Bone regeneration is an important issue in many situations, such as bone fracture and surgery. Umbilical cord mesenchymal stem cells (UC-MSCs) are promising cell sources for bone regeneration. Bone morphogenetic proteins and their bioactive peptides are biomolecules known to enhance the osteogenic differentiation of MSCs. However, fibrosis can arise during the development of implantable biomaterials. Therefore, it is important to control cell organization by enhancing osteogenic proliferation and differentiation and inhibiting fibroblast proliferation. Thus, we focused on the screening of such osteogenic-enhancing peptides. In the present study, we developed new peptide array screening platforms to evaluate cell proliferation and alkaline phosphatase activity in osteoblasts, UC-MSCs and fibroblasts. The conditions for the screening platform were first defined using UC-MSCs and an osteogenic differentiation peptide known as W9. Next, in silico screening to define the candidate peptides was carried out to evaluate the homology of 19 bone morphogenetic proteins. Twenty-five candidate 9-mer peptides were selected for screening. Finally, the screening of osteogenic-enhancing (osteogenic cell-selective proliferation and osteogenic differentiation) short peptide was carried out using the peptide array method, and three osteogenic-enhancing peptides were identified, confirming the validity of this screening. Although these materials have been widely studied for bone healing [Bone formation and regeneration are necessary phenomena in situations such as fracture due to osteoporosis, cleft lip and palate (CLP) and surgery . Osteoporosis is characterized by low bone mineral density, strength and micro-architectural deterioration, leading to an increased risk of fragility fractures . With an healing ,7, develMesenchymal stem cells (MSCs) are one of the most promising resources in regenerative medicine and tissue engineering. MSCs derived from different sources, such as bone marrow (BM), adipose tissue (AT) and umbilical cord (UC), have been extensively studied for bone cell therapy and tissue engineering . ParticuBone morphogenetic proteins (BMPs) are a family of growth factors known to induce bone formation and have been studied in the context of osteogenic differentiation in MSCs . Since tHowever, fibrosis can arise during the development of implantable biomaterials. Fibrosis is the final and common pathological outcome of many chronic inflammatory diseases. It is defined by excessive accumulation of fibrous connective tissue  in and around the inflamed and damaged tissue, resulting in fatal organ damage . For insThus, we focused on screening of osteogenic-enhancing peptides to enhance osteogenic proliferation and differentiation and inhibit FB proliferation as bone-regenerative biomaterials. For peptide screening, we used the SPOT synthesis peptide array method ,37. We dIn the present study, we developed a new peptide-screening platform to evaluate osteogenic enhancement (osteogenic cell-selective proliferation and osteogenic differentiation) and identified several osteogenic-enhancing short peptides. To develop the new osteogenic peptide screening platform, ALP activity, which is one of the most famous assays for initial osteogenesis, was adopted as the case study. Before screening, the conditions of the screening platform were defined using the W9 peptide, which is an example of the shortest sequence known to be an osteogenic differentiation peptide. Additionally, UC-MSCs were chosen to detect lower ALP activity. Next, in silico screening was first performed to define candidate peptides and to search the homology of several BMPs related to osteogenic differentiation in several species . The lenp = 0.068). The ALP assay showed no differences between Blank and RGD at all time points (p < 0.05). To define the more significant endpoint of the ALP activity assay, the ALP activity per unit cell number was calculated (p = 0.079). Considering that ALP is an osteogenic marker in the initial stage, the duration of the peptide array screening was defined as Day 7.To screen for osteogenic-enhancing peptides using a peptide array, it is necessary to determine the optimal day for evaluation. Thus, cell proliferation and osteogenic differentiation assays with UC-MSCs were carried out at Days 7, 14 and 21 on the peptide array. As controls, no peptide (Blank), RGD peptide and W9 peptide were used. RGD peptide is well known as a cell adhesion peptide, while W9 is known to be an osteogenic differentiation peptide. In the proliferation assay, no differences were observed between Blank and RGD at all time points a. For the points b. Howevelculated c. The vaFor in silico screening, several sequences of BMPs were downloaded from UniProt. The information regarding the BMPs used in this study is listed in To determine the candidate peptides for peptide array screening, 19 BMP sequences were aligned to define the important sequences that may be related to osteogenic differentiation. To select candidate peptides, we set two criteria depending on the following hypotheses: (1) homologous BMP sequences across several species with the ability for osteogenic enhancement; and (2) short peptide-like 9-mers with the ability for osteogenic enhancement. p < 0.05). In the screening for UC-MSC proliferation peptides, several peptides  showed higher proliferation than the no peptide Blank (p < 0.05). In contrast, peptide No. 11 showed significantly lower proliferation than that in the Blank (p < 0.05). These results indicate variation in proliferation because the peptides were derived from BMPs in each cell type and that several peptides, such as peptide No. 2  and Nos. 1, 6  could be considered as osteogenic cell-selective proliferation peptides.To identify the osteogenic-enhancing peptides in 25 candidate peptides, all peptides, including RGD and the Blank, were synthesized on cellulose membranes by fluorenyl-meth oxy-carbonyl (F-moc) solid phase peptide synthesis, as previously reported ,41. In tde Blank b. Howevede Blank c. Only pp < 0.05). In the screening for osteogenic differentiation peptides for UC-MSC, 15 peptides  showed higher differentiation than in the Blank . In UC-MSCs, 20 peptides  showed higher differentiation than the Blank (p < 0.05).When investigating ALP activity, it is important to consider ALP activity per unit cell number. The number of cells may be higher for the peptide defined as having higher ALP activity. To resolve this problem, all ALP values were divided by unit cell numbers. he Blank b, and diThese results indicate that several osteogenic differentiation peptides are derived from BMPs, and several of these peptides may be considered as osteogenic differentiation peptides, such as peptide No. 11 (showing higher ALP activity in both OBs and UC-MSCs).To identify osteogenic-enhancing peptides (osteogenic cell-selective proliferation (higher proliferation of OBs and UC-MSCs and lower for FBs) and osteogenic differentiation (higher ALP activity in OBs and UC-MSCs)), each peptide was scored based on its osteogenic cell-selective proliferation and differentiation-inducing capacity. In the present study, we developed a new PIASPAC method to screen for osteogenic-enhancing peptides. Upon aligning 19 BMPs  from eight species, 25 candidate 9-mer peptides were obtained from in silico analysis. Using the established PIASPAC method, three osteogenic-enhancing peptides were discovered in the osteogenic cell-selective proliferation assay and osteogenic differentiation assay.As shown in To select screening candidates, we hypothesized that osteogenic-related sequences (peptides) existed in regions showing high homology across the 19 types of BMPs from eight species. Several homologous regions were observed in the in silico screening process . FurtherAs a result of peptide screening, three peptides were found to be osteogenic-enhancing peptides from among the 25 candidate peptides . To idenIn this study, our results supported two hypotheses: (1) homologous sequences of BMPs across several species are involved in osteogenic-enhancing; and (2) short peptides, such as 9-mers, are involved in osteogenic enhancement. As a result, three short peptides with the ability of osteogenic enhancement were identified from homologous BMP sequences. Although these results supported our hypotheses, further studies are needed to evaluate mature osteogenesis in vitro and in vivo using alizarin red staining, calcium determination, hydroxyapatite determination and mRNA expression analysis related to the bone regeneration gene . However, human cells were used in the present study, so it is necessary to investigate the same experiment with other species’ cells, such as mouse or rat. Additionally, it is necessary to investigate other peptides other than the 25 candidate peptides, and the efficiency should be evaluated to compare using sequences of BMPs and that of another protein family. Moreover, it is necessary to investigate the inhibition of fibrosis in vivo to confirm the effect of osteogenic cell-selective proliferation. However, the concept of cell-selectivity is a novel and important idea for tissue engineering for controlling regeneration and its side effects, such as fibrosis. The peptide array platform developed in this study is a promising method for identifying cell-selective peptides and aiding the development of biomimetic materials.The BMP sequences were obtained from UniProt and were then aligned in the database. The graphical image was obtained using Jalview (ver. 2.9.0b2), which is a free program for multiple sequence alignment editing.2 and were used within 4 to 6 passages.Normal human osteoblasts  were maintained in the OGM Bullet Kit . Human mesenchymal stem cells from umbilical cord matrix  were maintained in mesenchymal stem cell growth medium . Normal human dermal fibroblasts  were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum  and 1% penicillin-streptomycin . The cells were maintained at 37 °C with 5% COA peptide array was designed and synthesized using F-moc synthesis following the manufacture’s protocol using a peptide synthesizer  with some modifications, as reported previously ,41. All 4 cells/cm2, UC-MSC: 2.9 × 104 cells/cm2). After 7 days of culture, WST-8   was added to the medium following the manufacture’s protocol for 1 h, and the absorbance of the supernatant was measured using an absorption plate reader  at 450 nm. Significant differences in the data between two conditions were determined by the Student’s t-test.The previously-described PIASPAC protocol was applied to assay the relative cell number of OBs, UC-MSCs and FBs with some modifications ,41. Viab4 cells/cm2, UC-MSC: 2.9 × 104 cells/cm2). After 7 days of culture, SIGMAFAST™ p-nitrophenyl phosphate tablets  dissolved in water were added to the medium following the manufacturer’s protocol and incubated for 30 min. The absorbance of the supernatant was measured by an absorption plate reader (iMark™ Microplate Absorbance Reader) at 405 nm. Significant differences in the data between two conditions were determined by Student’s t-test.The previously-described PIASPAC protocol was applied to assay the relative ALP activity in OBs and UC-MSCs with some modifications ,41. ViabIn this study, a novel peptide-screening platform using a peptide array was developed to identify osteogenic-enhancing peptides. For this screening, in silico analysis was performed to compare the sequences of 19 types of BMPs that are well known as bone regenerative molecules. As a result, 25 candidate 9-mer peptides were selected from homology sequences, including some known to be from an osteogenic differentiation region. Among the 25 short peptides, three peptides were found to be osteogenic-enhancing peptides through peptide array-based osteogenic cell-selective proliferation and osteogenic differentiation assays.


