content instead of a linear change will be observed if D2O has more than two types of complex interactions with HPMC [The opposite results obtained in our investigations for HPMC with and without SIF indicated that interchain hydrogen bonding was involved in the gelation process, leading to a salting-out effect. In the case of HPMC, the decrease of Tith HPMC . This trith HPMC  for hydrmax, ΔH and ΔS with various D2O content were extracted from the thermograms and the results are illustrated in 2O content and followed sigmoidal shape with the variations in D2O content. Three distinct regions were observed. When the molar ratio is below 0.4, ΔH and ΔS increased slightly. They underwent a rapid increase in the region where 0.4 < molar ratio < 0.6, and then gradually reached a plateau. To interpret this trend, it is necessary to investigate the thermodynamic parameters of HPMC containing SIF in H2O. There are several processes that might consume or release heat during thermogelation. For instance, heat was consumed to break intermolecular hydrogen bonding and water cages as well as for hydrophobic association. However, the formation of interchain hydrogen bonding is an exothermic process. It has been extensively studied and demonstrated that the positive ΔH (endothermic peak) was mainly related to the destruction of water cages around hydrophobic clusters of the polymer chains [In addition to Tr chains ,74,75. Tr chains ,67.2O, it was expected that more heat be consumed to break the strengthened intermolecular hydrogen bonding. On the other hand, this is compensated by the formation of interchain hydrogen bonding because the latter is prevalent in D2O. The increase of ΔH and ΔS in D2O compared to those in H2O can only be explained by consideration of the small molecules including water molecules and ions surrounding hydroxyl groups of the HPMC chains  and the Tmax curve of the SIF has a higher negative slope, indicating that it has a stronger salting-out effect as compared to that of the SGF. This result can be explained by the difference in the salting out capacity of anions since cations have much less effect on the thermogelation as compared to anions. The value of the viscosity B coefficient of Cl− is −0.005 L mol−1, which is less than those of OH− and H2PO4− [The more pronounced salting-out effect with increasing buffer content is because of the fact that buffers with higher concentrations can attract more free water molecules around the salt ions, resulting in fewer free water molecules available around the hydrophobic clusters and hydroxyl groups. The water cages around the hydrophobic clusters and the shell around the hydroxyl groups were further weakened with the increasing buffer content upon heating. Similarly, the re-formation of cage structure around the methyl groups was further deferred with high buffer content during the cooling process (data not shown). The Td H2PO4− . Anions The effects of buffer content on ΔH and ΔS are illustrated in max decreased slightly with increasing pH. This was caused by the slight increase in NaOH concentration because NaOH adjusted the pH. In the case of SIF with equal salt content, the similar trend was also observed. This was because the salting-out ability of OH– is more pronounced than that of Cl−. The corresponding ΔH and ΔS of HPMC in buffers with different pH values are illustrated in The effect of pH on the thermogelation is of interest since orally administrated drug loaded hydrogels are exposed to SGF (pH between 1.0 and 2.5) and followed by SIF (pH between 5.5 and 7.9) . As showmax was not significant, indicating that the gelation process was a temperature driven process rather than only driven by the heat input [In addition to the parameters mentioned earlier, the effect of polymer concentration on thermal behavior was examined (data not shown). The influence of polymer concentration ranging from 1 wt% to 10 wt% on the Tat input . On the onset but became turbid at temperatures (LCST) between the Tonset and Tmax measured by the micro-DSC. During cooling, the solution became transparent at low temperatures. However, an obvious hysteresis was observed during the cooling process. The results are consistent with those obtained using the micro-DSC. It should be mentioned that the LCST was relatively independent of concentration in the studied range. However, the LCST of diluted HPMC solution (3 wt% and less) was found to increase by a few degrees. For instance, the LCST of HPMC solution (1 wt% in DI water) was 69.0 °C. The reason for this is that the polymer aggregates are slow to aggregate to a size that can be detected by the UV-vis at low concentrations. The gelation behavior induced by temperature is known as the sol-gel transition with a LCST. The LCST moved to a lower temperature in the presence of SGF and SIF.Transmittance changes for HPMC aqueous and buffer solutions as a function of temperature are presented in et al. [et al. [The turbidity was attributed to the Rayleigh scattering from junctions formed