Tablets prepared from NFX-BNZ combined SNEDDS loaded on Syloid 3050 silicas demonstration near complete dissolution in the flow through cell apparatus compared to NFX and BNZ commercial tablets respectively (Lampit® and Rochagan®). NFX-BNZ-SNEDDS demonstrated nanomolar efficacy in epimastigotes and amastigotes of T. cruzi with acceptable selectivity indexes and demonstrated enhanced survival and reduced parasitaemia in acute murine experimental models of CD. Thus, the results presented here illustrate the ability for an easily scalable and personalised combination oral therapy prepared from GRAS excipients, enabling treatment access worldwide for the treatment of CD. Chagas disease (CD) is a parasitic zoonosis endemic in Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective when received at the early stages of the disease and it involved two drugs (nifurtimox (NFX) and benznidazole (BNZ)). Both treatments require multiple daily administrations of high doses, suffer from variable efficacy and insufficient efficacy in chronic CD, many side effects, and a very long duration of treatment that results in poor compliance, while combined available therapies that lead to reduced duration of treatment are not available and polypharmacy reduces compliance and increases the cost further. Here we present self-nanoemulsified drug delivery systems (SNEDDS) able to produce easily scalable combined formulations of NFX and BNZ that can allow for tailoring of the dose and can be easily converted to oral solid dosage form by impregnation on mesoporous silica particles. SNEDDS demonstrated an enhanced solubilisation capacity for both drugs as demonstrated by flow-through studies and in vitro lipolysis studies. High loading of SNEDDS to Syloid 244 and 3050 silicas (2:1  Trypanosoma cruzi (T. cruzi) transmitted via the triatomine bite wound or mucous membranes  as previously described . La. Law/w).trations , while ctrations  possiblytrations , NFX doereported ,60), andIn vitro lipolysis assays are useful to understand intestinal digestion of lipid-based formulations such as SNEDDS, but they do not account for pre-intestinal digestion (gastric lipolysis) which accounts for 10–25% of total lipolysis. Two phases were achieved during our lipolysis experiments . Given the use of a high HLB co-surfactant (Labrafil M 1944 CS), a quick formation of an o/w emulsion in the aqueous phase is expected. The lack of an oil layer can be explained by the fact that all excipients are digestible by lipases  and the 2/g respectively) and a high pore volume (1.6 and 1.7 mL/g respectively) with Syloid 244 having a low particle size (2.5–3.7 µm) vs. Syloid XDP 3050 (59 µm) [w/w) which allowed clinically translatable amounts of both NFX and BNZ to be loaded. However, Syloid 3050 resulted in powders with better flow  . Syloid w/w). This can be explained by stronger interactions of drug impregnated SNEDDS to Syloid 244 FP when each drug-loaded SNEDD is loaded individually that are abolished when drugs are present together potentially due to an intramolecular interaction (hydrogen bond) between BNZ and NFX allowing for higher affinity for the surface as also supported by TLC experiments . Both NFX and BNZ liquid SNEDDS illustrated high epimastigote killing with low IC50 values <2 μg mL−1. SNEDDS enhanced the solubilisation capacity of NFX and BNZ in media and release was quick explaining their high in vitro efficacy. On the other hand, solid SNEDDS prepared with Syloid 3050 illustrated a lower IC50 than Syloid 244 in both epimastigotes and amastigotes, which further indicates their potential to translate into optimal formulations for the treatment of CD. The combined NFX-BNZ-SNEDDS formulation enabled dose reduction for combined SNEDDS required to ensure 30 days post infection survival in treated animals (NFX and BNZ SNEDDS showed enhanced in vitro efficacy in comparison to drugs alone and reduced IC animals B and red animals C. Toxici animals B, howeveT. cruzi compared to fibroblasts and show a favourable target product profile for clinical translation while they were able to increase survival and reduce parasitaemia in a murine model of acute infection.Prepared SNEDDS enhance the solubilisation capacity of NFX and BNZ and provide an easy method to combine them into oral liquid and solid clinically translatable formulations with a predicted shelf-life of 1 and 2 years respectively under refrigeration. NFX-BNZ-SNEDDS with low levels of NFX and BNZ show excellent selectivity for


In systemic lupus erythematosus (SLE), a prototypic systemic autoimmune disease with complex etiology, a disbalance between Treg and pathogenic effector/memory CD4+ T cells develops during disease progression indicating that gradual loss of control over T cell activation is an important event in the immune pathogenesis. This progressive failure to adequately regulate the activation of autoreactive T cells facilitates chronic activation and effector/memory differentiation of pathogenic T cells, which are considered to contribute significantly to the induction and perpetuation of autoimmune processes and tissue inflammation in SLE. However, in particular in humans, little is known about the factors which drive the escape from immune regulation and the chronicity of pathogenic T cell responses in an early stage of autoimmune disease when clinical symptoms are still unapparent. Here we briefly summarize important findings and discuss current views and models on the mechanisms related to the dysregulation of T cell responses which promotes chronicity and pathogenic memory differentiation with a focus on the early stage of disease in lupus-prone individuals.In the normal immune system, T cell activation is tightly regulated and controlled at several levels to ensure that activation occurs in the right context to prevent the development of pathologic conditions such as autoimmunity or other harmful immune responses. CD4 Predominantly derived from a predetermined T cell subpopulation in the thymus, CD4+FoxP3+ Treg mainly recognize auto-antigens via their T cell receptor and are required to regulate the activation and expansion of auto-reactive T cells and other harmful immune cells in the peripheral lymphatic organs  is a severe multisystem autoimmune disease with complex pathogenesis which is characterized by immune dysregulation and chronic inflammation of various organs caused by the breach of immune tolerance predominantly towards nuclear autoantigens, such as double-stranded deoxyribonucleic acid (dsDNA). SLE primarily affects young women of childbearing age and clinical manifestations can range from relatively mild skin and joint involvement to life-threatening disease including renal, neurologic or cardiac inflammation , 2. Tisseactions , 6. Furte states . Despitee states . Regulatc organs –13. Givec organs , 15. Indactivity –18. Thesactivity –24.Given the pivotal role of T cells and their dysregulation in SLE pathogenesis, it can be hypothesized that chronic T cell activation and memory differentiation in early disease are key events for the initiation and progression of autoimmunity in SLE.+ T cells is associated with SLE disease severity (PTPN22 (R620W), encoding for a tyrosine phosphatase that regulates T cell signaling, is associated with aberrant receptor signaling function on effector and memory T cells as well as B cells  and for the low numbers of CD4+FoxP3+ Treg   , 53, whi (CaMK4) , 55. CRE (CaMK4) , 57, whi+FoxP3+ Treg population and the suppression of Th17 cells, and was capable to decrease disease activity in patients with active SLE , a serine-threonine kinase localized in the outer mitochondrial membrane, has been identified as a central regulator of T cell lineage specification, serving as a physiological sensor of mitochondrial dysfunction and ATP depletion in T cells . mTOR trtive SLE –65.Similarly, also the serine-threonine kinases Rho-associated protein kinases (ROCK) 1 and 2 play an important role in SLE pathogenesis. Generally, ROCKs regulate migration, activation, and differentiation of T cells and are crucial for controlling cytoskeletal components including the ezrin/radixin/moesin (ERM) proteins , 66. ERM+ T cells from SLE patients and positively correlated with disease activity (via the IRAK1 pathway (Interferons (IFNs), in particular type I IFNs, play a central role as initiators of the pathogenic immune response in SLE. Nucleic acids released from apoptotic cells and immune complexes trigger the production type-1 IFNs by tissue resident plasmacytoid dendritic cells. Type-1 IFNs exert stimulatory effects on a variety of immune cells including T cells through activation of the STAT1 pathway –73. Consactivity , 75. In  pathway , indicatWhether these abnormal signaling events are acquired during disease progression or genetically determined, however, remains to be determined.+, proliferating CD4+FoxP3- conventional T cells (Tcon) is strongly increased in patients with active SLE and correlates with disease activity , which are considered to represent chronically activated memory/effector CD4+ T cells, where shown to be expanded and to produce IFN-γ in patients with moderately active SLE (+ effector memory T-cell frequencies indicated more persistently active disease over time. Active SLE is further characterized by a decline in CD4+FoxP3+CD127lo Treg that express high levels of CD25 (CD25hi Treg), a subset of Treg with a high suppressive capacity, and by an imbalanced proliferation between Treg and Tcon in favor of an enhanced Tcon proliferation  and CD4+FoxP3+ Treg during disease progression in NZBW mice including young clinically healthy mice as well mice at the disease onset and with established disease (+ Tcon from lymphoid organs of young, clinically healthy mice at an age between 8-12 weeks already showed signs of increased activation and memory formation evidenced by higher frequencies of CD69+ and CD44+ among CD4+ Tcon compared to healthy BALB/c mice. Frequencies and numbers of activated and memory CD4+ Tcon and IFN-γ producing Th1 cells further increased substantially during progression to disease onset and active disease. A lower prevalence of CD4+FoxP3+ Treg, which had an intact suppressive function, was already detectable in young pre-diseased NZBW mice compared to BALB/c mice. In parallel, these mice also had higher frequencies of CD69+ and CD44+ Treg suggesting that Treg activation with the attempt to counteract the increased Tcon activation occurs already early in disease development. Phenotypically, Treg from young mice still expressed normal levels of CD25 and IL-2 production by CD4+ T cells was also not impaired in young mice indicating that, in contrast to the later disease stages, lack of IL-2 may not be responsible for the low prevalence of Treg which instead is rather genetically determined (+ T cells was also found to be significantly impaired (The NZBW mouse model is considered to authentically resemble most features of human SLE , 36. Theondition . We have disease . CD4+ Tctermined . This istermined . Alternatermined . A decreimpaired , indicatimpaired . Similarimpaired .