Tuberculosis (TB) is a major source of mortality in urban India, with many structural challenges to optimal care delivery. In the government TB program in Chennai, India’s fourth most populous city, there is a 49% gap between the official number of smear-positive TB patients diagnosed and the official number registered in TB treatment within the city in 2014. We hypothesize that this “urban registration gap” is partly due to rural patients temporarily visiting the city for diagnostic evaluation.We collected data for one month (May 2015) from 22 government designated microscopy centers (DMCs) in Chennai where 90% of smear-positive TB patients are diagnosed and coded patient addresses by location. We also analyzed the distribution of chest symptomatics  and diagnosed smear-positive TB patients for all of Chennai’s 54 DMCs in 2014.At 22 DMCs in May 2015, 565 of 3,543 (15.9%) chest symptomatics and 71 of 412 (17.2%) diagnosed smear-positive patients had an address outside of Chennai. At the city’s four high patient volume DMCs, 54 of 270 (20.0%) smear-positive patients lived out-of-city. At one of these high-volume DMCs, 31 of 59 (52.5%) smear-positive patients lived out-of-city. Out of 6,135 smear-positive patients diagnosed in Chennai in 2014, 3,498 (57%) were diagnosed at the four high-volume DMCs. The 32 DMCs with the lowest patient volume diagnosed 10% of all smear-positive patients.TB case detection in Chennai is centralized, with four high-volume DMCs making most diagnoses. One-sixth of patients are from outside the city, most of whom get evaluated at these high-volume DMCs. This calls for better coordination between high-volume city DMCs and rural TB units where many patients may take TB treatment. Patient mobility only partly explains Chennai’s urban registration gap, suggesting that pretreatment loss to follow-up of patients who live within the city may also be a major problem. India has the world’s largest tuberculosis (TB) epidemic, with nearly one-quarter of the global burden of incident TB patients annually, a considerable proportion of whom are treated in the Government of India’s Revised National TB Control Programme (RNTCP) . The RNTRNTCP statistics for cities in India indicate potential challenges around patient mobility and linkage to care. For India’s largest cities, with the exception of Pune, more patients are diagnosed with smear-positive TB every year than the number of smear-positive patients who are registered for TB treatment within those cities—a phenomenon we refer to as the “urban TB registration gap”  3]. Che. Che3]. What explains this large proportion of “missing” urban TB patients? We have two hypotheses for the urban TB registration gap. First, we hypothesize that this gap is partly due to pretreatment loss to follow-up —that is, that some patients diagnosed at TB designated microscopy centers (DMCs) subsequently do not start TB therapy or choose to take therapy in the private sector, where their subsequent status is usually not registered by the public health system.Our second hypothesis is that the urban registration gap is partly explained by patients coming from rural areas who temporarily visit cities to access TB diagnostic services. This movement into cities for medical evaluation could be due to poor access to government health services in rural areas , patientThe question of where TB patients in cities are coming from also intersects with an interrelated question of where TB patients are being diagnosed in these cities. In 2014, the Chennai RNTCP had 54 TB DMCs located at tertiary, secondary, and primary health facilities. Understanding the relative distribution of new TB diagnoses across these facilities—whether diagnoses tend to be centralized at a few high patient volume tertiary centers or widely distributed among numerous primary and secondary health centers—also has implications for strengthening urban TB services. With the emergence of higher-quality but relatively expensive diagnostic modalities, such as Cartridge Based Nucleic Acid Amplification Tests (CB-NAATs), understanding where most TB patients are being diagnosed may guide initial placement of these new diagnostic tests at specific centers, in order to have the biggest impact—at least until resources are available to scale-up CBNAATs more broadly to reach all patients who might benefit from these tests.In this paper, we first analyze patient address data from DMCs in Chennai to understand where chest symptomatics  and newly diagnosed smear-positive TB patients are coming from. Second, we evaluate the pattern of where TB patients are being diagnosed in Chennai, to understand the relative importance of tertiary, secondary, and primary health centers for detecting TB patients.Chennai is India’s fourth largest city, with a population of 7 million people. RNTCP DMCs in Chennai evaluate 63,000 to 67,000 patients with sputum microscopy and diagnose 5,600 to 6,300 smear-positive cases annually [In 2014, there were 54 government DMCs in Chennai where patients could get evaluated for TB by sputum smear microscopy. As per RNTCP protocol, newly diagnosed TB patients are subsequently referred to one of 25 TB Units in the city, or to TB Units elsewhere in Tamil Nadu state, to start therapy. Each TB Unit consists of a cluster of directly observed therapy (DOT) centers in a specific locale, usually based out of the city’s primary health centers (PHCs). Patients are referred to the TB Unit closest to their homes for ease of travel to a DOT center to undergo observed dosing for TB medications three times a week.Based on the analysis of data from Chennai’s 54 DMCs, we found that >90% of smear-positive TB patients were diagnosed at just 22 of these DMCs. To understand where TB patients live, we therefore copied the complete patient registers for the month of May 2015 from these 22 DMCs. For purposes of study feasibility, we exclude the remaining 32 DMCs from the analysis, since together they accounted for <10% of the city’s smear-positive diagnoses.From August to October 2015, research investigators visited each of the 22 DMCs across Chennai and collected data for all register entries, including patient address data, which were entered into a REDCAP database. For addresses within Chennai, the pin code was entered to be able to classify patient locations within general areas of the city. Addresses located outside of Chennai were classified based on the district in Tamil Nadu in which the town or village is located. Patient demographic data (age and gender), prior TB history, sputum smear results, line probe assay results, and HIV status (if known) were also entered into the database. The final de-identified dataset and data dictionary are available in the Four of the 22 DMCs in Chennai for which we collected data diagnosed the majority of smear-positive patients in the city: Government Thiruvatteeswarar Hospital of Thoracic Medicine , the Institute of Thoracic Medicine, Government Stanley Hospital, and Chennai General Hospital . We will hereafter refer to these four centers as the “high-volume DMCs”. We conducted a sub-analysis of address locations for chest symptomatics and smear-positive patients at these high-volume DMCs, given their disproportionate patient volume.Using a dataset with de-identified patient information, JMP Pro 12 statistical software was used to generate descriptive statistics on the specific areas of Chennai or districts within Tamil Nadu where chest symptomatics  and diagnosed smear-positive patients  live. We excluded 336 patients who were already on TB therapy and who had provided sputum samples for follow-up evaluation and 153 patients who had no address listed, of whom 11 were diagnosed smear-positive patients. This left 3,543 patient entries in the dataset for analysis. We tested for significant differences in proportions using the Chi-squared test.www.opendatacommons.org/licenses/odbl). The OpenStreetMap open-access license is available in the We used ArcGIS 10.3.1 software by Esri® to generate a heat map of Chennai based on the pincodes where chest symptomatics and smear-positive patients reside. Pincodes, or “postal index numbers”, are six-digit codes used by the Indian postal system to divide the country into different geographical administrative areas. We procured a commercially available pincode map from MBI International  of Chennai, which has 298 distinct pincodes. An ArcGIS basemap was used for all maps  Institutional Ethics Committee (FWA00005104) on December 29, 2014 and the Brigham and Women’s Hospital (Partners) Institutional Review Board (FWA00000484) on January 13, 2015.In May 2015, excluding follow-up cases and patients without district or city address listed, a total of 3,543 chest symptomatics were evaluated for suspected pulmonary TB using sputum microscopy and 412 were diagnosed with smear-positive TB at the 22 DMCs in Chennai. The demographic characteristics of these patients are presented in Of these 3,543 chest symptomatics, 565 (15.9%) had addresses listed outside of Chennai . Of the For both chest symptomatics and diagnosed smear-positive patients from outside of Chennai, the majority came from a few rural districts and towns relatively close to the city—most prominently Tiruvallur district (about 45 km from Chennai) but also Vellore (136 km away), Kancheepuram (70 km away), Tiruvannamallai (180 km away), and Villupuram (160 km away) . Only 24For chest symptomatics and diagnosed smear-positive patients with addresses within Chennai, about 15%—17% of patients did not have a pincode reported . The remOut of 1,920 chest symptomatics screened for TB at the four high-volume DMCs, 420 (21.9%) had out-of-city addresses , which iWe also disaggregated data on the address locations of chest symptomatics and diagnosed smear-positive patients by each of the four high-volume DMCs . ChennaiNotably, of all 565 chest symptomatics with addresses located outside of Chennai 420 (74.3%) were evaluated at the four high-volume DMCs. Chennai General Hospital alone evaluated 265 (46.9%) of all chest symptomatics with out-of-city addresses. Of the 71 diagnosed smear-positive patients with an address outside of Chennai, 54 (76.1%) were diagnosed at the high-volume DMCs and Chennai General Hospital alone diagnosed 31 (43.7%) of all smear-positive patients with outside addresses.In 2014, out of all 54 city DMCs, the four high-volume DMCs evaluated 27,779 (40.1%) of the total 69,200 chest symptomatics who underwent TB screening, and these high-volume DMCs diagnosed 3,498 (57%) of 6,135 total smear-positive patients. The next 18 medium- or low-volume DMCs screened 27,274 (39.4%) of the total 69,200 chest symptomatics and diagnosed 2,022 (33.0%) of 6,135 total smear-positive patients . The remThe smear-positive case detection rate—the proportion of chest symptomatics screened who had a positive sputum smear—varied substantially across DMCs, from as high as 20.6% at Government Thiruvatteeswarar (Otteri) Hospital of Thoracic Medicine to as low as 0.2% at some primary health centers and the Institute of Child Health. The four high-volume DMCs had a pooled smear-positive case detection rate of 12.6%. The subsequent 18 DMCs with medium- to low-volume had a pooled smear-positive case detection rate of 7.4%, which is significantly lower than the case detection rate at the high-volume DMCs (p<0.0001). The 32 lowest-volume DMCs had a pooled smear-positive case detection rate of 4.3%, which is significantly lower than the case detection rate in both the four high-volume DMCs and the 18 medium- or low-volume DMCs (both