in the gelation process. LCST is always considered as an indication of phase separation . The relet al.  suggesteet al. . In our  [et al.  in theirViscoelastic properties of HPMC solutions were investigated to further understand the gelation mechanism. SIF was chosen as a typical example since HPMC in SGF and in SIF exhibited similar behavior. Additionally, changing pH did not generate significant difference on the properties of HPMC, as evidenced earlier. The concentration dependence of the quasi-equilibrium modulusexponent . In thisut by Li  in his wAs seen in  network ,85.The temperature dependence of studied . It was Rheological measurements showed thatonset and Tmax measured by micro-DSC. The reason for such a rapid increase inonset is the kinetics of the sol-gel transition, where heat was absorbed to break the water cages and the shell surrounding the hydrophobic clusters and hydroxyl groups, respectively. This was followed by hydrophobic association and hydroxyl groups association as illustrated in It was observed that the rapid increase ofi.e.,To compare the viscoelastic properties of the polymers in different SIF contained solutions, frequency dependence for HPMC (10 wt%) was conducted at 65 °C.y Sarkar . It shouThe total number of higher hydrophobic substitutes increased with increasing SIF content in a sigmoidal manner, resulting in a similar trend in the changes of the crosslinking density. However,Among various dosage forms in medicine, tablets still account for 80% of the drug delivery systems administered to human due to ease of manufacture, convenience of dosing and stability as compared with liquid and semi-solid dosage patterns . For theVarious parameters influence the drug releasing profile of HPMC tablets. Porosity of the formed tablets, different particle sizes and distribution of HPMC powder and physicochemical properties of various grades of HPMC are some of the critical factors that influence the behavior of a drug-loaded HPMC tablet in body fluids ,89,90,912 pressure. The average weight of the formed tablets was 1.3 g. The tablets were of cylindrical shape with their average diameter and height measured at 18 mm and 5–8 mm respectively. The tablets were stored all the time in a dry box before use.For this purpose, tablets of three grades of HPMC with different properties were fabricated. To begin with, HPMC-A, -B, and -C in their powder form were dehydrated in vacuum at 60 °C for 24 h. The tablets were prepared using the direct-compression method without any extra additives or binders. The forming condition was set to 1-min compression under 400 kg/cmg of the three samples was measured using the TA modulated DSC 2920 in the range of 20–220 °C with the ramp rate of 10 °C/min under N2 environment maintained at flow rate of 50 mL/min.Upon taking 10 mg of pre-dried HPMC powders A, B, C each time and sealing the sample in the standard aluminum sample pan, Tr represents the swelling ratio, Wt andWt0 respectively represent weights of the tablet at a certain time and its original dry weight. The values were averaged over three weight readings for each tablet for minimizing measurement errors.Three full-sized tablets of each of HMPC-A, -B and -C were weighed accurately before being placed individually in a porous paper bag. The bag was used to facilitate handling of the tablets as and when required and simultaneously insure that the solvent could diffuse without any obstruction during the swelling process. A 50 mL of deionized water, SGF, and SIF were loaded in separate plastic tubes with a properly marked scale line. One set of media was used to soak one grade of HPMC tablets. Three rounds of experiments were carried out for each of HPMC-A, -B and -C tablets. All solutions were maintained at the near body temperature of 37 °C. In order to assess the water uptake at various time intervals, the swollen tablets were taken out quickly, had their surfaces wiped using a tissue paper and weighed immediately. The swelling ratios were calculated using Equation (6) as below:Since HPMC is a derivative of cellulose obtained by adding hydroxypropyl and methyl groups to substitute primary and secondary hydroxyl groups, three factors, namely, methyl content, hydroxypropyl content, and molecular weight control the final properties and behavior of HPMC . AlthougDue to methyl (hydrophobic) and hydroxypropyl (hydrophilic to some extent) substitution, the network of hydrogen bondings (H-bondings) in cellulose is disrupted. The hydrophobic interactions between methyl groups and water can lead to self-aggregation of HPMC chains. The aggregation is accelerated with aid of interactions with water (H-bonding) at elevated temperature or in a highly plasticized state, which leads to the gelation of HPMC in most cases. HPMC shows more affinity to water molecules, leading to higher water solubility of the polymer. The opposing effects of the hydrophobic methyl groups and hydrophilic hydroxypropyl groups grant HPMC the swellability in the aqueous media. By adjusting the