+ T cell life span from activation and proliferation to differentiation is strictly regulated by cellular metabolism (B6.Sle123 and in SLE patients demonstrated that both aerobic glycolysis and mitochondrial oxidative phosphorylation are elevated in CD4+ T cells (As described previously in humans, murine SLE T cells also exhibit rewiring of their TCR, in which expression of the CD3ζ chain is reduced . This retabolism , 89. A r T cells . The pat T cells , 91. The T cells , 93.Taking current knowledge into consideration, we propose a simplified model that may explain the gradual and progressive failure to adequately regulate the activation of autoreactive T cells in the immune pathogenesis of SLE which facilitates chronic activation of pathogenic T cells and promotes the generation of a robust autoimmune memory Figure 1While the role of T cells and their dysregulation in established SLE is currently relatively well understood, little is still known about the mechanisms and molecular pathways which drive the escape from immune regulation and the chronicity of pathogenic T cell responses in the very early stage of disease. Continuing research efforts in this field provide the unique opportunity to identify novel therapeutic targets that might be capable to prevent the occurrence of clinical manifestations or to induce long-lasting remission.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.German Research Foundation, GRK2633.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: This study aimed to investigate whether home exercise programs informed by wearable activity monitors improved walking ability of patients with peripheral artery disease (PAD). Methods: A systematic literature search was performed to identify randomised controlled trials (RCT) testing home exercise that were or were not informed by wearable activity monitors. The primary outcome was the change in walking distance measured by a six-minute walking test or treadmill test over the course of the trial. Network meta-analysis (NMA) was performed using the gemtc R statistical package. The risk of bias was assessed using Cochrane tool for assessing risk of bias in RCTs (RoB 2.0). Results: A total of 14 RCTs involving 1544 participants were included. Nine trials used wearable activity monitors to inform the home exercise program tested, while five trials did not use wearable activity monitors to inform the home exercise program tested. Overall quality assessment showed 12 trials to be at low risk of bias and two trials at high risk of bias. Home exercise programs informed by wearable activity monitors significantly improved walking distance compared to non-exercise controls  but not compared to home exercise programs not informed by wearable activity monitors . Conclusions: Home exercise informed by wearable activity monitors improve walking ability of patients with PAD. It is, however, unclear if activity monitoring informed exercise programs are more effective than exercise programs not using activity monitors. Exercise therapy is an evidence-based treatment for peripheral artery disease (PAD) recommended in all current practice guidelines which can be delivered by central facility supervised or home programs ,2,3. MosThere are a range of established ways for self and remote monitoring of the intensity and volume of exercise using devices worn by patients which could potentially replace direct supervision and improve efficacy of home exercise programs. This includes accelerometers or pedometers worn to monitor exercise volume and intensity, which have been used to provide data to inform motivational counselling to promote exercise ,9,10. RaThis review was performed according to the Preferred Reporting the Items for Systematic Review and Meta-Analysis (PRISMA) statement . The stuchange = √[sd2baseline+ sd2Final − ]. Pearson correlation coefficient was (Corr) calculated using individual participant data from one trial . Pooled SD was calculated using the formula [Average of SD = √((N1 − 1) × SD1^2 + … (Nn − 1) × SDn^2)/((N1 + … Nn) − n), where n is the total number of studies [Data were extracted on a customised spreadsheet by three authors . The primary outcome of this study was improvement in walking performance defined as mean change in 6MWD or TMWD at the completion of follow-up. The change in walking distance for participants was calculated using outcomes reported at baseline and completion of follow-up and reported separately in the following groups: Home exercise programs informed by wearable activity monitors, home-exercise programs not informed by wearable activity monitors and non-exercise controls. Home exercise programs informed by wearable activity monitors included any home exercise program which used any kind of sensors or wearables to monitor the physical activity and/or adherence, and use the information to support the home exercise program. Home exercise programs not informed by wearable activity monitors were defined as any home exercise program that did not use any kind of wearables during the follow up period to inform the exercise programs. Controls were defined as participants not taking part in any kind of structured exercise program. In studies that did not report the change score, mean difference (MD) was calculated as the difference in mean values between the specified follow-up and baseline. Standard deviation of change scores was imputed using the formula : sdchangne trial  as used ne trial  was emplle size) . In stud studies . The folThree authors  independently assessed the risk of bias of all included studies using the Cochrane tool for assessing risk of bias in RCTs (RoB 2.0) . The trip-value of <0.05 was considered as statistically significant. All statistical analyses were performed using R program version 4.2.1.The Bayesian random-effects NMA was performed using the R statistical package “gemtc” by assuming consistency, variance scaling factor of 2.5, non-informative weakly prior distribution of standard deviation between trials . Markov The literature search identified 1920 studies, of which 1663 unique records were assessed . UltimatWhere reported, the baseline risk factors were not significantly different between participants allocated to the home exercise programs and control groups . Pooled The structures of the exercise programs tested in the included RCTs and how activity monitoring data were used to manage the programs are summarised in One possible advantage to activity monitoring could be to encourage adherence to the prescribed exercise program. There was, however, limited reporting of this within the included trials . Only seThe risk of bias of the included studies is shown in The network model of all treatment arms from all included trials were tested for feasibility of network meta-analysis, and the model convergence was achieved with 100,000 iterations. The Gelman diagnostics showed a PSRF of 1.000445 suggesting the reliability of the network model. The network diagnostics using trace and density plots showed that the models converged and were valid to use .All treatment arms (n = 1526) were connected through 9 arms that tested home exercise programs informed by wearables activity monitors (503 participants), 6 arms that tested home exercise programs not informed by wearable activity monitors (316 participants) and 14 arms with non-exercise control groups (707 participants). The main finding of the NMA was that by comparison to the non-exercise controls, home exercise programs informed by wearable activity monitors significantly improved walking distance . Home exercise programs not informed by wearable activity monitors did not significantly improve walking distance by comparison to non-exercise controls  . There wp = 0.167), length of the exercise programs (ranging between 6 to 52 weeks)  or number of facility visits (ranging between 0 to 26 times)  did not significantly influenced walking distance outcomes.Meta-regression of trials testing home exercise programs informed by wearables suggested that presence or absence of telephone counselling  [p < 0.001) [p = 0.0001) compared to controls. Participants were also categorised into groups receiving incentives with a weekly payment of SGD 15 (Singapore dollar) if they logged between 50,000 and 70,000 steps per week or SGD 30 if they logged 70,000 or more steps per week. When activity monitoring was accompanied by a cash incentive to the participant (mean earning SGD 620) or charity incentive (mean earning SGD 320), the MVPA improved by a mean of 15 MVPA bout min per week  and 32 MVPA bout min per week , respectively, at 12 months compared to the control group [The benefit of using activity monitors has been examined in populations without PAD. A previous meta-analysis of six RCTs involving 693 participants with coronary heart disease suggested that self-monitoring of physical activity improved daily steps taken by a mean of 2503  . The lar< 0.001) . Anotherol group .In addition to improving uptake of exercise, wearable activity monitors may facilitate exercise programs which are more cost-effective than centre-based programs. Centre based supervised exercise therapy has been estimated to cost SGD 14,590 per patient for a program which involved three directly supervised 1-h sessions per week for 26 weeks, supplemented by a subsequent 12-month telephone-based program designed to maintain adherence to exercise . ActivitSeven of the 14 trials reported variable information about adherence to the exercise programs ,27,29,30This NMA included largely high quality RCTs with most considered at low risk of bias. A number of limitations of this NMA should be acknowledged. This includes the small number of RCTs identified, the few patients included and the heterogeneous nature of the tested exercise programs. Due to the lack of head-to-head trials available, nodesplit analysis could not be performed to test for inconsistency. Study design and key clinical risk factors, including age, sex, BMI and ABPI, were similar between included trials, suggesting the homogeneity of the included population samples. In addition, follow-up in the included trials varied between 6 and 52 weeks. A previous meta-analysis of RCTs suggested that home exercise programs improved walking distance for duration of less than 12 months but there was no evidence this lasted beyond this point . DigitalIn conclusion, this study suggests that home exercise programs informed by wearable activity monitors improve walking distance in patients with PAD. It is unclear, however, whether exercise programs using activity monitoring are more effective than programs not using activity monitoring.