p<0.0001).In 2014, total of 3,276 smear-positive patients  registered for TB treatment in Chennai in the 25 TB Units . The disOur findings suggest that the TB “urban registration gap” in Chennai may be partly explained by the temporary movement of patients from rural areas and towns into the city for diagnostic evaluation, especially at high-volume DMCs, which represent major tertiary hospitals or TB specialty facilities in the city. About one-sixth of all chest symptomatics and smear-positive patients reported home addresses located outside of Chennai city, mostly in nearby districts in Tamil Nadu state. In the RNTCP, regardless of where TB patients are diagnosed, they are referred to receive treatment at the DOT center closest to their homes, to facilitate the monitoring strategy of thrice-weekly facility-based directly observed therapy. As such, most patients with addresses outside of Chennai were likely referred back to their rural districts to start treatment and would not show up in Chennai’s TB treatment statistics. Indeed, 2013 RNTCP statistics suggest that in Tiruvallur—the district where the most out-of-city patients came from—only 1,815 patients were diagnosed with smear-positive TB at DMCs within the district, while 2,333 smear-positive TB patients were registered for TB treatment within the district [However, the 17% of diagnosed smear-positive patients with out-of-city addresses only explains about one-third of Chennai’s urban registration gap. It is possible that we underestimate the number of patients who live outside of Chennai, because some patients who migrate to Chennai for diagnostic evaluation may stay with relatives or friends in the city and report local addresses within the city when submitting sputum samples. However, it is unlikely that the phenomenon of out-of-city patients reporting local addresses within the city would explain the entire urban registration gap.As such, our findings have potentially important implications for the problem of pretreatment loss to follow-up  of newly diagnosed TB patients. First, since out-of-city patients only explain a small proportion of the urban registration gap, it is possible that the remaining gap is explained by pretreatment loss to follow-up of newly diagnosed TB patients who live within Chennai. Second, we anticipate that out-of-city TB patients might be at increased risk for pretreatment loss to follow-up, due to the potential challenges involved in traveling long distances to the city to get diagnosed and then having to return to rural areas to start treatment. We are currently conducting a cohort study that involves intensive tracking of newly diagnosed smear-positive TB patients to evaluate the problem of pretreatment loss to follow-up in Chennai in greater detail. We anticipate that the forthcoming results of that study, along with the findings of high patient mobility in this current study, will provide a more complete explanation for the urban registration gap.Pretreatment loss to follow-up is a major concern for TB control, as these patients are at high risk for death and may transmit TB to others , 9. ReceIn the current study, about three-fourths of patients from outside of Chennai sought care at high-volume DMCs in the city, likely because these hospitals are well known. For example, Chennai General Hospital has one of the state’s best-known medical colleges. It is also located across the street from the city’s largest railway station, making it easily accessible to out-of-city patients. About half of all chest symptomatics and smear-positive patients at Chennai General Hospital had out-of-city addresses. A recent study at a TB specialty treatment center near Chennai  found that many patients bypass their local TB services to come to these facilities because of name recognition, perceptions that they will receive higher quality care, and referral by other TB patients . SupportWhile Chennai’s RNTCP has regular meetings to facilitate communication between staff at TB facilities within the city to ensure appropriate referral and tracking of patients, these same routine communication mechanisms are not available between DMCs in Chennai and TB Units in other districts. Major tertiary hospitals and TB specialty facilities in urban centers like Chennai need to develop robust services for linking rural TB patients to care and need to develop processes for tracking these patients to ensure they reach rural treatment sites.Novel strategies to improve patient tracking might leverage India’s Unique Identification (UID or Aadhar) system, which includes biometric information  and is estimated to have enrolled about 85% of India’s population. A UID-based biometric-driven patient registration system at all TB facilities at a state or national level would avoid duplication of patient information and help to track patients who are referred to sites both within and outside of the city to ensure they are linked to care. Such a system would help to accurately estimate true patient losses across multiple steps of the TB cascade of care . Also, tMapping the addresses of patients located within Chennai suggests that a considerable proportion of TB patients are coming from a few pincodes in North Chennai. In theory, this finding could be due to detection bias, since North and Central Chennai have more TB diagnostic services and public hospitals than South Chennai. Additionally, maps of TB patients were not able take into consideration the population size within each pincode, which would allow for an examination of TB rates given different population distributions across the city.However, these North Chennai pincodes have a high population density and considerable overcrowding, and they are among the poorest areas of the city. In addition, our findings regarding the higher burden of TB in North Chennai is consistent with results of a recent city-wide TB prevalence survey conducted from 2010–2012 . Our finWe find that diagnosis of TB patients in Chennai is highly centralized, with just four high-volume DMCs diagnosing 57% of all smear-positive patients and the 22 highest-volume DMCs diagnosing 90% of all smear-positive patients. In contrast, there was a relatively equitable distribution of TB patients starting treatment across the city’s 25 TB Units. On the one hand, this finding highlights a need for the RNTCP to improve the general public’s awareness of local TB diagnostic services, especially for rural patients migrating hundreds of kilometers in some cases to seek diagnostic evaluation in Chennai’s tertiary hospitals or TB specialty facilities, when their local facilities should be able to readily diagnose TB.On the other hand, the highly centralized flow of patients offers unique opportunities for strengthening TB care delivery, because strategic interventions at these critical high-volume DMCs could rapidly impact a large proportion of the city’s TB patients. For example, at the time of our study, there was only one light microscopist evaluating sputum samples at each of the four high-volume DMCs, despite the fact that these DMCs diagnose nearly six times the number of smear-positive patients as the 32 lowest-volume DMCs. Increasing the number of light microscopists at each of these DMCs could facilitate same-day TB diagnosis and treatment initiation , which mIn addition, strategic placement of new diagnostic tests such as CB-NAATs for upfront testing of chest symptomatics at these high-volume sites could rapidly increased diagnosis of smear-negative and drug-resistant TB patients. A recent systematic review suggests that there is considerable underdiagnosis of smear-negative TB in the RNTCP, as the majority of patients do not complete the multi-step workup for empiric TB diagnosis . CurrentThe smear-positive case detection rate may be higher at DMCs in tertiary hospitals or TB specialty facilities  because some patients may have been referred to these hospitals by other public or private sector providers due to the severity of their illness. As such, these patients may be more likely to have TB or may have more severe disease due to health system-related delays in diagnosis . AlternaOther major cities in India have a considerable urban TB registration gap (as described in the Introduction), so future research should investigate whether patient mobility is a common phenomenon in other cities. The last Indian study we could find that addressed this question, conducted in Bengaluru in 1963, found that about 17% of diagnosed TB patients resided outside of the city, which is similar to the current study .Future research should investigate whether centralization of diagnostic volume at major tertiary hospitals or TB specialty facilities is also common in other cities. Mumbai is one of the few cities to disaggregate its TB reporting to the RNTCP into 22 different city areas, rather than reporting statistics for the city as a whole . These dOur study has a few important limitations. First, to make this study feasible, we were not able to audit the May 2015 records for 32 of the 54 DMCs in Chennai; however, these 32 DMCs diagnosed only about 10% of the city’s smear-positive patients given their low patient volume and case detection rates. Second, about 4% of all DMC register entries for patients did not list an address, so we could not include them in this analysis. It is possible that both of the above limitations might bias us towards overestimating the proportion of out-of-city chest symptomatics and smear-positive patients. However, we more likely underestimated the proportion of out-of-city patients, because some patients from other districts may list a local relative or friend’s address within Chennai, even if they are only in the city temporarily.In this study, we found that about one-sixth of all TB patients diagnosed at DMCs in Chennai have home addresses located outside of the city, and this finding is inadequate to explain Chennai’s entire urban registration gap, suggesting that there may be a considerable pretreatment loss to follow-up rate in the city. We also found that diagnosis of TB patients is highly centralized, with four high-volume DMCs diagnosing the majority of smear-positive TB patients in the city. The RNTCP should ensure strong coordination between high-volume DMCs in Chennai and the rural TB Units where these patients are likely to take treatment. Novel strategies such as a Unique Identification-based, biometric-driven patient registration system might help with this goal of improving patient tracking and linkage to care.In addition, introduction of new diagnostic tests such as CB-NAATs at major tertiary hospitals and TB specialty facilities for upfront screening of patients could have an impact on a large proportion of the city’s TB patients. Re-distribution of RNTCP personnel in different DMCs depending on the burden of TB patients diagnosed each facility may result in more efficient utilization of resources and better quality of care.Future studies should aim to understand why rural patients are coming to Chennai for diagnostic evaluation rather than going to TB facilities in their local districts, potentially through qualitative interviews. In addition, a needs assessment for these out-of-city patients may help to understand how TB care can be improved for this population. Finally, the urban registration gap is a phenomenon in several cities and future studies of patient addresses and pretreatment loss to follow-up in other cities may help improve urban TB care in India.S1 Appendix(XLSX)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.