hydrophobic-hydrophilic balance with the suitable ratio of hydroxypropyl and methyl groups, the swellability of HPMC in the aqueous media can be modulated.g of HPMC-A, -B and -C were found to be 178, 188 and 163 °C respectively. These results indicate that the polymer chains are almost immobile at 37 °C, the temperature used in the current experimental studies. Therefore it is impossible to have the formation of systematic aggregation taking place spontaneously except the inherent presence of H-bond network.The Tg of HPMC is reduced drastically. This results in reducing energy barriers that restrict the mobility of the functional groups, side chains and polymer main chains. This allows the polymer chains to change the conformation with greater freedom.When HPMC tablets are soaked in aqueous media, a large amount of water is absorbed into the porous polymeric matrix. The absorbed water acts as a plasticizing reagent and reduces the H-bond inter/intra-chains interactions. As a result, TAs depicted in Sr represents the swelling ratio, t means the soaking time and, a and b are the process related constant parameters. Equation (7) may be expressed as a linear curve byIt is known that the swelling process is governed by a power law  as in EqHPMC-A, -B and -C show similar trends irrespective of the type of media they are soaked in. The swelling patterns in the three media also show a similar trend with similar swelling rate and final swelling ratio. These results testify that the variants like pH and ionic strength of media have little effect on the swelling behavior in the adopted time scale . The difMany factors including particle size and distribution of powder, porosity of the formed tablets, interaction between polymer and solvent, influence the rate at which the fluid media is absorbed into HPMC tablets. However, the determining factors should be the physicochemical properties of HPMC, especially when a large amount of fluid media is absorbed in the swollen state. Hydrophobic interactions between methyl groups and water are the main drivers for the spontaneous swelling process. The self-aggregation behavior of different grades of HPMC is linked to their different chain structures. The ordered network formed is determined by the density of methyl groups in the polymer-fluid system at the swollen state. The possible mechanism involved in the fluid uptake and swelling processes is shown in The size of the formed network cells and the density of the physical cross-linking sites not only determine the strength of the gel layers but also control the disintegration behavior of the direct-compressed tablets with time in the fluid media. Obviously, HPMC-A had the weakest network that disintegrated much earlier than HPMC-B. HPMC-C exhibited the strongest network and stable shape even after absorbing considerable amount of the fluid media.2 pressure for 1 min at room temperature in tablet maker. The ready tablets were soaked in a 50 mL SIF solution maintained at 37 °C adopting the same procedure as described in the earlier section. Starting from the first half an hour until the 60th h, a 0.25 mL of drug-mixed solution was taken out every 30 min and diluted to 3 mL aqueous for UV measurements to determine the concentration of Indomethacin in the fluid media derived from the immersed tablets. A series of aqueous solution samples with different concentrations of Indomethacin were prepared separately and tested for UV absorption at 265 nm wavelength, and the relationship between the UV readings versus the drug concentrations was set as a standard curve. By comparing the results of UV tests on the collected samples with the standard curve, the release profiles for Indomethacin loaded HPMC tablets were obtained.Indomethacin in powder form was mixed with each of the three types of pre-dried HPMC powders in 1:20 ratio, respectively. Each mixture was then compressed at 400 kg/cmFrom the release profile in SIF shown in It is known that the gel barrier is the determining factor of translocation of drug transportation from HPMC matrix . The fasBased on the drug release profile, a detail description of the possible mechanism of the process can be depicted as shown in In the early stage, the drug-loaded tablets absorb a large amount of SIF into the porous matrix. This causes the outer layer to hydrate and convert the part into gel with the assistance of water molecules as plasticizing agents. As time progresses, the outermost layer dissolves either partially or fully into the media, allowing more water molecules to diffuse into the inner core region, which is intact so far, by forming a new gel front. The drug that remains rapped within the disintegrated part is released faster than that from those separated portions of the HPMC gel. The thickness of the gelation layer is rather thin at the early swelling stage, which is also the reason for the faster drug release. As more and more aqueous media is absorbed into the tablets, the gelation layer gets thicker and thicker until it reaches