Several diverse proteins possess similar binding sites. Protein binding site comparison provides valuable insights for the drug discovery and development. Binding site similarities are useful in understanding polypharmacology, identifying potential off-targets and repurposing of known drugs. Many binding site analysis and comparison methods are available today, however, these methods may not be adequate to explain variation in the activity of a drug or a small molecule against a number of similar proteins. Water molecules surrounding the protein surface contribute to structure and function of proteins. Water molecules form diverse types of hydrogen-bonded cyclic water-ring networks known as topological water networks (TWNs). Analysis of TWNs in binding site of proteins may improve understanding of the characteristics of binding sites. We propose TWN-based residue encoding (TWN-RENCOD), a novel binding site comparison method which compares the aqueous environment in binding sites of similar proteins. As compared to other existing methods, results obtained using our method correlated better with differences in wide range of activity of a known drug (Sunitinib) against nine different protein kinases .  Sunitinib is a small-molecule, multi-targeted kinase inhibitor that was approved by the U.S. Food and Drug Administration (FDA) for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor We compared binding site of nine protein kinases namely tyrosine-protein kinase Kit (KIT), Tyrosine-protein kinase ITK/TSK (ITK), cyclin-dependent kinase 2 (CDK2), vascular endothelial growth factor receptor (VEGFR2), p21-activated kinase (PAK6), mammalian STE20-like protein kinase 3 (MST3), platelet-derived growth factor receptor A (PDGFRA), hematopoietic progenitor kinase 1 (HPK1) and phosphorylase kinase subunit gamma-2 (PHKG2) using different binding site comparison methods. We selected PocketMatch Water molecules surrounding the protein surface are fundamental in mediating protein folding, structure, function and ligand binding Here, we introduce TWN-based residue encoding (TWN-RENCOD), a novel binding site comparison method. Unlike our previous TWN works 22.1In the TWN-RENCOD method , initial2.2Sunitinib bound X-ray crystal structures of nine human protein kinases namely KIT (PDB: 3G0E) 2.3All MD simulations were performed using GROMACS package, version 5.1.4 2.4Water molecules form a number of hydrogen-bonded cyclic water-ring networks recognized as TWNs a, b) = interaction potential energy between water molecules a and bvqj = partial charge on the j site (0.417e)qij = distance between qi and qjrA = repulsive force between i and j C = attractive force between i and j Potential functions considered in TWNs involve a rigid TIP3P water model. The Lennard-Jones and Coulomb potentials are used to model the interactions between water molecules. The interaction potential energy between water molecules is estimated according to the equation below.−1 is considered. Comprehensive details of TWN method are provided in our previous publications To determine hydrogen bond between water molecules, energy criterion of − 2.25 kcal mol2.5The kinase–ligand interaction fingerprints and structure (KLIFS) database contains a consistent alignment of 85 kinase ligand binding site residues 2.6The 3-membered ring TWNs were firstly analyzed within the binding site of a protein in all the MD trajectories. Then, distances between backbone atoms  of each residue within the binding site of protein and centroid of each 3-membered ring TWN were calculated. A distance matrix was created based on the calculated distances. This matrix was further converted into a class type matrix according to the distance range . Same procedure was followed for creating distance matrix for the other protein. As similar TWNs in a particular location of the binding sites of proteins existed in different MD trajectories of proteins, MD trajectories of the second protein were reordered according to MD trajectories of the first protein. Reordering was done depending on the minimum difference in the distance values of each MD trajectory of both proteins. Afterwards, reordered distance matrix of the second protein was also converted into the class type matrix and compared with class type matrix of the first protein to calculate similarity between proteins. Similarity was defined as the ratio of matched class pairs to the total number of class pairs.2.7Binding site sequence identity and similarity were calculated with the ‘Align Sequences’ module of Discovery Studio 2022 . Sequence identity and similarity percentage values were converted into decimal values. The ‘Align Sequences’ module aligns two or more protein sequences using the Align123 algorithm, a progressive pairwise alignment algorithm modified from the CLUSTAL W program Additionally, binding site similarity was calculated using three other programs namely PocketMatch 2.8Binding energy of Sunitinib against each protein kinase was estimated with ‘Calculate Binding Energies’ module of Discovery Studio 2022  using the CHARMm forcefield. This module calculates binding energy according to the equation below:3Sunitinib exhibits a broad range of inhibitory activity against nine representative protein kinases .Table 1IThe X-ray crystal structures of these protein kinases co-crystallized with Sunitinib are available in the RCSB PDB Binding sites of kinases were compared to understand the multi-target activity of Sunitinib. Firstly, we calculated binding site sequence identity and similarity values between proteins , and stuWe decided to compare binding sites using other available methods. A number of protein binding site comparison methods are available today, which can be classified into three categories namely residue-, surface- and interaction-based methods The aqueous environment is a pervasive factor which determines the structure, function and dynamics of proteins. Furthermore, water molecules within binding site of a protein play a crucial role in mediating the protein-ligand interactions. Proteins may also influence the organization of water molecules around the protein surface. Investigation of binding site water molecules can provide valuable information about the binding site characteristics. Water molecules often form hydrogen-bonding networks in the protein binding sites. Our research group previously developed an algorithm known as TWN to analyze water networks In addition to the Pearson's correlation, we computed Kendall and Spearman correlation coefficients using R software . Kendall and Spearman computations also showed similar performance as demonstrated by Pearson's computation . FurtherThe present study highlights the importance of water molecules in protein binding site and suggests that existing methods are insufficient to compare protein binding sites. Water network based comparison of protein binding sites can provide new insights about the characteristics of binding sites. Thus, the proposed method can be used to compare binding sites of similar proteins for the drug repurposing and for the development of new molecules for target proteins. However, it should be noted that unlike PocketMatch, ProBiS and IsoMIF programs, TWN-RENCOD is not suitable for the analysis of large number of proteins as this method involves MD simulations. Furthermore, TWN-RENCOD is developed to compare binding sites of similar proteins.4We present TWN-RENCOD, a new method to compare protein binding sites. The proposed method computes binding site similarity based on water networks observed in binding sites of proteins. As compared to various existing protein binding site comparison methods, TWN-RENCOD showed much better performance in explaining the variation in activity of Sunitinib against several protein kinases. Outcomes of this study suggest that comparison of protein binding sites using TWN-RENCOD can provide novel insights about the characteristics of binding sites which can be exploited for drug discovery and development.10.13039/501100003725National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF2020R1A2C100691511).(NRF2020R1A2C100691511).This research was funded by the Basic Science Research Program through the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Obesity and metabolic syndrome are of increasing global concern. In order to understand the basic biology and etiology of obesity, research has turned to animals across the vertebrate spectrum including zebrafish. Here, we carefully characterize zebrafish in a long-term obesogenic environment as well as zebrafish that went through early lifetime caloric restriction. We found that long-term obesity in zebrafish leads to metabolic endpoints comparable to mammals including increased adiposity, weight, hepatic steatosis and hepatic lesions but not signs of glucose dysregulation or differences in metabolic rate or mitochondrial function. Malnutrition in early life has been linked to an increased likelihood to develop and an exacerbation of metabolic syndrome, however fish that were calorically restricted from five days after fertilization until three to nine months of age did not show signs of an exacerbated phenotype. In contrast, the groups that were shifted later in life from caloric restriction to the obesogenic environment did not completely catch up to the long-term obesity group by the end of our experiment. This dataset provides insight into a slowly exacerbating time-course of obesity phenotypes. Metabolic syndrome is becoming an increasingly prevalent global health concern. Metabolic syndrome is an umbrella term used for various pathologies surrounding long-term obesity such as hypertension or type 2 diabetes  and whetZebrafish have emerged as an important vertebrate model system for several reasons. Not only are genes largely conserved but there is also a high degree of physiological and anatomical similarity. Genetic models of obesity have been established in zebrafish such as overexpression of agouti related peptide  or semapConsequently, a variety of metabolic complications due to an obesogenic environment have been described in zebrafish. Further, a recent study in zebrafish has found that maternal overfeeding has a negative effect on early obesity markers in larvae of the next generation, comparable to evidence in mammals . TherefoConsequently, we carried out a systematic characterization of DIO as well as CG. We compared CR with DIO and tested whether CG of fish which were shifted from CR to DIO at different timepoints showed an exacerbated long-term DIO phenotype. Zebrafish are externally fertilized and feed for the first 5 days-post fertilization (dpf) on maternally provided egg yolk. We therefore restricted food from 5dpf onwards and induced CG by overfeeding comparable to the DIO group. We induced CG at three timepoints