One prominent event associated with colorectal adenoma-to-carcinoma progression is genomic instability. Approximately 85% of colorectal cancer cases exhibit chromosomal instability characterized by accumulation of chromosome copy number aberrations (CNAs). Adenomas with gain of chromosome 8q, 13q, and/or 20q are at high risk of progression to cancer. Tumor progression is also associated with expansion of the extracellular matrix (ECM) and the activation of non-malignant cells within the tumor stroma. The glycoproteins versican and lumican are overexpressed at the mRNA level in colon carcinomas compared to adenomas, and are associated with the formation of tumor stroma.The aim of this study was to characterize versican and lumican protein expression in tumor progression and investigate their association with CNAs commonly associated with adenoma-to-carcinoma progression.Tissue microarrays were constructed with colon adenomas and carcinomas that were characterized for MSI-status and DNA copy number gains of chromosomes 8q, 13q and 20q. Sections were immunohistochemically stained for lumican and versican. Protein expression levels were evaluated using digitized slides, and scores were finally dichotomized into a positive or negative score per sample.P = 0.0001) and in high-risk adenomas and carcinomas combined compared to low-risk adenomas . Versican staining in the tumor stroma was more often present in high-risk adenomas combined with carcinomas compared to low-risk adenomas  and was associated with the presence of gain of 13q .Lumican and versican expression were both observed in neoplastic cells and in the tumor stroma of colon adenomas and carcinomas. Lumican expression was more frequently present in epithelial cells of carcinomas than adenomas (49% versus 18%; Epithelial lumican and stromal versican protein expression are increased during colorectal adenoma-to-carcinoma progression. Colorectal cancer (CRC) develops in a multistep process that starts with the formation of a colon adenoma. These are benign lesions arising from the intestinal epithelium, of which only a small subset progresses into CRC ,2. The pIn addition to changes in neoplastic cells, the formation of a tumor-specific microenvironment contributes to tumor progression. One of the most prominent changes in colorectal adenoma-to-carcinoma progression is the expansion of the extracellular matrix (ECM) accompanied by activation of non-malignant cells within the tumor stroma. This is reflected by an altered histological presentation hallmarked by (myo)fibroblast proliferation, macrophage and lymphocyte infiltration, and outgrowth of blood vessels . The intLUM) is a member of the small leucine rich proteoglycan family and has a role in fibrillar network formation. Lumican is thought to modify the fibrous tissue surrounding a tumor, thereby inducing a tumor-specific ECM [VCAN, also known as CSPG2) belongs to the family of large chondroitin sulfate proteoglycans and has hyaluronate binding properties [Previously we performed a genome-wide mRNA expression study, which revealed genes whose expression was upregulated in carcinomas compared to adenomas . A subseific ECM . Versicaoperties . Versicaoperties ,20. Prevoperties . In the Tissue micro arrays (TMAs) were constructed previously  using a Tumor samples were previously characterized for microsatellite instability (MSI) status and DNA copy number aberrations (CNAs) of chromosomal regions 8q, 13q and 20q . For assTissue sections were deparaffinized and rehydrated and endogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol. For the versican staining, antigens were retrieved by microwaving for 30 minutes at 90 watt in 10mM citrate buffer solution (pH 6.0). Primary mouse anti-versican antibody  was incubated at a 1:300 dilution in PBS containing 1% BSA and 0.1% Tween-20  at 4°C overnight, and subsequently detected by a HRP-coupled anti-mouse polymer  and incubation with Diaminobenzidine (DAKO). For the lumican staining, antigen retrieval was done by autoclaving in Tris (10mM)/EDTA (1mM) buffer (pH 9.0). The primary rabbit anti-lumican antibody  was incubated at a dilution of 1:50 in antibody dilutend (DAKO) overnight at 4°C. Staining was detected by incubation with a HRP-coupled anti-rabbit polymer  and incubation with Diaminobenzidine (DAKO). All sections were counterstained with Mayer’s haematoxylin.The stained TMA sections were automatically scanned with a digital pathology system  equipped with a 20x objective with a numerical aperture of 0.75 . Epithelial staining and stromal staining were scored separately into 4 categories; negative, weak, moderate or strong staining. For lumican, the highest score over multiple cores from each tumor was used for further analysis. For versican, because of its strong staining, the lowest score of multiple cores for each tumor was used for further analysis. Final evaluation was performed by dichotimizing the data into a negative and a positive group. Due to loss of cores for technical reasons during the staining procedure, results could be evaluated for fewer than the 82 carcinoma tissues and 82 adenoma tissues included. For lumican staining 72 colon adenomas and 65 colon carcinomas were evaluated, for versican 75 colon adenomas and 71 carcinomas .Pearson’s Chi-square test, or Fisher’s exact test whenever appropriate, was applied to evaluate associations between categorical variables; t-testing was applied for investigation of associations between the staining categories and age of the patients. All statistical tests were two sided, and Protein expression of lumican and versican was observed in neoplastic epithelial cells as well as in the tumor stroma of both adenomas and carcinomas, representative examples are shown in versus 18%; P = 0.0001, All epithelial and stromal scores for lumican and versican were dichotomized into a positive  and a negative group. Overall, epithelial lumican staining was present in 33% of all lesions and stromal expression was observed in 75% of cases . Lumicanet al., [Next, lumican and versican protein expression levels were compared to their mRNA expression levels in corresponding tumor samples. Previous work revealed lumican and versican as significantly higher expressed at the mRNA level by carcinomas compared to adenomas et al., ,15). For. Foret aversus 16%; P = 0.005, versus 36%; P = 0.07, data not shown). When high-risk adenomas were combined with carcinomas, stromal versican staining was more frequently observed than in low-risk adenomas  are not among the most commonly gained regions in adenoma-to-carcinoma progression [Accumulation of chromosomal aberrations is a potentially driving process in colorectal carcinogenesis and associated with high risk of adenoma-to-carcinoma progression; these chromosomal aberrations result in up- or down-regulation of many genes, via direct and indirect processes. The chromosomal regions of the HMGA2, an oncogene that is highly expressed in about 50% of carcinomas [A critical step in adenoma-to-carcinoma progression is invasion of tumor cells in the surrounding stroma while remodeling it into 'tumor stroma'. Lumican might have a dual role in cancer cells and stromal cells with opposite effects on cancer progression. For example, cell migration can be inhibited by lumican through prevention of the activation of focal adhesion kinase . In addircinomas , which crcinomas . These rrcinomas . Howeverrcinomas . Furtherrcinomas . A prevircinomas . This maIn the current study, versican was increased in the tumor stroma of high-risk adenomas and carcinomas compared to low-risk adenomas, suggesting that versican plays a role in remodeling of tumor stroma in adenoma-to-carcinoma progression. Versican has been found to promote tumor development and metastasis through stimulation of cell proliferation and by increasing cell motility in several cancer types ,33. In cIn conclusion, in the present study protein expression of both lumican and versican was upregulated in high-risk adenomas and carcinomas compared to low-risk adenomas, indicating that they may play a role in adenoma-to-carcinoma progression. Further studies are required to elucidate the specific roles of these proteins and the mechanisms by which they influence adenoma-to-carcinoma progression.S1 FigBox plots with dot plots of mRNA expression (determined by oligonucleotide microarrays) in colorectal adenomas and carcinomas ,15. For (TIF)Click here for additional data file.S1 TableMSS: microsatellite stable, MSI: microsatellite instable, gain: chromosomal gain.(XLSX)Click here for additional data file.