the fully swollen stage, which is the fully saturated and expanded state of the gel. With more water coming in, the fully swollen tablets show different routes in the later stage of drug releasing process. In the case of HPMC-A, the tablets disintegrate into various microgel particles containing drug in small amount at a rather early stage (10 h or so). Due to the smaller sizes of the microgel particles that float in the media in large numbers, the loaded drug is able to easily pass through the thin gel layer and dissolve into the media around. This has resulted in the fastest release rate among the three grades of HPMC. As far as HPMC-B is concerned, after achieving the fully swollen state, the tablets disintegrated into much larger pieces compared to HPMC-A. The much thicker gel layer and smaller specific surface area led to a slower drug release rate after 25 h. During the whole time scale, HPMC-C tablets kept its content integrated; the tablets became fully gelated with a small residual core. The thickest gel barrier hindered the diffusion of the drug trapped within the matrix into the media freely, resulting in the slowest drug release rate.m was found to in the same sequence of the Hofmeister series and attributed to their ability to influence water structure. Microcalorimetry results disclosed that the effect of monovalent salts on sol-gel transition was more cooperative than that of multivalent salts. All in all, the gel strength increased in the presence of salting-out salts, while it decreased slightly in the presence of salting-in salts.The investigations revealed that most salts (except NaI and NaSCN) added to aqueous solutions of HPMC lead to salting-out effects and therefore promote thermogelation of HPMC. NaI and NaSCN, however, showed salting-in effects and suppressed the thermogelation. The effectiveness of anionic species in reducing TSimilar to the salts, thermal behavior of HPMC hydrogels was influenced by surfactants. With the addition of SDS lower than 6mM, SDS existed as dissociative ions within the solution and the gelation of HPMC/SDS mixtures occurred at lower temperatures as compared to pure HPMC hydrogels. The addition of SDS higher than 6mM concentrations significantly affected the HPMC gelation; not only the peak shape of the DSC thermograms changed from a single mode to a bimodal but also each thermogram covered a wider range of temperature with reduced height of the first peak. The addition of SDeS in different concentrations had a salt-in effect on HPMC gelation without having any effect on the shape of corresponding thermograms although the thermograms became narrower with increasing the concentration of SDeS. SHS did not significantly affect the sol-gel transition of HPMC hydrogels except for apparently increasing the heat capacity. The anionic  surfactants increased the energy barrier of the sol-gel transition due to their priority binding to the hydrophobic parts of HPMC inducing polar outshells, which hinder the free access to HPMC chains at elevated temperature. The non-ionic (Triton X-100) surfactant showed much less influence on the gelation of HPMC solution. Difference in the chemical structure and electrostatic interaction between the surfactant and HPMC molecules determined the thermal energy requirements for sol-gel transitions in the ternary mixtures of surfactant/HPMC/water.The thermoreversible sol-gel transition of HPMC in simulated intestinal/gastric fluids provided an endothermic peak upon heating and an exothermic peak upon cooling using a micro-DSC. Enthalpy and entropy changes were linearly proportional to the HPMC concentration. Similar phenomena were observed for samples in SGF and SIF. Both SGF and SIF do not change the sol-gel transition mechanisms of HPMC, but the temperature at which these sol-gel transitions took place is decreased with the increasing SGF/SIF content. At the same time, enthalpy and entropy changes involved in the sol-gel transition increased. This salting-out effect was the result of the competition for free water molecules between the salt ions and the HPMC chains. Rheological measurements showed that the gel strength of semi-dilute HPMC solutions increased abruptly during the sol-gel transition and eventually reached a plateau upon heating. However, the gel strength decreased gradually upon cooling. Fluorescence experiments confirmed that the sol-gel transition of HPMC solution was driven by hydrophobic association.The swelling behavior of tablets of all three grades of HPMC in water, SGF, and SIF, all showed very similar trends. It was observed that the percentage substitution of methyl side groups and molecular weight of the polymer determine and control the various patterns of the fluid uptake and the swelling process for the tablets. The drug release profiles for the Indomethacin-loaded tablets showed a reverse trend as compared to the fluid uptake process. However, similar to the fluid uptake process, drug release was also controlled by the size and density of the gel network.