associated with different growth rates in wildtype animals: 1) a juvenile phase (one month) which exhibits naturally high growth rates (CG1), 2) three months old fish, a stage when growth begins to slow (CG3) and 3) nine months of age when growth mostly stagnates in wildtype fish (CG9). We investigate growth parameters, fat distribution and metabolic genes in order to establish whether zebrafish a) show signs of compensatory or catch-up growth, b) show signs of metabolic syndrome after a long-term obesogenic diet in support of and beyond previous studies and c) whether early food restriction exacerbates the effects of long term DIO and pre-disposes fish for or exacerbates metabolic syndrome.Zebrafish of the Ekkwill strain were raised and maintained at 28°C under a 14 h light / 10 h dark cycle and in tanks with continuous water exchange and recycling  . EmbryosFish were raised as previously reported in groups of 5  or 50 fishes  in 3 L tanks . In our We carried out a control experiment for CG1 where we compared changing the amount of food at a constant density versus changing density at constant food levels . For thiChanging food levels, constant density: 5 fish per 2.5 liter (L) fed eitherDIO: 100 ml paramecia daily (5–13 days post fertilization (dpf)) and 2x 10 drops of artemia (14 dpf onwards).CR: daily feeding of 10 ml paramecia (5–13 dpf) and 2x 1 drop of artemia (14 dpf onwards).CG1: CR conditions from 5-30dpf, DIO conditions 30dpf and olderConstant food levels, changing density: feeding under DIO conditions as above, density eitherDIO: 5 fish per 2.5L tank for the extent of the experimentCR: 50 fish per 2.5L tank for the extent of the experimentCG1: 50 fish per 2.5L tank from 5dpf until 30dpf which were split into 10 times 5 fish per 2.5L tank at 30dpf onwardsAll zebrafish experiments were approved by the national animal care committee  and the University of Cologne. Animals were sacrifices with an overdose of Tricaine MS-222 according to .For determination of body length and body weight, fish were anesthetized in 0.13% Tricaine (w/v). Body length was measured from the anterior tip of the mouth to the base of the caudal fin  using millimeter paper (to 0.5 mm). For determination of body weight, anesthetized fish were dried on Whatman paper and measured to one decimal point in milligrams. Per measurement 10 fish  were measured individually per condition.We calculated specific growth rate (SGR) in %weight per day according to  calculatTotal RNA was purified from pool of 3 brains or individual bodies with the PureLink RNA Mini Kit  after homogenization of the frozen material with mortar and pestle on liquid nitrogen and RNA extraction with Trizol . cDNA was synthesized with Superscript II Reverse Transcriptase  according to the manufactor`s instructions.agrp [insa [rps23 [Quantitative real-time PCR (qRT-PCR) was performed in quadruplicates with an Applied Biosystems 7500 Fast Real-Time PCR system , SYBR Select Master Mix  and primers for agrp  and insarp [insa . Relativa [rps23  as an inAfter determination of body weight, fish were put in single tanks (1 L) to acclimate and were starved for at least 24 hours. Feeding experiments took place at room temperature in a separated room without any disturbance to avoid stressing the fish. At least 5 fish were tested per condition.Drosophila melanogaster (D. melanogaster) were used for quantification of food intake because they can survive several hours in water without changing weight and keep moving making them an attractive prey for zebrafish. D. melanogaster provided by the Roth laboratory at the University of Cologne were used to raise larvae for the feeding experiment. Third instar larvae of D. melanogaster were washed out of the feeding medium, counted, dried using Whatman paper and weighted. Larvae with a weight of at least 10% of the fish`s body weight were given to the fish. During the following 8 hours, every 1–2 hours a counted number of fresh larvae were given to the fish. After one and after eight hours, fish were put in single tanks with fresh water and remaining larvae were counted and weighted. Weight was measured to one decimal points in mg using a precision scale. Food intake during the first hour was calculated in fed larvae in % bodyweight, while food intake during eight hours (whole day) was calculated in fed larvae/100 mg body weight.Because there is no standard method available for quantification of food intake in adult zebrafish, a new method for quantification of food intake in adult zebrafish was established. Instead of commercial fish food, living third instar larvae of wild-type For μCT imaging, adult zebrafish were fixed and decalcified in Bouin`s solution at room temperature for 7 days, stored in PBS and imaged using a micro-computed tomography (μCT) device . Zebrafish were placed individually in 1.5ml Eppendorf tubes using and an ultra-focus scan over the whole body was performed in a full-rotation in step-and-shoot mode. 322 projections  were acquired per subscan with an x-ray tube voltage of 60 kV, power 0.166 mA, aluminum filter 0.25 mm,exposure time of 363 ms, 6 averages and a object-source distance of 86 mm. All CT images were reconstructed at an isotropic voxel size of 18 μm using a Feldkamp type algorithm (filtered back-projection).Fat-containing regions were appear hypo intense in μCT data and were segmented using Imalytics Preclinical . The head and tail regions were removed by cutting out the tissue posterior to the operculum and anterior to the dorsal fin. The rest of the trunk (without the viscera) was subsequently bisected, decalcified in 0.5 M EDTA, embedded in the same way as mentioned for the livers. 8–10 μm transverse sections of the trunk and livers were obtained using a Leica CM1850 cryostat. The slides containing sections were air dried for 60 minutes at room temperature and fixed in ice cold 4% PFA for 10 minutes. After fixation, the samples were washed thrice in PBS for 5 minutes each and treated with 100% propylene glycol for 5 minutes. Meanwhile, the Oil Red O (ORO) (Sigma-Aldrich) stock solution (0.5g Oil Red O in 100 ml propylene glycol) was filtered and prewarmed at 60°C. The samples were stained with ORO for 8–10 minutes at 60°C. The slides were differentiated in 85% propylene glycol for 5 minutes, rinsed twice in distilled water and counterstained with Gill’s haematoxylin for 30 seconds. Excess stain was removed by washing with running tap water for a minute, rinsed in distilled water and mounted with Aqua-Mount® (Avantor).For the quantification of subcutaneous adipocytes, ORO and haematoxylin-stained transverse sections of three levels were selected, total numbers and areas per side around the pleural ribs (PR) region was measured using ImageJ. Average adipocyte cell size along both sides of the pleural rib in each section was calculated using area divided by cell number. The single adipocyte cell sizes were further validated using ImageJ. For each condition, two fish and three consecutive sections were analysed, and the mean was recorded.After determination of body weight, fish were put in single tanks (1 L) to acclimate and were starved for at least 24 hours. Determination of oxygen consumption was performed at room temperature (19–20°C) in a separated dark room without any disturbance to avoid stressing the fish. Individual fish were placed in respiratory chambers and allowed to acclimate for at least 30 min with continuous water flow before measurements started.2) > 0.9 were used to calculate routine and standard metabolism. Mean of oxygen consumption rate during 24 hours was defined as routine metabolism [2 / kg/ h.The experimental set-up consisted of 4 respiratory chambers  with ministirrers inside which are placed in a water bath. Fresh air-bubbled water is provided using a Minipuls 3 flushing pump  which is controlled by a computer. Dissolved oxygen levels were measured using Oxy 4 mini , a multichannel oxygen measuring system which connected via optical cables with oxygen sensors  which are glued inside the chambers. Oxygen levels were recorded every second during a 5 min phase in each interval . Flushing is sufficient to exchange more than 99% of the water . Oxygen tabolism , while mtabolism , 69. BotAfter determination of body length and body weight, fish were sacrificed, cut in small pieces and digested with trypsine on ice for 40 min. Intact mitochondria were isolated according to . The mitOxygen consumption rates were measured with Oxygraph-2k  at 28°C using 150–200 μg crude mitochondria as described before . In shorBlood sugar levels were determined using the glucose meter Accu-Chek Aviva  as described . In shorStatistics were carried out in GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego, California USA. The particular test used is indicated in each section. P-values less than 0.05 were considered significant.https://doi.org/10.1594/PANGAEA.941313 while the CT scans are available under https://doi.org/10.1594/PANGAEA.940201.We have made the primary data available through GFBio  and the We compare calorically restricted (CR) fish with overfed / diet induced obese (DIO) fish and fish that were shifted from CR to an obesogenic environment after 1, 6 or 9 months of CR  = 3.363*log(SL) - 2.329, R2 = 0.9575  = 4.134*log(SL) -3.404, R= 0.9575 . Therefoad libitum food access. We first looked at the expression levels of agouti related protein (agrp) which is known to be an indicator of hunger in animals including zebrafish [agrp expression in fish brains in the CR group compared to the DIO group, suggesting increased hunger in the calorically restricted group. When we tested CG9 fish one to two weeks after the transfer from CR to DIO conditions we found that agrp levels were significantly reduced compared to CR but had not yet returned to baseline compared to DIO . We validated the procedure with fed and fasted animals since there should be a difference between these two groups due to the thermic effect of food. Consistent with previous publications –79, we fWe tested for mitochondrial function in CR and DIO fish. As there were significant size difference between CR and DIO fish we also included a size-matched DIO group which reached this size at a significantly younger age (1.5 months versus 9 months). Therefore, the CR and DIO groups are matched in age and the CR and size-matched DIO groups in size. We found that the maximal rate of oxidative phosphorylation did not differ between age matched or size matched calorically restricted or overfed fish  period and standard length in life. This result suggests that either zebrafish do not respond with compensatory growth upon food restriction or that there is a critical period where growth stunting conditions lead to compensatory growth with an increased growth rate and that this period is already over 5 days post