Neurotensin (NTS)-polyplex is a nanoparticle system for targeted gene delivery that holds great promise for treatment of Parkinson’s disease and various types of cancer. However, the high instability in aqueous suspension of NTS-polyplex nanoparticles is a major limitation for their widespread clinical use. To overcome this obstacle, we developed a clinical formulation and a lyophilization process for NTS-polyplex nanoparticles. The reconstituted samples were compared with fresh preparations by using transmission electron microscopy, dynamic light scattering, electrophoretic mobility, circular dichroism and transfection assays in vitro and in vivo. Our formulation was able to confer lyoprotection and stability to these nanoparticles. In addition, transmission electron microscopy (TEM) and size exclusion-high performance liquid chromatography (SEC-HPLC) using a radioactive tag revealed that the interaction of reconstituted nanoparticles with fetal bovine or human serum did not alter their biophysical features. Furthermore, the formulation and the lyophilization procedure guaranteed functional NTS-polyplex nanoparticles for at least six months of storage at 25 °C and 60% relative humidity. Our results offer a pharmaceutical guide for formulation and long-term storage of NTS-polyplex nanoparticles that could be applied to other polyplexes. The KP, the natural ligand NTS and the hemagglutinin-derived HA2 fusogenic peptide (FP) are the molecules that functionalize NTS-polyplex NPs. NTS and FP are covalently linked to poly-l-lysine to form a conjugate known as NTS-carrier. Every molecule that decorates NTS-polyplex NPs stimulates a specific cell function and they act sequentially, to transfer the pDNA, containing the gene of interest (transgene), to the target cells. Thus, NTS in NTS-polyplex NPs enables selective transgene transfer via internalization of NTSR1 that is expressed in the plasma membrane of target cells -G-C(Acm)-G-C(Acm)-CONH2(N2S2-RKKRRQRRR-c(RGDyK))), was obtained from Dr. Ferro-Flores  to form radioactive NTS-polyplex NPs in the PF25 at the optimum molar ratio that were analyzed with a Waters HPLC System . It’s worth mentioning that this 99mTc-KP  was proven to have comparable properties  to those of KP used in the rest of experiments, as shown by electrophoretic mobility and transfection assays (data no shown). The stationary phase was a Protein-Pak 300 SW column and the mobile phase was water at a flow of 1 mL/min. To monitor the stability of the complex and the assembly of radiolabeled KP, pDNA and NTS-carrier, a Waters Photodiode UV-Vis detector was used in series to a gamma radioactivity HPLC detector .Considering the parameters listed in myo-inositol, pantothenic acid, pyridoxine and thiamine) [2, MgCl2 and NaH2PO4, at isotonic concentration and pH 7.4. We chose glucose because the mammalian brain depends on this monosaccharide as main source of energy and its metabolism is fine regulated in the brain. These characteristics make glucose ideally suited for intracerebral administrations of NPs in comparison to reducing disaccharides as sucrose.NTS-polyplex NPs were used freshly prepared in EF25 (DMEM with 25 mM glucose). EF25 is composed by 7 inorganic salts, 14 amino acids, 8 vitamins and 1 sugar. For human use, the Food and Drug Administration (FDA) has not approved as inactive ingredients 1 of these inorganic salt (ferric chloride), 1 amino acid (cysteine) and 6 vitamins  . So, ourAccording to previous reports on nonviral systems ,24,30,31Student’s t test . These results demonstrate that in PF25, NTS-polyplex NPs exert a comparable and even higher transfection efficiency in vitro than in EF25. The increased transfection efficiency of NTS-polyplex NPs in PF25 can be caused by the low zeta potential (+14.1 mV) of this formulation as compared with that of EF25 (+25.3 mV). A low zeta potential reflects a higher electrostatic interaction between NTS-polyplex components . Thus, a higher interaction of these components might lead to a more efficient assembling of NTS-polyplex NPs and to a major availability for cell uptake. Thus, the saline composition and the glucose-DNA ratio of 241.8 in PF25 could confer adequate solubilization of NPs.To determine and compare the biological functionality in vitro of NTS-polyplex NPs assembled in EF and PFs, we carried out transfection assays with the plasmid pEFGP-N1 in N1E-115 cells and determined GFP expression by using epifluorescence microscopy and flow cytometry. In the case of EF280 and PF280, we observed cell death because of hypertonicity resulting from the high glucose concentration , which wsubstantia nigra of Wistar rats and compared the results with those from transfections using EF25. The confocal microscopy analysis at day 3 after transfection showed a large amount of substantia nigra cells that express GFP, in a similar fashion as with the transfection using EF25. In contrast, no GFP-expressing cells were observed in the substantia nigra injected only with the respective formulation   because n = 10). Then, we evaluated the lyoprotective ability of PF25 by determining biological functionality and physical properties of reconstituted NTS-polyplex NP lyophilisates. Lyophilisates were reconstituted with water for injection after 2 days of storage at 4 °C and 60% of relative humidity.A cake of fine white powder was obtained in lyophilisates. Humidity determined by Karl Fisher method was <1%; reconstitution mean time was 9 s; and pH of reconstituted lyophilisates was 7.5  when compared with the transfection efficiency of freshly prepared NTS-polyplex NPs. These results demonstrate that NTS-polyplex NPs in PF25 conserve their transfection ability after reconstitution of lyophilisates, even with higher transfection efficiency than freshly prepared NTS-polyplex NPs.To evaluate the effectiveness of PF25 to maintain the functionality in vitro of reconstituted lyophilisates of NTS-polyplex NPs, transfection assays in N1E-115 cells were made. The results were compared with EF25 freshly prepared. Epifluorescence microscopy analysis showed a significant population of N1E-115 cells that express GFP when transfected with NTS-polyplex NPs from reconstituted lyophilisates or freshly prepared in EF25 A. The trsubstantia nigra cells . All these data show that 25 mM glucose in EF25 and PF25 is an efficient stabilizer and lyoprotectant for NTS-polyplex NPs.In agreement with the results in vitro, confocal microscopy analysis showed that a single injection of reconstituted NTS-polyplex NPs caused GFP expression in a great population of rat ra cells B when cora cells B. No GFPTo evaluate the structural integrity of NTS-polyplex NPs after lyophilization, TEM was used. Individual fresh samples of NTS-polyplex NPs prepared either in EF25 or in PF25 presented a typical toroid shape that results from the compaction degree of all components of NTS-polyplex NPs as reported previously for other polyplexes ,35,36,37These results suggest that PF25 and the lyophilization process contributed to the protection, stability and dispersion of NTS-polyplex NPs.n = 6). NTS-polyplex NPs in PF25 had a mean diameter of 141.7 nm and a mean zeta potential of 14.1 mV (n = 6). There was no significant difference in the size of NPs in both formulations . However, there was significant difference in zeta potential of NTS-polyplex NPs in PF25 when compared with that in EF25  (n = 6) was 138.5 nm and 0.2 mV, respectively. There was significant difference in zeta potential of freshly prepared and reconstituted NPs (both in PF25) . In contrast, there was no significant difference in particle size  when compared with freshly prepared NTS-polyplex NPs ; whereas mean PdI for reconstituted NTS-polyplex NPs (PF25) was 0.47. There was significant difference with PdI of freshly prepared NPs in PF25 , which suggests a more homogeneous population and thus a higher stability of reconstituted NTS-polyplex NPs; this correlates with TEM results. On the other hand, a peak of 10 nm was observed in freshly prepared NPs, either in EF25 or PF25, which could be attributed to free NTS-polyplex components, as it was absent in the reconstituted samples (PF25). The intensity of reconstituted NPs distribution curve also increased five-fold  was used. In addition, the zeta potential of the same samples was determined by measuring electrophoretic mobility using a Laser Doppler Velocimetry. The mean diameter of NTS-polyplex NPs freshly prepared in EF25 was 142.8 nm and the mean zeta potential was 25.3 mV  . This coplex NPs . Howeverive-fold . The neaSecondary structure of freshly prepared and reconstituted NTS-polyplex NPs was determined using CD. This procedure could be useful for quality control due to its simplicity, quickness and low cost. PF25 facilitated the characterization of band patterns of NTS-polyplex NPs because this vehicle does not contain the components of EF25 that interfere with this spectroscopic analysis.In the first place, we confirmed that the band pattern in pDNA spectrum was similar to that previously reported ,39. The The displacement of bands in the spectra of both pDNA-KP and NTS-polyplex NPs reveals the electrostatic interactions; firstly, between pDNA and KP and then, between pDNA-KP complex and NTS-carrier A. IntereSerum proteins are the first biomolecules that interact with NTS-polyplex NPs when used in gene therapy protocols and therefore can affect the physical features in detriment of their transfection efficiency. Therefore, we used TEM to monitor the stability of reconstituted NTS-polyplex NPs in PF25 at different times of interaction  with FBS .These micrographs show that reconstituted NTS-polyplex NPs conserve their physical integrity and no large aggregates were formed even after 60 min of interaction with FBS.99mTc-KP, pEGFP-N1-99mTc-KP complex and 99mTc-NTS-polyplex in PF25 ) ) . NTS-polThus, these results demonstrate that PF25 is a suitable vehicle of NTS-polyplex NPs for gene therapy applications in the clinic.Despite the advances in academic research, the clinical translation of nanovectors remains challenging, particularly from the manufacturing and regulatory perspectives. The transition of nanovectors from encouraging preclinical research to clinical trials and finally regulatory approval has not yet occurred.The dimensions and structural complexity of NTS-polyplex NPs require specific characterization and analytic techniques, as well as a robust manufacturing process to ensure the production of high quality, safe and effective products.The preclinical success in the treatment of Parkinson’s disease and cancer ,9,10 andBesides lyophilization, it is important to consider the effect of ingredients of a formulation on the nanovectors properties. For example, low salt concentration in PF25 contributes to decrease aggregation of NTS-polyplex NPs. It has been reported that increased salt concentration reduces the hydrate layer around the particles and promotes their aggregation ; and a hThe use of lyoprotectants like saccharides in the formulation is essential to conserve NPs structure and functionality ,23,43. TT’g [T’g) for 3 h prior to the sublimation phase of lyophilization. This also counterbalanced the absence of glassy matrix formed by high (hypertonic) concentration of saccharides, which also has the role of immobilizing NPs during the freezing step of lyophilization.It has also been reported that polyplexes with neutral surface charge are more instable, aggregate more easily in solution and form precipitates ,46,47. NT’g . This waThus, the designed lyophilization protocol, in concert with glucose included in PF25, was able to preserve the physical characteristics of NTS-polyplex NPs, as demonstrated by secondary structure and particle size analyses. The freshly prepared NTS-polyplex NPs (both EF25 and PF25-formulated) showed a PdI quite large , which resulted from a bimodal particle size distribution . One peaLyophilization also contributed to the increase in transfection efficiency of NTS-polyplex NPs in PF25 as compared with that in freshly prepared NPs. Stability, dispersion and neutralization of those NPs are features of reconstituted lyophilisates as shown by TEM micrographs and zeta potential results. These features can be attributed, in the first place, to the immediate immobilization of NTS-polyplex NPs by the freezing after assembling. In addition, the replacement of water molecules by glucose functional groups in lyophilization might conserve the structure and prevent agglomeration of NTS-polyplex NPs. Finally, the efficient electrostatic neutralization of NTS-polyplex components might also avoid their agglomeration after reconstitution.99mTc-NTS-polyplex NPs with human serum, as shown by SEC-HPLC analysis. However, after 30 min of interaction, two small fractions of higher molecular weight were separated. Three hypotheses can explain the presence of these two populations. The first hypothesis is that the ionic strength of human serum promotes the self-aggregation of NTS-polyplex NPs. The second hypothesis is that these NPs interact with human serum proteins to form high molecular weight aggregates. And the third hypothesis is that NTS-polyplex NPs undergo conformational change or degradation after interaction with human serum proteins or due to its ionic strength. Spectroscopic and chromatographic analysis beyond the scope of this work can provide further insight into the nature of these populations in order to propose a model for the dynamics of NTS-polyplex NPs after systemic administration.Once administered intravenously, polyplex aggregation and disassembly might be caused by unwanted interactions with blood serum, biological tissue and immune cells. Increase in size of polyplex systems due to aggregation in systemic circulation could lead to capillary bed capture in the location or first encounter and restrict polyplex migration across size-dependent endothelial gaps . Surface99mTc-NTS-polyplex was demonstrated by SEC-HPLC at 30 and 60 min after administration, as shown by the peak corresponding to the retention time of 10 min. Considering that only the disassembly of NPs at the kidney glomerular basement membrane would allow transit of the NPs components into the urine, a hypothesis that could explain our findings is that NPs reform in the urine after disassembly and filtration, as demonstrated