fertilization. Indeed, there is controversy in the mammalian literature about the effect of early growth restriction on compensatory growth and the most solid evidence involves food restriction during the peri-natal period, particularly by food restricting dams during pregnancy or pre-weaning [The other main direction of this study aimed to characterize CG in zebrafish. We wondered whether we could utilize the fish to model aspects of metabolic disease, particularly a predisposition to develop metabolic disease later in life after caloric restriction or undernourishment in early life / childhood. We found that body weight and length catch-up and do not compensate. Historically the two terms are often interchangeable, but Malcolm Jobling has pointed out key differences, specifically that compensation involves a period of increased growth rate above normal growth rates while catch-up growth involves animal growth at normal growth rates after a period of depressed growth . Importa-weaning . In the -weaning , 88. Our-weaning . HoweverBased on the literature, one would predict that in the case of compensatory growth, the shift from CR to DIO would lead a long-term exacerbation of metabolic effects while in the case of catch-up growth one would expect effects similar to DIO or slightly behind the DIO group still catching up. Our growth curves support catch-up and not compensatory growth, and we indeed observed that none of the CG groups showed metabolic endpoints with an exacerbated phenotype compared to the DIO group. Indeed, some of the CG groups did not show a complete catch up process to the DIO group yet. While the early shift (CG1) caught up completely with the DIO group endpoints, the CG3 group mostly did and the CG9 group never caught up completely. The growth curves approximated each other, but especially in the metabolic endpoints there were still significant differences which are suggestive of a sequential process in the development of metabolic endpoints. For example, the CG3 group did catch up to the DIO3 group in adipocyte size but not number suggesting that SAT adipocytes undergo hypertrophy before the undergo a renewed period of hyperplasia. In mammals, hypertrophy during obesity is considered to be indicative of metabolic complications while hyperplasia is considered to be protective . Fish apIn summary, it is notable that fish exhibit similar trends in the storage of lipids in an obesogenic environment compared to mammals and that fish of the Ekwill strain show several adaptive responses to DIO. However, significant variation exists between people in terms of response to obesity. Similarly, a recent paper showed differences between two strains of fish in fasting induced central gene expression changes . It woulS1 Fig standard length;  fish weight and  body mass index of fish undergoing CG3  of CG 9 ; error bars indicate STDEV, ns indicates a lack of significant difference between the CG and the DIO group at that timepoint as indicated by a 2-Way ANOVA followed by Tukey’s multiple comparison test.(TIF)Click here for additional data file.S2 Fig Growth curves of fish kept at different feeding conditions and densities; (A) Body lengths of fish raised in low density (5 fish per tank) but different feeding regimes; fish raised with ad libidum feeding conditions which is able to induce diet-induced obesity (DIO) show significantly increased linear growth compared to fish raised under caloric restriction (CR); fish raised under caloric restriction before 1 month of age but with ad libitum conditions afterwards (CG) show briefly increased growth rates and compensate for differences in body length suggesting that these fish exhibit compensatory growth (CG); (B) Body lengths of fish raised with different densities while every tank received the same amount of food resulting in different feeding conditions; fish raised in low density (5 fish per tank) with ad libidum feeding conditions (DIO) show significantly increased linear growth compared to fish raised in high density (50 fish per tank) and therefore under caloric restriction (CR); fish raised in high density  before 1 month of age but in low density  afterwards (CG) show briefly increased growth rates suggesting that these fish exhibit CG; n = 10 for each condition;; error bars indicate STDEV, ns indicates a lack of significant difference between the CG and the DIO group at that timepoint as indicated by a 2-Way ANOVA followed by a multiple comparison test.(TIF)Click here for additional data file.


Purpose: Cancer is the leading challenge to human health since the dawn of early Egyptian manuscripts, where they found tumour from fossils in the modernized 20th century. Increasing rate of incidence and death from cancer in the past few years is thought provoking. Among all type of cancers, breast cancer is very common among women and diverse in character. Drug resistance is the challenging aspect for traditional chemotherapy.Methods: Data was collected from online platform without any time restriction. After screening and evaluation, 66 articles were considered for this study. This review is a summarized collection of information from published studies on human genes associated with drug resistance in breast cancer treatment.Results: Analysis of these findings highlights the importance of MAP kinase and ABC gene families in creating resistance barriers. Genes involved in cell cycle alteration, apoptosis, and hippo pathway were also linked with drug resistance particularly in breast cancer.Conclusion: The exact mechanism of chemotherapy resistance is still unresolved and unexplained the drug resistance seen in breast cancer patients were multifactorial. Drug induced up regulation or down regulation of genes contributes unusual protein expression and ultimately leads to resistance. The ultimate focus of this review is to identify the genes having pivotal role in chemotherapy resistance in breast cancer. Breast cancer is one of the world’s commonest cancers with a collection of neoplastic diseases that are molecularly and clinically complicated and comprise different sub types with unique characters. Breast cancer is rarely seen in males. Being a highly prevalent cancer type, it is observed in both developing and developed countries with different causes and factors of progression. Adopting new life styles, industrialization, globalization, increased life expectancy, pollution etc are the leading cause of cancer. 2012 Global Cancer statistics reveals that among the eight million diagnosed cases of cancer all over the world, one million constitute of breast cancer. According to 2015 cancer statistics almost 231 000 US women were diagnosed and about 40 000 patients were died with breast cancer. As per 2018 statistics, every year around 1.2 million new cases of breast cancer were diagnosed. The GLOBACON report of 2018 concluded breast cancer as the most frequently identified cancer type and leading cause of cancer related death in majority of the countries. Major part of breast related cancer are linked with the expression of estrogen receptor and their treatment is related with the disease prognosis.Abnormal growth of cells due to uncontrolled cell proliferation in body is called as cancer. commonly adopted treatment strategies for breast cancer are chemotherapy, radiation therapy and hormone therapy. Among them chemotherapy is widely accepted as traditional treatment method, in spite of its obstacle due to chemotherapy resistance. Important mechanisms lead to drug resistance includes alteration in expression of ABC transporters gene family, damage of topoisomerase enzyme, mutation in DNA repair genes, induced apoptosis by genetic imbalance, alteration in signalling pathways of NF alpha etc. All these worsen the condition of breast cancer making chemotherapy a failure in most cases. The cells which already acquired drug resistance show cross resistance to anti proliferative nature of anti-oestrogen drugs which have crucial role in breast cancer treatment. In this review, we are discussing on various genes involved in chemotherapy resistance pathways of breast cancer.Rate of breast cancer mortality can be reduced up to a limit by early diagnosis; timely treatment and management. Alteration in MAPK pathway cause poor tamoxifen response in ductal carcinoma and estrogen expression in lobular breast cancer. Copy number alteration and mutation acquired by drug resistant cells affect the metallotropic glutamate receptor and MAPK pathway. MAP deregulation induces tamoxifen resistance. Likewise the hyperactivity of MAPK is closely linked with GRM1 gene mutation. The GRM1 gene is considered as an oncogene in epithelial cells. Un controlled expression of the KLF4 gene, plays an important role in the transition of G1 to S phase through P53. Phosphorylation of MAPK pathway and estrogen receptor kinase and P38 activation were induced by KLF4 knock down causing tamoxifen resistance. Hyperactivity of MAPK signalling by the c- ras, b- raf, MEK ½mutation is commonly seen in many human cancer cells including breast cancer. MAPK/ERK pathway regulates cell cycle and cell proliferation having a crucial role in cancer treatment especially in triple negative breast cancer cases. Heterodimerization of RAF kinase cause the activation of ERK pathway and drug resistance. Demodulation of Jak, stat 3 and Akt signalling along with MAP kinase pathway cause paclitaxel resistance by STAT3 in breast cancer, and also the basal activity of JAK1/STAT1 and JAK1/STAT3 signalling is higher in chemo resistant cells. The Genes of STAT3 Inhibition involved in apoptosis activation is down regulated in chemotherapy resistant cells.Mitogen activated protein kinase (MAPK) is a group of protein which communicates and transfers signal from the cell surface to the nuclear environment in response to external stimuli.ABCD1, C1, and G2 transporters are extensively studied ABC transporters gene family involved with drug resistance.Efflux proteins are involved in the transportation of drugs and toxic substance from inside the cell to the outside ATP dependent drug efflux pumps may sometimes reduce drug uptake. ABCG2 gene is commonly known as BCRP which is a part of ATP binding cassette transporters that play a major role in cellular transportation by the expense of ATP across concentration gradient inducing drug resistance mechanism. various anti cancers agents’ effluxed by altered expression of p-glycoproteins are considered most studied and explained mechanism of drug resistance. P-glycoprotein is an example of adenosine triphosphate binding cassette protein, which expressed by the mutation of genes in ABC family and drug doxorubicin, acquires resistance through the mutation of this gene family. This in turn because drug effects leading to insufficient accumulation of doxorubicin in nucleus, showing only about 0.4% doxorubicin enter cell after internalization.ABCB1 gene expression, paving the way for multiple drug resistance through drug efflux. Many study reveals that the cells treated with epirubicin and anthracycline, show ABCG2 activation and expression in breast cancer cell lines by self-renewing capacity. ABCG2 gene has an important role in gefitinib resistance. Which is used for EGFR over expressed breast cancer like erlotinib. These drugs interrupt EGFR signaling in the target cells. Drug induced over expression of proteins by ABCG2 genes acquire resistance to the drug gefitinib in EGFR expressed breast cancer cells. At low concentration gefitinib act as a substrate for ABCG2 protein, inducing gefitinib resistance through Akt nuclear EGFR (nEGFR) pathway.ABCI gene family induce drug efflux causing resistance to methotrexate, and up regulation of GPR120 gene cause alteration in GRP120 protein mediated signaling, inducing resistance to epirubicin in chemotherapy, and Akt NFKB signaling is responsible for GRP120 mediated resistance to epirubicin. Intracellular epirubicin accumulation is directly associated with ABC transports expression ABCG1 and ABCG2 gene up regulation by NF-kB p65 cause blockage of GPR120 signaling.FASN and ABCG2 expression is decreased by GPR120-si-RNA and GP6120 expression is related with the levels of AACC1, ABCC2, FASN and FFAs. All these are directly or indirectly related to ABC gene up regulation and multiple drug resistance.Resent research points out multiple drug resistance induced by oxidized low density lipoproteins and very low-density lipoproteins. Down regulation of p-glycoproteins by these lipoproteins induce alteration in  MDR1 over expressions by p53 alteration cause chemo resistance in doxorubicin and Tuxol. Which significantly contributes changes in P-glycoprotein levels there by inducing resistance. Acquired and intrinsic cross resistance to vinca alkaloid derived drugs and anthracycline are related to p glycoproteins induced MDR1 expression. Deletion of the mir 125b gene in chromosome 19q and microRNA 451 regulate MRD1expression in anthracycline induced resistance.ABC gene family members MDR1 have a significant role in drug resistance. FOXA1 is an inhibitor of AR signaling by detecting it is binding to the cells, whose activity completely overlaps estrogen receptor binding site in an AR positive cells lines and cause tamoxifen resistance. GRLH2 binding site is closely associated with FOXA1 binding site. Tamoxifen resistance result in LYPD3 protein level elevation, which is considered as a target for GRHL2. One of the reasons for HER2 mediated drug resistance is by the amplification or deletion of Topoisomerase II alpha gene TOP2A located in 17q21 near to ERBB2 oncogene. Topoisomerase II alpha is the molecule target for topo II inhibitors, potent anti-cancerous drug.Chemotherapy cause changes in genes involved in cell proliferation by holding the cell division, where FOX protein family have a significant role in regulating cell cycle. Studies report over expression of the androgen receptor as a reason for chemotherapy resistance, contributes uncontrolled cell proliferation in triple negative breast cancer patients. Two routes are involved in the activation of apoptosis cascade. The intrinsic pathway also known as mitochondrial pathway that releases cytochrome C by activation of tumor necrosis factor in response to ligand binding. Autophagy, mitotic catastrophe, necrosis, and senescence are non-apoptosis mechanism involved in cell death.Apoptosis pathways have a significant role in cancer treatment, and any alteration in this pathway is a major obstacle for effective treatment.CASP3 gene is located in the 4q34 with size 2635 bp, this mediates apoptosis in response to chemotherapy, but losing caspase 3 activity cause cell survival and induce drug resistance through apoptotic pathway in breast cancer.K1F14 is a tumor inhibitor gene that prevents cell migration and induce cell apoptosis and its knockdown is found increases breast cancer subjects.KIF14 plays an important role in drug resistance by enhancing cell proliferation through Akt1 pathway which with decreased activation lowers the level of Akt and affects the P13K/ Akt signaling that controls the apoptosis pathway. It also decreases the levels of tumour suppressor gene p63, promoting Akt phosphorylation. Docetaxel is one of the frequently used drugs for breast cancer and decreased KIF14 expression increase docetaxel resistance.FOXM1 is a cell proliferation gene which encodes a protein regulating cell cycle gene and also an apoptosis inhibitor. XIAP produces protein for preventing apoptosis and survivin is produced by BIRC5, whose expression covers upregulation in drug resistance. XIAP and BIRC5 induce taxane and anthracycline resistance. Survivin, induce multiple drug resistance to drugs in tumors associated with endothelial cells causing resistance to drugs like paclitaxel and temozolomide by preventing apoptosis of caspase 7 and 4. The genes XRCC2 and BRCA2 involved in homologous recombination and DNA damage repair. This confers cisplatin induced drug resistance by maintaining DNA damage.XRCC2 stimulates RAD51 levels and Elevated RAD51 level is linked with high recombination rate and increases resistance to DNA altering drugs.XRCC2 expression is directly linked with drug resistance by different mechanisms.O6 methyl guanine methyl transferase is a DNA repair enzyme, whose increased activity causes resistance to drug such as temozolomide, streptozotocin, procarbazine and dacarbazine. This induces DNA lesions.ASK1 gene is a MAP3 K member which plays a vital role in breast cancer cell apoptosis under stress conditions. Raf 1 mediated inhibition of ASK1 cause drug resistance in endothelial cells through basic fibroblasts growth factor. Pro apoptotic activity of this gene is inhibited by BFGF, which leads to the end of apoptosis.ASK1 gene’s level is related with CLDN6 expression and over expression of CLDN6 is linked with breast cancer chemo resistance through GSTP1 its activity is regulated by p53. in the treatment of hormone sensitive breast cancer, tamoxifen resistance is a clinical obstacle. The major genes involved in tamoxifen resistance by X box binding proteins. Which is directly related with estrogen receptor alpha function. First three amino acids of the protein coding gene involved in the activation of estragon receptor in estrogen deficiency. Tamoxifen also targets the mitochondrial genes like SIRT3 and AMPK. The protein encoded by SIRT3regulats ATP generation, aging and carcinogenesis. It has a significant role in inhibiting apoptosis and recent studies show the evidence of this gene in drug resistance. this drug up regulates the SIRT3 gene function and AMPK gene phosphorylation. Where AMPK has a crucial role in drug resistance by producing self-renewal cancer stem cells. AMPK also act as a downstream regulator of tumour suppressor genes like P53 and LKB1. Tamoxifen regulates estrogen modulation through non genomic and genomic signalling , LAST 1/2 (large tumour suppressor), YAP (yes associated protein), TAZ , RASSF1. Hippo pathway is activated by MSTA1/2. Transcription of YAP and TAZ are inhabited by the phosphorylation and down regulation of LAST1/2. Hippo pathway activation is closely related with breast cancer stem cell formation. Breast cancer stem cell’s improper TAZ and YAP activity cause multiple drug resistance. An anti-Microtubule drug taxol cause LAST2 knockdown. LAST2 play vital role in up regulating estrogen receptor, and this cause resistance to tamoxifen, Studies shows that high level of TAZ were observed in breast cancer cells.Hippo pathway is identified first in Drosophila Melanogaster, and later identified in mammals. Core components are Kinase  Studies suggest that MVP protein have a particular role in drug resistance through the activation of MAP kinase pathway. MYP is the primary component of vault complex. It is involved in chemotherapy resistance by phosphoinositide-3-kinase/AKT signalling and EGFR induced MAPK pathway associated with ABCC1 and ABCB1. NOTCH1 down regulate MVP expression.Vaults are largest nucleoprotein particle ever discovered which have a size of ~42 × 75 nm and ~13 MDa mass. It is first described in 1986.GRMI, KLF4 etc have clear link with drug resistance in breast cancer. KLF4 knockdown cause tamoxifen resistance and STAT3cause paclitaxel resistance. Another important members involved are ATP-binding cassette (ABC) transporters gene family which include ABCB1, ABCG2 and Multiple drug resistant gene family like MDR1 genes. Chemotherapy resistance induced by preventing apoptosis is mediated by genes like FOXM1, XIAP, BIRC5, XRCC2, BRCA2, CLDN6, ASK1, SIRT, AMPK, TWIST etc. Genes associated with hippo pathway and vault proteins associated genes induce drug resistance by various mechanisms. Conventional cancer chemotherapy is severely restricted by multidrug resistant tumor cells due to changes in the level or activity of membrane transporters that mediate energy dependent drug efflux and proteins that influence drug metabolism. The extensive use of chemotherapeutic agents for cancer treatment has led to many patients being cured. Sadly, many cancers do not yet respond to chemotherapy, and other cancers which initially respond later become resistant. The use of biologics and gene therapy is a highly active area of research to address the problems of chemo resistance. Combination therapy, a type of treatment that incorporates two or more therapeutic agents, is one of the cornerstones of cancer treatment. Similar to the monotherapy strategy, the amalgamation of anti-cancer drugs increases effectiveness because it addresses main pathways in a synergistic or additive way. (Different genes play a crucial role in resistance to multiple drugs . Genes rive way. . The drugs combinations which are currently exist were refined from clinical experiences and it comprises a small part of therapeutic space. Computational approach of the biological science to discover new collaborative drug combinations for treating breast cancer will help in overcome multiple drug resistance, rather than preventing drug resistance, it also prevents cell’s mitotic activity, suppress the proliferation of cancer stem cells, reduce the rate of tumor formation and induce apoptosis. The five- year survival rates for most metastatic cancers are still quite small, so developing a new anti-cancer medication is costly and extremely time- consuming. Optimization of therapeutic efficacy by taking into account the therapeutic synergy that is immune to multiple drug dosing and scheduling can be carried out to overcome the acquired drug resistance in breast cancer patients. No ethical issues are associated with the publication of this review article. Not applicable.