by Zuckerman et al. [Urine clearance of components of n et al. , suggestgT, making the storage temperature a determinant factor for the chemical and physical degradation rates. Physical degradation rates of pharmaceuticals, such as crystallization of amorphous compounds, are also related to molecular mobility. In addition, aggregation, one of the most common degradation cause of lyophilized biomolecule formulations, involves collisions between molecules and is closely related to molecular mobility [gT may have led to chemical and physical degradation. The decrease in transfection efficiency after 6 months of storage at 25 °C and 60% RH can be attributed to a probable molecular mobility after a long storage time, even though storage temperature and RH were adequate. An accelerated stability study suggests that in tropical weathers, it would be recommended to storage lyophilisates under refrigeration. All these findings suggest that PF25 is an appropriate vehicle for the clinical use of NTS-polyplex NPs that also confers a considerable shelf life at room temperature after lyophilization.Finally, accelerated stability tests of NTS-polyplex lyophilisates in PF25 demonstrated that these preparations remain stable and functional, with transfection efficiency comparable to freshly prepared NTS-polyplex NPs, for at least 6 months at 25 °C and 60% relative humidity. Degradation of NTS-polyplex NPs after 3 months at 40 °C and 75% RH can be attributed to molecular mobility in these conditions. Previous reports have shown that the storage stability of amorphous pharmaceuticals in the solid state is largely affected by changes in molecular mobility at mobility . Althougw/w) as low as 250 was able to confer lyoprotection and stability to NTS-polyplex NPs, in concert with adequate conditions for freezing and/or lyophilization. Our experimental design could be applied to the clinical formulation of other polyplexes.This work describes analytical methods, instrumental techniques and a lyophilization process to develop a pharmaceutical formulation for NTS-polyplex NPs that showed to be safe and provide high transfection efficiency within a long shelf life. According to our findings, a glucose-pDNA ratio (


Nurse-coordinated care (NCC) improves the achievement of low-density lipoprotein-cholesterol (LDL-C) targets after an acute coronary syndrome (ACS). We hypothesised that NCC improves achievement of LDL-C targets through more intensive medication titration.We used data from Randomised Evaluation of Secondary Prevention by Outpatient Nurse Specialists (RESPONSE), a multicentre randomised trial on the efficacy of NCC in 754 ACS patients. Follow-up data were collected at 6 and 12 months. To enable comparison between the various types and dosages of statins, we used the average lipid-lowering potency  as an indicator of lipid-lowering medication intensity.Most patients in NCC intervention and usual care groups (96%) had started lipid-lowering therapy during the index hospitalisation. At 6 months, titration activities (up or down) were applied in 45% of NCC patients compared with 24% of patients receiving usual care (p<0.001), and a difference was also seen at 12 months follow-up . In patients not on LDL-C target at baseline, titration activities at 6 months were recorded in 63% and 30% of NCC and usual care patients respectively (p<0.001), with increased titration activities in both groups at 12 months .NCC is associated with more frequent and intense lipid-lowering medication titration to reach LDL-C targets as compared with usual care alone. Further, merely starting the guideline-recommended dose is insufficient to reach the guideline-recommended LDL-C target level.TC1290 (Netherlands). Nurse-coordinated care improves the achievement of low-density lipoprotein-cholesterol (LDL-C) targets after an acute coronary syndrome (ACS).Nurse-coordinated care is associated with more frequent and intense lipid-lowering medication titration to reach LDL-C targets as compared with usual care alone.Merely starting the guideline-recommended dose is insufficient to reach the guideline-recommended LDL-C target level. Nurse-coordinated care, combined with guideline-based titration recommendations, improves ACS patient outcomes.Among patients with coronary heart disease (CHD), treatment of risk factors is the cornerstone of secondary prevention.Nurse-coordinated care (NCC) has shown to be a promising strategy to improve secondary prevention, and is currently recommended in the 2016 European prevention guidelines.To address this gap in knowledge, we investigated the process of medication titration in the treatment of LDL-C in NCC. We used data from the Randomised Evaluation of Secondary Prevention by Outpatient Nurse Specialists  (RESPONSE) trial (see below). As the lifestyle risk factors were comparable in both groups in the study, the previously reported improvement of the proportion of patients on target for LDL-C in the NCC intervention group could not be explained by lifestyle changes. Additionally, participating nurses in this trial reported that the NCC intervention allowed them more frequent contact with patients and the opportunity to monitor targets more carefully.<2 years), and (4) were diagnosed with heart failure New York Heart Association class III or class IV.We used data from the RESPONSE trial, a multicentre randomised clinical trial including 754 patients from 11 centres in the Netherlands.Nurses participating in the NCC programme were registered nurses with at least a 4 years bachelor’s degree in nursing. They had experience in cardiovascular care and were trained in motivational interviewing. Patients in the NCC group visited the outpatient clinic up to four times during the first 6 months after inclusion, in addition to outpatient clinic visits to their cardiologist . During each nurse visit, cardiovascular risk factors were assessed, lipid profiles (including LDL-C) were reviewed, medication therapy evaluated and patient compliance with medical treatment and lifestyle recommendations was encouraged. To achieve the target lipid levels, the nurses were also encouraged to titrate medication in collaboration with the treating cardiologist.<2.5 mmol/L, as recommended by the national CVD prevention guideline at that time.Data on clinical and demographic characteristics and CHD risk factors were collected at baseline and at 6 and 12 months after randomisation. Baseline measurements were performed within 8 weeks after ACS. Patients were enrolled at an average of 4 weeks (SD 2.7) after the ACS. Data on medication use was collected at baseline, 6 months and 12 months follow-up. The data on lipid-lowering medication included number of lipid-lowering medications and, for each medication, the generic name, dosage and frequency. When LDL-C was not on target during the four NCC visits, nurses documented when medication was changed during the NCC visit, and if the treating specialists were consulted and/or patients were referred to treating specialists. All venous blood measurements were taken after a minimum of 8 hours of fasting. The target for LDL-C level was Our main outcome of interest was the proportion of patients with up-titration or down-titration activities in the NCC compared with usual care, assessed by changes in lipid-lowering medication intensity at 6 months and 12 months, relative to baseline medication intensity. The 6 months follow-up visit was performed directly after completion of the NCC intervention , while between 6 and 12 months follow-up, no specific interventions took place in either group. To account for the use of different lipid-lowering agents and dosages, the intensity of each prescription was expressed as a potential average lipid-lowering potency  ranging from 13 to 70.As the Dutch guideline for cardiovascular risk management recommends starting with simvastatin 40 mg daily when patients are diagnosed with ACS,2 test for categorical variables. Differences between characteristics of up-titrated and down-titrated patients were analysed by the χ2 test. The p values presented in Comparisons between groups were performed using χIn order to include the NCC intervention effect at 6 months, we plotted ALLP changes between baseline and 6 months. We assessed if patients in the NCC group who were (not) on target at baseline received greater intensity changes than those in the usual care group by estimating the interaction between treatment arm and (not) being on target at baseline in a linear regression analysis. These analyses were performed using Stata V.13.1 .To check for selective dropout, we used a logistic regression model and regressed a binary variable indicating missingness  on the following variables as predictors of missingness under the hypothesis that if all ORs were close to 1, selective dropout due to these predictors is unlikely: age, gender, education level, index event, history of CVD, alcohol, smoking at baseline, diabetes mellitus and their interaction with randomisation group.Our population consisted of 754 patients with a mean age of 58 years (SD 10.1), 80% were men. The majority (73%) had no history of CVD prior to the index hospitalisation. As previously described, baseline patient characteristics did not differ between the NCC and usual care groups.The proportion of patients with up-titration or down-titration of lipid-lowering medication from baseline to 6 and 12 months follow-up was higher in the NCC group as compared with the usual care group . ReflectIn patients not on LDL-C targets at baseline , most tiAt baseline, the proportion of patients on lipid-lowering medication was high in both the NCC (96%) and the usual care group (96%), and 68% of all patients were on LDL-C target at baseline . SimvastThere were no differences in demographic or clinical characteristics as age, gender, level of education, index event or cardiovascular risk factors of up-titrated and down-titrated patients (data not shown). However, up-titrated patients had dyslipidaemia more frequently as compared with patients with no titration , and up-titrations were associated with allocation to the NCC group .Down-titrated patients had dyslipidaemia less frequently as compared with patients with no titration . Down-titration was also more frequently seen in patients allocated to the NCC group as compared with patients with no titration .Our study demonstrates that NCC in patients with ACS is associated with more frequent lipid-lowering medication titration and with higher ALLP values to reach LDL-C targets as compared with usual care alone. These titrations took place in a relatively short amount of time (four visits in 6 months after an ACS), but changes made in the first 6 months in lipid-lowering medication were also observed 6 months after completion of the NCC programme, and were reflected in a higher proportion of patients reaching targets for LDL-C. Our study took place in a context of high prescription rates of lipid-lowering medication (96% in both groups at baseline). Despite these high prescription rates, the target for LDL-C (2.5 mmol/L) was not reached in a considerable number of patients in both groups . Our study shows that there is considerable room for individual tailoring of lipid-lowering medication therapy, with more both up-titration and down-titration in medication intensity in the NCC group. While lifestyle modification could account for some changes in LDL-C levels, it is unlikely that this can explain the differences in the higher proportion of patients on target in the NCC group, as lifestyle risk factors were comparable through the study up until 12 months follow-up.Large proportions of high-risk cardiovascular patients have been shown to discontinue their statin therapy, emphasising the need for healthcare providers to discuss medication use with their patients.According to the European Society of Cardiology (ESC) guideline, reducing dosage is an effective approach for enhancing medication adherence.Patients allocated to the NCC group reached the target level of LDL-C in a short period of time after discharge. This is likely to be beneficial, as several trials have demonstrated important reductions in major cardiovascular events from lowering cholesterol, especially LDL-C.Secondary prevention based on nurses’ collaboration has the potential to improve patient care. While healthcare organisations differ widely across Europe, the ESC prevention guidelines recommend a multidisciplinary team for secondary prevention including physicians and nurses. In some countries, secondary prevention is mainly the task of physicians, while in others, specially educated and trained nurses play a more prominent role.The ESC guideline target for LDL-C changed from 2.5 mmol/L to 1.8 mmol/L after the completion of inclusion of patients in the RESPONSE trial.Moreover, it should be noted that our data on medication use were based on self-report by professionals, and not corroborated with additional questionnaires regarding adherence or pill counts. While side effects were discussed with patients during NCC consultation, we did not specifically collect data on such side effects. This might be a valuable part of follow-up research. Furthermore, we did not correct for possible confounders such as lifestyle factors in our analysis. The development of a model with the hypothesised pathways between LDL-C on target and NCC interventions, including all potential confounders of this relation, could potentially help to more fully investigate the association between NCC titration and LDL-C on target. Such causal mediation analysis may be used to investigate the causal role of titration activities relative to other factors associated with NCC in future trials.In conclusion, among patients hospitalised for ACS, NCC resulted in more intensive medication titration compared with usual care alone. The greater proportion of patients on LDL-C target at 6 and 12 months follow-up is likely explained by the more intensive titration of lipid-lowering medication in NCC patients compared with usual care alone. Merely starting the guideline-recommended dose is insufficient to reach the guideline-recommended LDL-C target level. NCC, combined with guideline-based titration recommendations, can improve ACS patient outcomes and should become part of routine daily practice.