Penile Cancer in Genitourinary Oncology as part of the Frontiers in Oncology has explored hot topics in this rare disease that needs further development. Authors from Asia, Europe, North and South America have explored important diagnostic, prognostic and therapeutic aspects.The Research Topic Deng et al.].Lack of significant progress in the treatment and management of penile cancer patients in the United States in recent decades is suggested by the increasing trend in incidence-based mortality (IBM) with no significant improvement in the 5-year relative survival rate observed using population-level data from the SEER database [Gu et al.].A large multicenter Chinese study including 340 penile cancers has identified that phimosis has decreased its prevalence through the years studied while HPV infections rose, becoming a more important etiological factor. HPV types 16 and 18 comprised 88.6% of infections detected and when added type 33, 91% of infections studied are accumulated .Other Chinese group sequenced 35 penile cancer patients’ complete exome in order to define a correlation between gene copy number alterations and the disease prognosis. The 5-year survival rate of patients with Gao et al.].Lymph node status is the most important prognostic factor of penile cancer, and examined lymph node (ELN) count and lymph node density (LND) were independent prognostic factor for overall survival when analyzing 528 patients in the Surveillance, Epidemiology, and End Results cohort from 2010 to 2015 and 156 patients in a Chinese cohort (2006-2016). Using the ROC curve, the recommended cutoff values of ELN and LND were 13 and 9.3%, respectively (P <0.001) [Wu et al.].Based on the indication for complementary treatment and the already known poor prognosis of extra lymph node extension (ENE) in penile squamous cell carcinomas, a Chinese group studied 234 patients who underwent bilateral inguinal lymph node dissection surgery and has developed a nomogram based on pathological staging and clinical-laboratory data including platelet-lymphocyte ratio and squamous cell carcinoma antigen, capable of predicting the risk of ENE presence [Yang et al.].Also, deep inguinal lymph node metastasis (ILNM) was the most accurate factor for predicting pelvic lymph node metastasis (PLNM) in a different cohort of 189 Chinese patients. Once involvement of deep ILNs indicates poor prognosis, authors propose that patients with deep ILNM should be staged as pN3 and referred for pelvic lymph node dissection [Nemitz et al.].Radioisotope-guided dynamic sentinel node biopsy (DSNB) simultaneously or secondarily after penile surgery has potential to limit morbidity. Among 41 Germany patients, the morbidity rate was 15.8% per inguinal region and intervention was only required in six groins (7.9%). While DSNB is limited by false-negative results, it might be reduced when performed simultaneously to primary tumor resection . Other two patients with advanced penile squamous cell carcinoma who were administered chemotherapy combined with sintilimab also showed sustained partial and complete response for one and two years, respectively [Mei et al.].Two patients successfully treated with pembrolizumab are reported, one with high tumor mutational burden and complete response for 38 months and other with PDL-1 expression in penile tumor tissue with partial response for 18 months [Lebentrau et al.].In a European survey, guideline adherence for most treatment recommendations increases with growing annual penile cancer caseload. The probability of a guideline-adherent recommendation increased with each patient treated per year. The type of hospital care (academic vs. non-academic) did not affect guideline adherence in any scenario [Pinho et al.]. This research topic selection highlights the current penile cancer landscape, from new technologies with prognostic ability, with the lymph node dissemination still as the Achilles’ heel, to the great therapeutic potential of immunotherapies for the near future.Brazilian authors brought a compilation on non-coding RNA and its role in penile cancer, as well as the opportunity to use paraffin samples, allowing retrospective studies. Micro RNAs, pi-RNAs, and long non-coding RNAs (LncRNAs) are already known as corelated to disease and are possible tumor biomarkers with potential diagnostic and prognostic targeting in the near future [LR: Supervision; KB, DC, LD: Manuscript writing/editing. All authors contributed to the article and approved the submitted version.Ministério da Ciência e Tecnologia/Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq, processo 304747/2018-1.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phosphorylated tau (p-tau) species in bodily fluids are among the most reliable molecular biomarkers for differential diagnosis and progression monitoring of Alzheimer’s disease (AD) . The ATNWe quantified endogenous p181tau in CSF samples of APPPS1 transgenic (tg) mice  that do not develop neurofibrillary tangles or extensive neuron loss . CSF p18We then used an immunoassay which allows the quantification of tau phosphorylated at threonine 217 with or without adjacent phospho-epitopes  , 5 see a. On aveThese observations imply that p-tau increases in CSF are not exclusively triggered by Aβ deposition but can also be induced by the deposition of Danish amyloid (ADan). Thus, it is tempting to speculate that CSF p-tau increases are a general phenomenon of secondary tauopathies, as opposed to primary tauopathies. Interestingly, AD and FDD but also Familial British Dementia, yet another cerebral amyloidosis with concomitant neurofibrillary degeneration ), all shSupplementary file1 (DOCX 101 kb)Below is the link to the electronic supplementary material.


This study describes the feasibility and patient satisfaction for an integrated treatment to address multiple health outcomes in a sample of older adults living in a low-income independent residence facility and their own homes in the community. Specifically, 30 older adults were offered the opportunity to participate in a feasibility study of different components of Activate for Life treatment targeting balance and physical strength (Otago Exercise Program), breathing retraining (Gentle Yoga and Yogic Breathing), and mental health . Three treatment combinations were compared in a randomized repeated measures design to determine if adding components to the existing Otago program were feasible and if this affected patient satisfaction. Arm1: the Otago strength and balance program alone (n = 10); Arm 2: Otago + Gentle Yoga and Yogic Breathing (n = 10); and Arm 3: Otago + Gentle Yoga and Yogic Breathing + Behavioral Activation (we named this combination ‘Activate for Life’ n = 10). Dependent measures included recruitment rate, session completion characteristics, and satisfaction with the program. Overall, study and treatment components proved feasible, and participants reported high satisfaction with all 3 Arms.


Achieving adequate nutrition in preterm infants is challenging. The post-discharge period may be critical for influencing growth and cognitive outcomes. We studied the effects of post-discharge nutrition on childhood cognition.Preterm-born children were randomized at ~36 weeks corrected age (CGA) to either preterm formula (PTF) or term formula (TF) until 6 months, or PTF until 40 weeks CGA, then TF until 6 months (crossover group). Childhood cognition was assessed using the short form Wechsler Intelligence Scale for Children III, allowing computation of full-scale intelligence quotient (FSIQ) and four-factor index scores; verbal comprehension, freedom from distractibility (FDI), perceptual organization (POI), and processing speed (PSI).p = 0.03) compared to crossover. Faster weight gain from term to 12 weeks CGA was associated with 5-point higher FSIQ (p = 0.02) and four-point higher POI (p = 0.04). Infant head growth was positively associated with FSIQ  and FDI .Ninety-two children were recruited (mean 10.1 years). FSIQ did not differ by group. PTF-fed children had 10-point higher PSI (While there is no long-term impact of post-discharge macronutrient enrichment on childhood cognition, greater weight and head growth in specific epochs is associated with better outcomes. Further studies are needed to determine optimal early diet in preterm infants.Achieving adequate nutrient intakes in preterm infants before and after hospital discharge is challenging.Nutrient intakes prior to discharge affect later cognitive and metabolic outcomes.Follow-up of a randomized controlled trial shows no cognitive benefit in later childhood from a more nutrient-dense formula compared to standard formula after hospital discharge.Growth in the first year of life is strongly correlated with childhood cognition and emphasizes the importance of nutrition in early life. Macronutrient intakes in the first week of life alone are associated with higher neurodevelopmental scores at 18 months of age,3 and data strongly support a positive dose–response relationship between the amount of mother’s own milk an infant receives and later neurodevelopment.4 Studies have shown higher verbal intelligence quotient (IQ) in children born preterm who received higher macronutrient intakes in the first four postnatal weeks and these advantages correlate with the size of specific neuroanatomical brain regions using magnetic resonance imaging in adolescents.5 These data emphasize the critical importance of establishing adequate nutritional intakes in early life.Preterm birth represents ~10% of all births worldwide and is associated with increased risks for adverse metabolic and cognitive outcomes over the life course.6 Enteral milk feeds take several days to establish, and while the benefits of mother’s own milk are unequivocal, unfortified breastmilk will not provide sufficient nutrients to meet the needs of most very preterm infants.7 It is therefore common for preterm infants to accumulate nutrient deficits during the hospital stay, and many preterm infants are discharged home on a lower weight and length centile compared to their birth centile.8Preterm infants present many challenges in establishing appropriate nutrition. While parenteral nutrition is considered a standard of care in most very preterm infants (<32 weeks gestation), it may take several days to achieve adequate intakes.9 Meta-analysis of trials of infants who are formula feeding after discharge show greater weight gain in infants receiving macronutrient enrichment, but no consistent benefits on neurodevelopment in infancy.10 Widely used measures of infant development such as the Bayley Scales of Infant Development (BSID) may lack the sensitivity to detect differences due to nutritional interventions in early life11 and assessment of cognition in later childhood is therefore important. The aim of our study was to conduct long-term follow-up of a randomized controlled trial (RCT) of macronutrient enrichment in formula-fed preterm infants following hospital discharge. Follow-up in infancy had shown greater growth at 12 weeks post-term and 18 months of age in the infants who received a macronutrient enriched formula on discharge, but no differences in infant developmental outcome using BSID II.12 We aimed to determine the effect of differences in the diet on cognitive outcomes in later childhood and to explore any associations between growth in infancy and later childhood cognition.Studies have therefore explored whether increasing macronutrient intakes post-hospital discharge improve outcomes. Breastfeeding after discharge is strongly recommended and a small number of studies have explored macronutrient supplementation in these infants, but these show no consistent evidence of benefit.13 Briefly, preterm infants with a mean (±SD) gestation of 31 (±2) weeks and birthweight of 1.37 (±0.3) kg without significant neurological abnormalities or ongoing respiratory disease at discharge were recruited. Parents who had chosen to formula feed were approached prior to hospital discharge at a mean age of 36 corrected weeks, and their infants randomized to one of three different diets. The three diet groups were: (A) preterm formula (PTF) from discharge to 6 months corrected age; (B) term formula (TF) from discharge to 6 months; or (C) PTF from discharge to term corrected age, and TF until 6 months corrected age . Growth was assessed regularly using standard techniques until 2 years corrected age and we excluded children who were lost to follow-up before 18 months corrected age. Full details on nutrient compositions are available elsewhere,13 but in summary, PTF had an energy density of 80 kcal/100 mL and protein 2.2 g/100 mL, and TF contained 67 kcal/100 mL and 1.6 g/100 mL. For this follow-up study, parents were contacted by letter and children were reviewed at ~10 years of age by one of two psychologists  blinded to original group allocation and infant growth outcomes. Research Ethics approval (Newcastle and North Tyneside Research Ethics Committee) and signed parental consent were obtained.We studied children recruited as preterm infants from a single tertiary neonatal unit in Newcastle upon Tyne, UK between 1993 and 1997, and full details of the original trial are provided elsewhere.15 Full-scale IQ (FSIQ) was used as a measure of overall cognitive ability and four-factor index scores were determined that relate to specific elements of cognitive functioning. The verbal comprehension index (VCI) evaluates verbal reasoning ability, freedom from distractibility index (FDI) assesses working memory and attention, Perceptual Organization (POI) measures visuospatial reasoning and Processing Speed Index (PSI) tests the speed and accuracy of information processing. An FSIQ score of 80 or above is considered within the normal range, with scores of 70-80 and 55-69 indicating ‘borderline’ and mild cognitive impairment, respectively.Cognitive ability was assessed using a short-form version of the Wechsler Intelligence Scale for children 3rd edition (WISC-III) that allows the computation of all four-factor index scores. Each index has a mean score of 100 and SD of 15.16 To ascertain growth during a specific epoch, weight, length, or OFC change in SDS were calculated by subtracting the SDS at the start of the period from that at the end. We used maternal higher education  as a crude proxy for parental education, need for mechanical ventilation in infancy as a proxy of illness severity, and determined socioeconomic status (Townsend index) using a nationally validated deprivation score based on residential locality.17Weight, height and occipitofrontal head circumference (OFC) was measured using standard techniques and standard deviation scores (SDS) calculated using the British 1990 growth references.t test (parametric) or Mann–Whitney U (nonparametric) tests. Individual linear regression models were generated using FSIQ and the four-factor index scores as separate dependent outcomes to assess associations between diet group or epoch of infant growth and subsequent cognitive outcome. Multivariate regression models were used to quantify associations after adjustment for potential confounding factors. Variables included in the models were sex, gestation, birthweight SDS, Townsend index, and maternal education level. Levels of significance were set at p ≤ 0.05, and statistical analysis was performed using STATA version 11.0.Baseline characteristics were compared using p = 0.56) or gestational age (p = 0.67). The three randomized groups did not differ significantly in demographic or clinical characteristics  of these at a median age of 10.1 years (Table p = 0.04)  Table . Althoug4) Table .Table 2Cp = 0.02), and 4-point higher POI for every unit increase in weight SDS between term and 12 weeks (p = 0.04) (Table p = 0.04) and the freedom from distractibility index (FDI) increased by six points (p = 0.003) for every unit increase in OFC SDS. This impact of OFC growth over the first year of life remained significant for the FDI even after adjusting for weight and length gain  was significantly associated with a 5-point higher FSIQ (,19 and we did not detect an impact of the early randomized dietary group. Importantly, we have shown a strong association of early infant growth and later cognition, which was significant for weight gain in the first 12 weeks. Our data also show that head growth in the first year was significantly associated with both FSIQ and the FDI subset, which remained significant even after adjusting for weight gain. These data are in keeping with other studies in preterm infants demonstrating that weight gain between term and 4 months corrected age was associated with higher developmental scores at 18 months20 and another study showing that each unit increase in weight SDS between term and 1 year of age was associated with a 1.9-point IQ advantage at 8 years of age.21 In combination, these growth data emphasize that the first year of life remains a potentially important period for optimizing brain development through dietary management.22 Although there is no evidence of a long-term cognitive benefit for enriched formula after hospital discharge, it is important to note that our children were generally healthy preterm infants, and it is possible that there may be an advantage for higher-risk preterm populations. Some smaller studies show a benefit for macronutrient enrichment in high-risk infants after discharge,23 and this might be especially important where infants are unable to upregulate feed volumes to meet caloric demands.We assessed cognitive outcome in children born preterm who were formula fed and recruited to a randomized trial of post-discharge diet. Overall, their mean FSIQ was ~7 points below standardized mean scores, similar to other follow-up studies in preterm infantsStrengths of our study include the randomized controlled design, detailed measures of early growth, and 10-year follow-up of cognition by trained psychologists blinded to original group allocation and early growth measures. However, our study has important limitations. It was not powered to detect long-term differences in cognition, and while we were able to follow-up >80% of the original cohort 10 years later, it is possible that those followed up are not representative of the entire cohort or other very preterm infants. Furthermore, long-term follow-up studies reflect nutritional practices many years ago, which may be less relevant for contemporary populations. Our study included relatively healthy preterm infants who were not breastfed, yet there is increasing data to show a persisting benefit of breastfeeding after discharge in preterm infants. The associations between infant growth and later cognition are important, but they are observational in nature and may be biased by residual confounding or due to reverse causation, for example, by genetic factors. Our small group sizes precluded analysis by sex, even though the addition of sex into the model did not change the results.In summary, preterm infants are frequently discharged from the neonatal intensive care unit on a weight centile lower than that at birth, but feeding formula enriched with macronutrients compared to standard milk formula does not appear to improve long-term cognitive outcome in otherwise healthy infants. The strong associations between infant growth and later cognition are important and suggest that nutritional exposures and growth in the first year of life require closer attention.Supplementary Table 1


Processes that disrupt the distribution and transport of mitochondria along axons will likely give rise to peripheral neuropathies. Axonal transport of mitochondria has been extensively studied in a range of mouse models of CMT, along with changes to neurite networks and neuronal excitability. In fact, mouse models have been generated for most CMT-causing genes and have helped reveal a number of the pathomechanisms underlying the disorder.Brain, Van Lent and co-workersMore recently, the generation of patient-derived iPSC (induced pluripotent stem cell) lines and their differentiation into less accessible cell types such as neurons has become an established approach for studying disease mechanisms. Since 2015, several studies have been published where CMT iPSC lines have been derived from patient fibroblasts or peripheral blood mononuclear cells.et al.R94Q, NEFLP8R, HSPB8K14N, HSPB1G84R and HSPB1P182L—including those that cause the most common axonal forms of the disorder, as well as from two healthy controls. An isogenic control line was also generated by correcting the MFN2R94Q mutation via CRISPR/Cas9 mutagenesis, enabling natural variability to be filtered out. All iPSC lines were differentiated into iPSC-derived motor neurons as well as sensory neurons.Van Lent NEFL-mutated motor neurons), Van Lent and colleaguesMFN2 and NEFL mutations showed an increase in burst rate, the other mutations led to a decrease in activity.After first confirming previously published gene and mutation-specific features in patient-derived motor neurons  moved from the cell body to the axon terminal (anterograde transport) and from the axonal terminals to the cell body (retrograde transport). This may explain why axonal transport defects seem to be a common disease pathway in CMT, and why targeting axonal transport may be beneficial for a broad range of genetic forms of CMT.MFN2 and NEFL mutant sensory and motor neurons, suggesting that common molecular targets are present in both neuronal types (New treatment approaches aim to improve axonal transport with HDAC6 inhibitors by restoring tubulin acetylation.al types . This il