In this study, we investigated the variability of vascular hysteresis loop (VHL) shapes and the spatial heterogeneity of neovascularization and microvascular alterations using vascular architecture mapping (VAM) in patients with recurrent glioblastoma during bevacizumab mono-therapy. VAM data were acquired in 13 patients suffering from recurrent glioblastoma prior to and 3 months after bevacizumab treatment onset using a dual contrast agent injections approach as part of routine MRI. Two patients were additionally examined after the first cycle of bevacizumab to check for early treatment response. VHLs were evaluated as biomarker maps of neovascularization activity: microvessel type indicator (MTI) and curvature (Curv) of the VHL-long-axis. Early response to bevacizumab was dominated by reduction of smaller microvasculature (around 10 µm). In the 3-month follow-up, responding tumors additionally showed a reduction in larger microvasculature (>20 µm). VAM biomarker images revealed spatially heterogeneous microvascular alterations during bevacizumab treatment. Responding, non-responding, progressive, and remote-progressive tumor areas were observed. MTI may be useful to predict responding and non-responding tumor regions, and Curv to assess severity of vasogenic edema. Analysis of VHLs in combination with VAM biomarkers may lead to a new perspective on investigating the spatial heterogeneity of neovascularization and microvascular alterations in glioblastoma during antiangiogenic therapy. Thereby, GBs are among the most vascularized of all solid tumors showing a disorganized, abundant and aberrant vasculature with tortuous vessels of variable diameter, heterogeneous distribution2, and abnormal capillary bed topology3. GBs are therefore attractive targets for antiangiogenic therapies (AAT)4 and the VEGF-specific antibody bevacizumab was approved by the Food and Drug Administration (FDA) for this purpose in 20095. Administration of bevacizumab in humans has been shown that magnetic resonance imaging (MRI) based tumor volume criteria doesn’t make sense for response assessment in GBs6 even though its criteria have been updated by the Response Assessment in Neuro-Oncology (RANO) working group7. The major challenge is the decreased vessel permeability8, which results in diminished contrast agent extravasation9 but does not necessarily reflect biological tumor response10.Glioblastoma (GB) is the most aggressive primary brain tumor in adults with an elevated expression of vascular endothelial growth factor (VEGF) protein, which has been identified as a critical regulator of tumor angiogenesis, endothelial cell proliferation, and migration14. The physical basis for MRI-based vessel caliber estimation is the different sensitivity of gradient-echo (GE) and spin-echo (SE) MRI to magnetic susceptibility13. The highly susceptibility-sensitive GE signals are dominated by a broad range of larger microvessel diameters 11, with reduced sensitivity to the smaller microvascular range15. In contrast, despite its lower overall sensitivity to contrast agent-induced signal variations, SE signals exhibits a peak sensitivity to the microvasculature at a vessel diameter around 10 µm11, including capillaries and both small arterioles and venules. These differences in sensitivity to the vascular properties were shown in simulations studies14 and verified in practice16.Computer simulations as well as experiments in animals and humans have demonstrated that more advanced MRI techniques can estimate microvascular vessel caliber, thereby providing further insight into tissue microvascularityet al.13 were the first who reported about a “loop” in the parametric plot of the relaxation rates for GE  shapes in patients suffering from recurrent GBs during bevacizumab mono-therapy. VHL analysis was extended with MRI biomarkers adapted to the temporal shift phenomenon. This approach, termed vascular architecture mapping (VAM), was used for investigating the spatial heterogeneity of neovascularization activity and microvascular changes due to bevacizumab treatment.et al.19 and Xu et al.17. In areas with faster blood flow velocities, i.e. in voxels with arteriole dominated microvasculature . Curv was severely decreased, i.e. showed severely larger negative values, in peritumoral vasogenic edema when compared to ipsi- and contralateral NAB.Accordingly to our definition for the calculation of microvessel type indicator (MTI) values, a counterclockwise VHL-direction was associated with a negative signed VHL-area, i.e. a negative MTI, and vice-versa for the clockwise VHL direction. In MTI maps, negative values were assigned to cool colors and positive to warm colors, respectively. Consequently, maps of MTI enabled discrimination between supplying arterial  tumor areas with decreased neovascularization activity interpreted as response to AAT; (ii) tumor areas with unchanged and increased neovascularization activity interpreted as non-response to AAT; and (iii) areas with newly developed neovascularization activity interpreted as progression during AAT.U images clearly revealed a spatial segregation in responding and non-responding tumor areas. The posterior tumor parts showed an increase in MTI  and decrease in NU as indication for decreased neovascularization activity as response to anti-angiogenic treatment. The anterior part of the tumor, however, showed an unaltered high or even increasing neovascularization activity: highly negative or decreasing MTI values, and high or increasing NU values, respectively. The changes in the RU images were less distinct. Reduction in peritumoral edema was accompanied with a massive increase in the Curv parameter .Figure entclass1pt{minimaeas Fig. . Althougeas Fig. .Figure U maps, respectively. Additionally, the posterior part of the edema showed no regression but partly even a progression on the Curv map , which is accompanied with a further reduction in both A patient with response, progression, and remote progression in the contralateral hemisphere during AAT is depicted in Fig. U, and RU in these three different tumor areas for each patient individually are depicted in Fig. U, and RU were significant different between the 3-month follow-up compared to base-line: responding tumor areas showed an increase in MTI and a decrease in NU and RU; non-responding tumor areas and progression, however, showed a decrease in MTI and an increase in NU and RU. As realized from investigation of the individual cases, Curv was most sensitive to changes in peritumoral edema.All 13 patients included in this study showed areas with response to bevacizumab, nine patients additionally showed non-response, and eight patients additionally showed a progression of the GB during AAT. The changes for the MTI, NP = 0.042) higher MTI values at baseline in tumor areas which responded in the follow-up (MTI(baseline)response = −82.2 ± 60.4) compared to the tumor area which not responded in the follow-up (MTI(baseline)non-response = −147.8 ± 80.2). All the other VAM biomarkers showed no significant differences at baseline.Comparison of VAM biomarker values at baseline examination between tumor regions which showed response and non-response at the 3-month follow-up in order to check for potential predicting factors revealed significant (i) estimation of the microvascular compartments ; ii) estimation of the microvascular type (arterioles vs. capillaries vs. venules); and iii) estimation of the spatial heterogeneity of neovascularization activity and its changes during AAT by evaluating features i and ii via calculation of VAM biomarker images.Analysis of VHLs in combination with the VAM approach demonstrated three features for investigation of the microvasculature of GBs: et al.19 recently built on the hysteresis effect, which was originally described by Kiselev et al.13 more than a decade ago, and demonstrated that AAT of recurrent GBs using cediranib can improve microcirculation and reduce vessel calibers. This response was associated with prolonged patient survival. An analysis of the dynamic changes of the VHLs, however, was not performed in this study.Firstly, investigation of the two VHL-variables, especially of the maxima of 17 and was expanded by the temporal shift phenomenon between max[19. A detailed interpretation of the temporal shift, however, was not presented so far. Considering both the maxima and the direction of the VHL may be helpful to explain the temporal shift phenomenon. In a voxel dominated by larger (faster-flow) arterioles and capillaries the blood with the first-pass contrast agent bolus flows from arterioles with lager diameter  followed by capillaries with smaller diameter , i.e. the GE signal rises and peaks before the SE signal resulting in a clockwise rotating VHL and a separation of maxima. The same is true for a voxel dominated by (slower-flow) venules and capillaries: the contrast agent bolus flows from capillaries to venules and the SE signal rises and peaks before the GE signal. The temporal shift leads to a VHL with two or more reversal points between max[17. It is well-known that neovascularization in GBs is highly defective, resulting in a disorganized, abundant and aberrant vasculature with tortuous vessels2 and abnormal capillary bed topology3. This causes heterogeneity in blood flow and perfusion through the tumor vasculature20. Interestingly, VHLs with a temporal shift has already been shown in the previous studies from Kiselev et al.13  vs. slow-inflow (venules-dominated) assessed by rotational direction of the VHL (clockwise vs. counterclockwise). This was previously described with computer simulations under consideration of a tree model of microvasculaturesee Fig.  and Xu esee Fig. .22. The Curv parameter might be a marker for the severity of vasogenic edema and compression of small microvessels due to increased interstitial pressure in the edema. However, there is no verified histopathologic basis for an interrelationship so far. Correlations with DTI data, histologic parameters, and Monte Carlo simulations for VHL formation could be informative.Finally, the VAM approach allowing for investigation of the spatial heterogeneity of neovascularization activity and microvascular changes due to bevacizumab treatment by evaluating the first two features via calculation of biomarker images. Maps of the VAM biomarker which were adapted to the temporal shift phenomenon provided a complementary insight into the heterogeneous structure of microvasculature pathologies in glioma as consequence to angiogenetic activities initiated by the lesions. Furthermore, we detected changes in VAM biomarker images which can be interpreted as microvascular response, non-response, and progression during AAT. Bevacizumab-untreated tumor regions (at baseline) with lower neovascularization activity (less negative MTI) tended rather to show response to AAT in the follow-up than regions with higher neovascularization activity. This might be an indication for the known vascular niche which is thought of as protective microenvironment that enables glioblastoma stem cells to freely proliferate and remain undifferentiated, completely unaffected by any external influences25, however, does not meet these requirements and may necessitate performing the routine GE perfusion in addition or in a separate study, which consequently requires a further contrast agent injection. This might be a reason for the limited application in clinical routine of this promising technique. Additionally, most previous studies published used a double-dose for the combined GE-SE perfusion25, and/or applied a second injection for perfusion MRI25 or correction of leakage effects24. The efforts to upgrade the combined GE-SE perfusion sequence with the promising Simultaneous-MultiSlice (SMS) technique26 for full coverage may help to overcome these limitations. The interpolation to the target resolution, which potentially influences the magnitude of the measured susceptibility effect, and the short TE, which reduces contrast agent sensitivity and hence SNR, of the SE-EPI perfusion sequence are a further limitations. Applying a larger acquisition matrix would increase TR and consequently the temporal resolution. This is also true for a longer TE. The accuracy of the hysteresis quantification, however, is critically dependent on a high temporal resolution.This study has several limitations. First of all, the dual contrast agent injections approach in combination with two separate SE- and GE-DSC acquisitions is significantly more sensitive to both patient motions and variations in injection timing compared to a combined GE-SE sequence. Our strategies help to minimize the probability of these sources of error but cannot eliminate them entirely. Moreover, the injection of a double dose of contrast agent with two separate injections is required. On the other hand, our approach with separate SE- and GE-DSC sequences allowing the acquisition of VAM data with high SNR, high spatial resolution, and coverage of the whole brain, which is mandatory for routine GE-EPI perfusion. The combined GE-SE sequence used in previous studiesIn summary, analysis of VHLs in combination with VAM biomarkers is capable of both, assessing the topological and structural heterogeneity of tumor microcirculation, and monitoring the response to and the progression during bevacizumab treatment. This approach may lead to a new perspective on investigating microvascular changes in glioma and other brain pathologies as well as on therapy monitoring. However, validations of the biomarkers are required to enable rational interpretation.This study was approved by the Institutional Review Boards of the University of Erlangen and the University Clinic of St. Pölten. Analysis of patient data and all methods were performed in accordance with both the Declaration of Helsinki and the relevant guidelines and regulations. Written informed consent for both study participation and publication of information/images was obtained from all subjects. Between July 2015 and December 2016, fifteen patients  suffering from recurrent glioblastoma who received bevacizumab  as second-line mono-therapy were included in this study. From the 15 patients, two patients (13%) with severe neurological deficits were excluded due to patient motion between or during the MR scans.All MRI studies were performed on a 3 Tesla clinical MR scanner  equipped with a standard 12-channel head coil. MR examinations were performed 1–5 days prior to (baseline) and 3 months after bevacizumab treatment onset (follow-up). In two patients a MRI examination was additionally performed after the first cycle of bevacizumab (two weeks after onset) in order to check for early response to treatment.27 factor of 2, b-values of 0 and 1000 s/mm2; TA: 1.3 min), and pre-and post-contrast enhanced (CE) T1-weighted GE sequences .The clinical routine MRI protocol included an axial fluid-attenuated inversion-recovery (FLAIR) sequence , a single-shot diffusion-weighted echo-planar imaging (DW-EPI) sequence 18. To minimize the probability of these sources of error we used the following strategies as described previously28:For VAM we used a dual contrast agent injections approach in order to obtain DSC perfusion MRI data using SE- and GE-EPI sequences with high signal-to-noise ratio (SNR), high spatial resolution, and coverage of the whole brain. However, patient motions between the two scans as well as differences in the time to first-pass peak may significantly affect the data evaluationi) To prevent patient motion, special caution was made to properly fix the head and provide clear and repeated patient instructions before and during the MRI examination. Due to the fact that patient motions during or between the SE- and GE-EPI DSC perfusion scans may significantly affect a pixel-based analysis, we used the approach described by Hsu et al.18 to detect patient motion: During data preprocessing, dynamic masks for the contours from the SE- and GE-EPI perfusion raw data were created. By matching the contours, we checked for patient motion between and during the scans18. A mismatch of more than one pixel was defined as ‘severe patient motion’, which was an exclusion criterion of this study from further evaluation. This was necessary in two patients as mentioned above. For the remaining 13 patients we did not observe significant motions, i.e. contour mismatch was not more than one pixel, and thus no image coregistration was applied during data analysis.ii) To prevent time differences of the first-pass peak signal between the contrast agent boluses a peripheral pulse unit (PPU) was fitted to a finger of the patients in order to monitor heart rate and cardiac cycle. Special attention was paid to perform the two injections at the same heart rate and exactly at the same phase of the cardiac cycle . Due to the fact that timing differences between the SE- and GE-EPI DSC perfusion scans may significantly affect a pixel-based analysis, we additionally checked for any error in injection timing. During data preprocessing the arterial input functions (AIFs) for the SE- and GE-EPI DSC perfusion data were verified for correct timing, i.e. were checked for differences in the time to peak (TTP) between the two AIFs. For all 28 examinations included in this study we did not observe differences between the SE- and GE-AIFs of the respective examination.30. CE T1-weighted images in axial, sagittal, and coronal orientation were performed between two DSC perfusion acquisitions. The total acquisition time of the three CE T1w sequences was 8 minutes. Thereafter the second DSC MRI was obtained using the GE-EPI technique during a bolus injection under the same conditions as for the SE-EPI DSC acquisition.Geometric parameters  and measurement parameters were chosen identical for the SE- and GE-EPI DSC sequences: TR, 1740 ms; in-plane resolution, 1.8 × 1.8 mm; SE-EPI were spatially interpolated to a 128 × 128 matrix; slice thickness, 4 mm; 29 slices; GRAPPA, 2; 60 dynamic measurements; and TA: 2.0 min, respectively. TE was different between SE- (33 ms) and GE-EPI (22 ms). Both DSC perfusion MRI examinations were performed with administration of 0.1 mmol/kg-bodyweight gadoterate meglumine  at a rate of 4 ml/s using a MR-compatible injector  and with utmost caution for the injection time (see above). A 20 ml bolus of saline was injected subsequently at the same rate. The first DSC MRI was obtained by the SE-EPI technique since SE-EPI DSC perfusion MRI is less sensitive to contrast-agent leakage18. Next, AIFs for the SE- and GE-EPI DSC data were automatically identified in each slice using automatic segmentation of brain tissue sub-classes in DSC concentration-time curves. In short, concentration-time curves were assumed to belong to one of five tissue sub-classes , and multiple features  were used to assign each concentration–time curves into the most probable tissue class. The average of five concentration-time curves with the lowest first moment and the highest average peak height was then selected as the final AIF31. ΔR(t)2,SE and ΔR(t)2,GE were calculated from the signals of the SE- and GE-EPI DSC data using the following equation:0 is the baseline (prebolus) signal, and S(t) is the signal during the first bolus passage of the corresponding sequence. S0 was determined as the mean of the signals from the 4th to the 15th dynamic volume18. For S(t), the respective DSC perfusion MR data were corrected for remaining contrast agent extravasation33 and a minimum search between the first and the second bolus passage (caused by recirculation) was performed as described previously17 in order to separate the boluses. The truncated ΔR(t)2,SE and ΔR(t)2,GE curves of the first bolus were fitted to a previously described gamma-variate function34 and used for calculation of the VAM analysis was performed using custom-made in-house software developed using MATLAB . SE- and GE-EPI DSC data were imported into the software and checked for motion artifacts during or between the acquisitionsram Fig. , which wi) microvessel type indicator (MTI) as the signed area of the VHL; ii) microvessel density (NU) and iii) radius (RU) which were adapted of the temporal shift phenomenon; and iv) the curvature (Curv) of the long-axis of the VHL  tumor regions responding to bevacizumab with decreased neovascularization; ii) non-responding tumor regions with unchanged or increased neovascularization; iii) areas with newly developed neovascularization activity interpreted as progression during AAT; edema in the vicinity of iv) responding; v) non-responding; and vii) progressive tumor vii) as well as contralateral normal appearing brain tissue (cNAB) of predominantly white matter. Differences in VAM biomarkers between baseline and 3-month follow-up examination were determined using a Wilcoxon signed-rank test. In order to check for potential predicting factors, we compared VAM biomarker values at baseline examination between tumor regions which showed response and non-response at the 3-month follow-up, respectively, using a Mann-Whitney U test. P values of less than 0.05 were considered to indicate significance.Software  was used for statistical evaluation. The mean values for the four VAM biomarkers within seven regions of interest (ROI) were calculated. These ROIs were manually defined by an experienced neuroradiologist (A.D.) based on features seen in FLAIR images, CE T1w MRIs, and VAM images, respectively. ROIs were located in: