Genome organization is critical for setting up the spatial environment of gene transcription, and substantial progress has been made towards its high-resolution characterization. The underlying molecular mechanism for its establishment is much less understood. We applied a deep-learning approach, variational autoencoder (VAE), to analyze the fluctuation and heterogeneity of chromatin structures revealed by single-cell imaging and to identify a reaction coordinate for chromatin folding. This coordinate connects the seemingly random structures observed in individual cohesin-depleted cells as intermediate states along a folding pathway that leads to the formation of topologically associating domains (TAD). We showed that folding into wild-type-like structures remain energetically favorable in cohesin-depleted cells, potentially as a result of the phase separation between the two chromatin segments with active and repressive histone marks. The energetic stabilization, however, is not strong enough to overcome the entropic penalty, leading to the formation of only partially folded structures and the disappearance of TADs from contact maps upon averaging. Our study suggests that machine learning techniques, when combined with rigorous statistical mechanical analysis, are powerful tools for analyzing structural ensembles of chromatin. Chromatin folding, the dynamical process during which chromatin establishes its three-dimensional organization for proper function, is of critical importance. However, it is difficult to visualize and characterize due to challenges associated with live-cell imaging at high temporal and spatial resolution. Here, using a combination of deep learning and statistical mechanical theory, we demonstrate that great insight can be gained into the folding process by analyzing snapshots of chromatin structures taken across a population of cells. Though these static structures are not connected in time, prior research on chemical reactions suggests that fluctuation within the conformational ensemble provides valuable information for uncovering the reaction mechanism. Our analysis reconciles the seemingly contradictory results from different experimental techniques and supports the presence of multiple factors in organizing the chromatin. As single-cell experimental data are becoming routine, the approaches presented here could help with their interpretation to provide more insight into chromatin folding. Three-dimensional genome organization is expected to play a crucial role in transcription, DNA replication, and repair –5. SigniHi-C and related techniques have also provided insight into the dynamical folding process for the establishment of genome organization. In particular, the extrusion model was proposed to explain numerous features of chromatin loops and TADs observed in Hi-C contact maps , 17. It Many questions on genome folding remain outstanding and necessitate the development of additional experimental techniques and theoretical tools of interpretation. Recently, Zhuang and coworkers applied a super-resolution tracing method –34 to chHere we combine deep learning techniques with statistical mechanical tools to investigate the mechanism of genome folding. Specifically, we apply the variational autoencoder (VAE) , a deep In Ref , Zhuang We applied the deep learning framework, VAE, to carry out the dimensionality reduction for an ensemble of chromatin structures from both WT and cohesin-depleted cells. Compared to existing approaches, VAE not only compresses the data into a low-dimensional space with non-linear embedding, but also produces a deep generative model for estimating the statistical probability of each configuration –42. ThisAs shown in The biological significance of the one-dimensional coordinate is evident from its correlation with the fraction of TAD contacts , which iTo more closely examine the relationship between the VAE coordinate and TAD formation, we characterized the variation of average distance maps along the VAE coordinate. These maps were determined using chromatin structures from either WT or cohesin-depleted cells. The number of cells at various values of the folding coordinate are listed in e.g., RCAN1 and KCNE1) within this region and may impact their expression profiles.As shown in Remarkably, for cohesin-depleted cells , variatioff-pathway configurations at values less than -1.6. In these cells, chromatin exhibits a single domain at the end of the genomic region with a boundary quite different from that of WT cells. This domain must unfold before chromatin can transition into WT-like structures.Close examination of the distance matrices reveals additional subtlety of chromatin folding in cohesin-depleted cells. In particular, though both share similar TAD boundaries, the folded chromatin structures in cohesin-depleted cells are less compact and do not exhibit fine sub-TADs as those from WT cells. In addition, the VAE coordinate also uncovers folding coordinate in the following. It provides a fresh perspective on the heterogeneity intrinsic to single-cell imaging data , by evaluating the potential energy differences in the Cartesian space. As shown in PVAE(Q), and the contact energy, U(Q), is much worse ], favors unfolded chromatin configurations with negative folding coordinate values for cohesin-depleted cells. However, its difference from the homopolymer free energy introduced in the previous section, Therefore, both the tests on PVAE(Q)], shown in We note that the free energy, −log have introduced an approximate expression the mixing entropy as S(qo) = ΣijQij(qo) log[Qij(qo)] + (1 − Qij(qo)) log[1 − Qij(qo)]. Qij(qo) denotes the average contact probability between pairs i and j computed using all chromatin structures with a folding coordinate of qo. Using the above expression for S(qo), we computed F(qo) (A) and the corresponding probability distribution Figs (TIF)Click here for additional data file.S10 FigHere we show that the results obtained from processing the imaging data at 90kb resolution with a binarization cutoff of 400 nm are comparable to those shown in Figs (TIF)Click here for additional data file.S11 FigHere we show that the results obtained from processing the imaging data at 30kb resolution with a binarization cutoff of 300 nm are comparable to those shown in Figs (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The palpable heartbeat requires synchronous activation, and inactivation, of trillions of motors, compartmentalised in billions of cardiomyocytes, in a fraction of a second. To bring the biophysical concepts of myocardial contraction, in health and disease, closer to the practicing cardiologist we draw parallels with motor vehicles to discuss both recent advances describing how the heart modulates itself and a new era of myocardial therapeutics.Contraction requires two proteins, myosin and actin. Myosin (MYH7) is the molecular engine of the heart, converting the chemical energy of ATP into movement. Force production at the level of the cardiomyocyte occurs when millions of myosin engines combine, pulling each of ~50 sarcomeres per heart cell ~10% of their resting length closer . Mechani-1). Flexibility requires variable recruitment of myosin motors. Therein lies the challenge of regulation. If output is a numbers game, how do you pack a cell full of engines sufficient for a burst of activity but allow the whole system to idle most of the time? How do you hard-wire changes needed for prolonged elevations in activity like pregnancy, while retaining the ability to handle variations in pre-load that come from breathing? Distinct control mechanisms  are needed.The way myosin works dictates how the heart regulates force production. Myosin takes fixed steps (~10nm), that generate small amounts of force (picoNewton range), using an ATP hydrolysis mechanism that is slow (10s2+) housed in the thin filament troponin complex. This permits the interaction between motor and road in high systolic (1.0μM) calcium, but disengages it in low (0.1μM) diastolic calcium  in contractility are required. These molecular gears ensure durable changes in cardiac output, but are too slow for beat-to-beat control .The molecular accelerator/brake which underpins the Frank-Starling mechanism is housed in myosin itself. Myosin cycles between OFF (cannot bind actin), ON (can bind actin) and ACTIVE states . In diasHypertrophic cardiomyopathy (HCM) is an inherited disease, typically due to pathogenic variants of the thick (~70%) and thin filament (~20%). HCM cardiomyocytes expend more energy in systole and diastole. Since ATP consumption/force production comes from myosin, genetic changes facilitating longer or stronger interactions with actin, or which encourage more myosin heads to participate in any given contractile cycle, could cause this.MYH7 pathogenic variants reduce how much myosin is sequestered into the OFF state, or reduce sensitivity to the molecular signal coming from MYBPC3 (less brake) which promotes this transition [MYBPC3 pathogenic variants by contrast exert less influence on MYH7 (reducing the proportion in the OFF state) or bring thick and thin filaments closer together promoting actin/myosin interaction. Conversely some thin filament mutations increase the amount of Ca++, and the duration it is held, in the sarcomere [Most HCM-causing variants increase the proportion of ACTIVE myosin , resulting in both diastolic dysfunction and hypercontractility . MYH7 paansition . MYBPC3 A subset of inherited dilated cardiomyopathy (DCM) is due to pathogenic variants in the same myofilament genes, but with opposite molecular effects on contractility.Established negative or positive inotropes target the engine indirectly via the gears, without delivering a prognostic impact in various conditions aside from the proven, yet counter-intuitive, role of beta-blockade in chronic systolic heart failure. Controlling the engine directly via the accelerator/brake may be clinically useful, is now biologically plausible, and potentially free from some of the undesirable effects of existing inotropes, such as effects on heart rate.If most motors are idle, and only 10% needed for peak force, perhaps recruitment could be increased pharmacologically with clinical benefits in systolic heart failure? Conversely, in advanced systolic heart failure, if the control systems drive the engine to destruction, rather than push it harder, beta-blocker data might suggest a value of preventing the engine overheating by suppressing the motor? This hypothesis could be tested in HCM disease states which rev the engine continuously , and indSeveral broad classes of myofilament therapeutics exist. Myosin activators include Omecamtiv mecarbil. The phase III GALACTIC-HF trial in chronic systolic heart failure reported a modest reduction in heart failure hospitalisations without reducing heart failure deaths, or improving quality of life , while tThe contractile apparatus responds rapidly, and reversibly, to fluctuating pre- and after-load using molecular accelerators and brakes to control the engine at any particular gear. It is now possible to stimulate or suppress the engine directly with a novel class of myocardial therapeutics. This conceptual framework of clutch, gears, and accelerators or brakes should help clinicians understand and categorize future directions in sarcomeric therapies for systolic and diastolic dysfunction.


Diffuse gliomas are the most frequent brain tumours, representing 75% of all primary malignant brain tumours in adults. Because of their locally aggressive behaviour and the fact that they cannot be cured by current therapies, they represent one of the most devastating cancers. The present review summarises recent advances in our understanding of glioma development and progression by use of various in vitro and in vivo models, as well as more complex techniques including cultures of 3D organoids and organotypic slices. We discuss the progress that has been made in understanding glioma heterogeneity, alteration in gene expression and DNA methylation, as well as advances in various in silico models. Lastly current treatment options and future clinical trials, which aim to improve early diagnosis and disease monitoring, are also discussed. A brain tumour is formed when there is an abnormal clonal growth of cells within the brain. Brain tumours can be classified into malignant (cancerous) and benign (non-cancerous). Malignant tumours can be primary or secondary; the former originate from cells within the central nervous system (CNS), while the latter arise from cancer cells, which have metastasised from other tissues of the body to the brain via haematogenous spread. Two main cell types compose the CNS: neurons, which act as the communicators of the nervous system, and glia , which provide support and protection to neurons, maintain homeostasis and form myelin sheaths around them . In the Gliomas are the most frequent (75%) of all primary malignant brain tumours in adults ; becauseThe revised 2016 WHO Classification of Tumours of the CNS divides gliomas into low grade glioma  and high-grade glioma  based on integrated classic histological features and molecular biomarkers [Of all adult primary brain tumours, glioblastoma , has classified brain tumours based on cell type and grading (grades I–IV), using histopathological criteria which refer to similarities with putative cells of origin and presumed levels of differentiation . The pleIDH1 and 2), wildtype (non-mutant) versus mutant (IDH mutations are thought to occur early on in the genesis of a glioma [IDH1 Arg 132 (R132H). The mutated IDH enzyme reduces α-ketoglutarate (α-KG) to D-2-hydroxyglutarate (D-2HG) [In the WHO classification of 2016, “diffuse gliomas” (Grades II–IV) are classified in two groups based on a hallmark genetic feature, the mutational status of the isocitrate dehydrogenase genes (s mutant . IDH muta glioma  with the (D-2HG) . D-2HG i (D-2HG) ,16, poss (D-2HG) .IDH gene [TERT) can also be found within this group [90% of glioblastomas (grade IV), termed as primary or de novo, and roughly 30% of diffuse and anaplastic astrocytomas  contain the wildtype IDH gene ,19. Furtis group ,21, resuis group . After tis group . IDH-wilis group ,24.Seventy percent of diffuse and anaplastic astrocytomas (grades II-III), 100% of diffuse and anaplastic oligodendrogliomas, and 10% of glioblastomas ,19 are IATRX mutations to the tumour neurobiology has not been established to date, although there is some evidence to suggest it has a role in cell cycle regulation, histone regulation, and chromatin remodelling [The second group refers to the majority of grade II–III gliomas that carry mutations in the transcriptional regulators ATRX and TP53 but no 1p19q codeletion, encompassing the diffuse or anaplastic astrocytomas (II–III) and secondary glioblastomas (IV) . Decipheodelling .IDH mutations and 1p19q codeletion status) and phenotype as diagnostic criteria, most  tumours of this group fall into the category of oligodendroglioma or astrocytoma [IDH-mutant, ATRX-mutant, 1p/19q-intact) and oligodendroglial  mixed populations of tumour cells [A major achievement in the WHO classification of 2016 is the  omission of the previously ill-defined diagnoses of oligoastrocytoma and anaplastic oligoastrocytoma. By using both genotype , or how tumour cells overcome contact inhibition ,40. HoweThe use of cerebral organoids is becoming a more attractive approach when investigating human glioma biology. They can be derived from human pluripotent stem cells including human induced pluripotent stem cells (iPSC) and human embryonic stem cells (ESC) . CerebraCerebral organoids are a more complex and physiological system to study brain cancer compared to other in vitro and in vivo models. Cell lines and patient-derived tumour xenografts (PDTXs) used to study tumorigenesis have their limitations: genetic changes acquired during the in vitro cultures and two dimensional (2D) grown might make the findings obtained by using these methods unreliable; PDTXs models are expensive and require long term studies . CerebraG12V-IRES-tdTomato construct into the TP53 locus by homologous recombination using CRISPR-Cas9 technology, Ogawa et al. were able to cause simultaneous disruption of TP53 and gain of function of HRas proteins, generating tumour formation. Organoids positive for tdTomato reporter were shown to exhibit a greater proportion of proliferative cells positive for the marker Ki67, when compared to controls represented by untransduced organoids. Flow cytometry showed that at 8 weeks post-electroporation, 5.7% of the organoid culture was composed of tumourigenic cells (tdTomato positive), which greatly increased to 86.8% at 16 weeks post-electroporation. Tumourigenic cerebral organoids also demonstrate the ability to proliferate and invade otherwise healthy organoid [Recently, cerebral organoids derived from human ES cells following the protocol first described by Lancaster  have beeorganoid . This stOther studies have employed similar methods to study the invasive behaviour of multiple patient-derived GBM samples. Krieger et al. describe their use of human iPSC-derived cerebral organoids as scaffolds for the study of growth and invasion of GBM samples . The fluGJA1) encoding connexin-43, which allows gap junctions to form between GBM microtubes; Collagen Type IV Alpha 5 Chain (COL4A5) which encodes for a component of the extracellular matrix, and Glypican 3 (GPC3), an activator of the Wnt signaling pathway previously associated with invasiveness. This data demonstrate how co-culture of patient-derived GBMs with iPSC-derived cerebral organoids provides the cellular interactions necessary to induce a change in the transcriptome towards invasion. This mixed-organoid model and scRNA-seq also describes the heterogeneity between different patients’ tumours and provides a way to test the individual tumour’s susceptibility to targeted drug therapies on a clinically relevant time scale.Recently, Krieger et al. have investigated differentially expressed genes in GBM cultures in combination with/without normal organoid cultures derived from iPSC. By using single cell RNA-sequencing (scRNA-seq), they identify a subset of genes that were differentially expressed in patient samples that had been co-cultured with healthy organoids compared to patient samples with no co-culture or healthy organoids cultures alone. This gene set was upregulated across all GBM samples that had been co-cultured and included: Gap Junction Protein Alpha 1  genetically engineered mouse models (GEMMs) which involve altering specific genes known to influence glioma formation and progression and (2) xenograft models which utilize patient-derived cells/tumour fragments. Xenograft models are generated using either glioma cell lines or patient tumour samples cultured in vitro and later inserted directly into the mouse brain to induce tumour formation, or by injecting small fragments of human gliomas directly into the brain, thus bypassing the need for culturing the samples, which could lead to the loss/acquisition of genetic alterations due to adaptation of tumour cells to culture conditions .In genetically engineered mouse models (GEMMs), specific genetic alterations identified in human tumours are introduced in the germline  or in the somatic cells  of a mouse model to generate a de novo tumour. In these models, the genetic alterations of interest are targeted to a specific cellular population within the mouse tissue/organ, and to a specific developmental stage of the animal . GEMMs represent an important experimental tool to investigate the cellular and molecular events responsible for tumour initiation and progression, and to determine the minimum genetic alterations necessary for malignant transformation. Another important advantage represented by GEMM is that they have been established in immunocompetent animals, highlighting the importance of the microenvironment in tumour biology. GEMMs have been used for testing biomarker expression, drug delivery methods and new therapeutic strategies, and many studies using GEMMs have been successfully translated into clinical trials.There are numerous glioma GEMMs some of which are reviewed here .+/− and Trp53+/− to produce a double heterozygous mouse. This model produced mostly low-grade diffuse astrocytoma (WHO grade II), which in some cases progressed to a grade IV glioblastoma [Ras, EGFR, and Akt among others [A model of astrocytoma was developed by Reilly et al. by crossing two mouse strains, Nf1blastoma . There hg others . Some mog others . GEMMs og others . These dAlthough GEMMs are useful tools in understanding how acquisition of mutation(s) cause tumour formation, they also present some limitations: the genetic alterations generated are limited to a number of genes, thus they cannot fully represent the genetic complexity and heterogeneity that is often seen in resected human glioma samples. Moreover, their development is normally costly and time consuming.In a patient derived xenograft (PDX) model, human gliomas in the form of tumour cell models or en bloc biopsies are injected or transplanted into an immunodeficient mouse to prevent rejection of the transplant from the host. Several strains of mice with different levels of inhibition of the immune system can be used in these studies: athymic nude mice, in which T-cells populations are greatly reduced; severely compromised immune deficient (SCID) mice, in which both B and T cells are lacking; NOD-SCID mice, in which there is an impaired development of B and T lymphocytes and natural killer (NK) cells; and recombination-activating gene 2 (Rag2)-knockout mice, in which both the maturation of both B- and T-cells is impaired. In these mice, the tumour sample can be implanted heterotopically, into an area of the mouse unrelated to the original tumour site  or orthotopically, into the site of origin of the tumour . Tumours generated in xenograft models grow relatively quickly and retain the cellular and molecular characteristics of the patient’s tumour. Preservation of the intra-tumoural genetic heterogeneity allows an accurate representation of the sample of origin. Furthermore, PDX have the advantage of developing an in vivo vascular supply. For these reasons, they represent a useful clinical model to test the response of tumour cells to therapy, albeit for a limited number of in vivo passages in the mouse. After which a selective pressure for propagation starts that causes specific genetic and epigenetic signature changes in the cells, making the generated tumours less representative of the original tumour from the patient .In a heterotopic xenograft model, glioma cells are injected in a tissue where they will not experience the same stimuli and cell-cell interactions present in the microenvironment of the tissue of origin. Brain tumour samples will also lack input from brain-specific immune factors, such as microglia and macrophages, which can both affect and be effected by the tumour cells ,74. To rRecently, a particular xenograft model using patient-derived glioblastoma cells was generated by implanting cells via intravitreal injection into the mouse eye . This moXenograft models using human tumour samples can provide an excellent insight into human glioma biology, however these models rely on immunodeficient murine hosts. Given the more recent interest in the role of the immune system in cancer progression, these xenograft models present some limitations, as they cannot fully recapitulate the ‘true’ microenvironment in which immune and cancer cells interact.In ‘humanized’ NOD/SCID mice models a functional human immune system is generated, through engraftment with human primary hematopoietic stem cells (HSCs) typically derived from human foetal tissues, umbilical cord blood, or peripheral blood mononuclear cells (PBMCs). To facilitate engraftment, the human leucocyte antigen (HLA) of HSCs and PBMCs should be matched to the tissue being xenotransplanted . Human gRecently, studies of glioma samples transplanted into mice with intact immune systems have started to emerge. Semenkow et al. provided evidence of an immunocompetent mouse model that was able to more faithfully resemble the pathophysiology seen clinically in patients with GBMs, compared to transplants grown in immunodeficient hosts . To faciIntra-tumour heterogeneity describes a phenomenon in which different cells within the same tumour acquire distinct epigenetic and genomic alterations. This allows development of dissimilar morphological and phenotypic features and consequent divergent development from the progenitor cancer cell population. Intra-tumour heterogeneity has been proposed to be one of the major obstacles for conventional and targeted therapies to maintain remission ,88,89,90EGFR, PDGFRA, and MET using fluorescent in situ hybridisation (FISH) [PDGFRA, MDM4, and AKT3 loci, and deletion of the genomic locus containing PTEN. Furthermore, each tumour copy number alternations (CNA), were classified as ‘common’, ‘shared’, and ‘unique’, i.e., all tumour fragments, more than one but not all fragments, and only one fragment, having CNA respectively. The vast majority of CNAs (31/38), showed ‘shared’ and ‘unique’ alterations. It was deduced that ‘common’ aberrations should have occurred earlier in development, as all tumour fragments (four fragments per tumour) were extracted from the same tumour mass. While ‘unique’ and ‘shared’ alternations found in a subset of tumour cells should have occurred later on in the development. Therefore, it was implied that taking samples from multiple sites within the tumour, enables both spatial and temporal interrogation of mutations. ‘Common’ CNAs were found in chromosomes 7 and 10, of which the most frequent putative glioblastoma drivers, such as EGFR, CDK6, MET, and PTEN, are located. Using microarrays and hierarchical clustering, it was also found that distinct glioblastoma subtypes are present within the same tumour. By measuring the mitotic distance between cells within a fragment, they also demonstrated that different subclones exist within each fragment, and also that distinct cell lineages are present, illustrating a complex and dynamic hierarchy. The authors concluded that, the founder clone initially displayed amplification/gain of EGFR, CDK6, and MET, and loss/deletion of CDKN2A/B, PTEN, and PARK2. Later clones displayed altered gene expression and loss of chromosomes. The variation in gene expression in tumour development suggests that a single therapy is highly unlikely to be effective for each subclone, meaning that while it might be effective for one clone, the rest of the tumour will be able to survive by either having inherent resistance [Previously, intra-tumour heterogeneity was demonstrated at the level of n (FISH) . Sottorin (FISH) . This wan (FISH) . Thirty-sistance  or acquiEGFR truncations and in frame deletions (EGFRvIII). Additionally, using hierarchical clustering and principle component analysis four meta signatures  were comprised of clusters, which vary amongst the whole set of cells, enriched by genes related to each meta-signature. Quiescence markers were found to be significantly higher in non-cycling tumour cells. Clustering of genes anti-correlated to the cell cycle signature, 9/12 were within the hypoxia meta-signature. Reordering of the cells by hypoxic module score, showed clear gradients for two clusters (MGH28/31), implying that hypoxia as a factor of tumour microenvironment can play a role in gene expression. This finding emphasised previous findings reporting tumours containing niches with phenotypic changes including quiescence and self-renewal [A recent study by Patel et al., used scRNAseq to measure intra-tumour heterogeneity in patients affected by primary glioblastomas. Six thousand genes were analysed from 430 cells isolated from each tumour; this constitutes adequate coverage to characterise heterogeneous gene expression and delineate correlation between transcriptional and functional expression . Extensi-renewal  and adap-renewal . Finally-renewal . They id-renewal . MoreoveFailure of clinical trials to show survival benefit for “diffuse glioma” is well known ,98. The In a recent study, the critical differences in gene expression between IDH mutant astrocytomas (A) versus oligodendrogliomas (O) were deciphered, by combining 9879 single cell RNA-seq profiles from 10 IDH-A tumours, 4347 single cell RNA-seq profiles from 6 IDH-O tumours, and 165 TCGA bulk RNA profiles . DiffereTP53 mutated in astrocytomas has been shown to influence the nuclear factor kappa beta NF-kβ immunoregulatory pathway [A greater macrophage-like expression was found in tumours of higher histopathological grades (II vs. III vs. IV) and this was correlated with increased endothelial cell signature. TME composition might be partly influenced by tumour cell gene expression, i.e.,  pathway . In contThe TME plays an important role in gliomagenesis and may influence the tumour evolutionary process. Unsupervised transcriptome analysis of the TCGA data revealed four clusters of glioblastoma subtypes: classical (CL), mesenchymal (MES), neural (NE), and proneural (PN), each linked to specific genomic alternations . MES is Tumour-associated macrophages (TAM) and microglia are innate immune cells that can make up to half of the tumour mass in glioblastoma. Brain TAM and microglia retain plasticity and exist in a spectrum of different cellular states classified according to their environment: M1 proinflammatory and M2 cytoprotective . During + T cells [+ T cells were found to be significantly increased at recurrence in comparison with their primary tumours [The type of oncological treatment can also be affected by the TME. It has been postulated that TMZ treatment can induce aberrant mutations ,118. The T cells . CD8+ T  tumours . Hypermu tumours . Further tumours . Therefo tumours ,123.Glioblastoma metastasis outside the brain are rare , while tInvasion of glioma cells into normal tissue is a complex process, which is determined by complex interactions between glioma cells and the extracellular microenvironment (ECM); this involves expression and activity of proteolytic enzymes, such as matrix-degrading proteases (MMPs) and cathepsins to degrade ECM components, as well as interactions of tumour cells with normal astrocytes . By usinThe permeability of the blood–brain barrier (BBB) presents one of the biggest barriers in the design and testing of novel brain cancer therapies. The BBB is comprised of endothelial cells that are held together by tight junctions which is what gives the capillaries of the brain such low permeability . This laWang et al., made use of molecular dynamics (MD) simulations to simulate the bilayer of human brain microvascular endothelial cells (hBMECs), which forms the outer layer of the BBB . This moDisruption of the BBB in order to deliver chemotherapeutic agents to the brain is a potential avenue of study when considering how best to get drugs into the brain. The tight junctions of the BBB make the process of drug delivery difficult and so Goliaei et al. presented a molecular dynamics simulation using ultrasound and nanobubbles to disrupt the integrity of the BBB . To do tPrabhakar and Banerjee, have since gone on to demonstrate the biological potential of these nanobubbles in delivering drugs to cells in vitro. By combining nanobubbles, liposomes and the chemotherapy drug, paclitaxel (PTX) they show that bursting nanobubbles with ultrasound lead to a much higher percentage of PTX being released compared to the use of nanobubbles without ultrasound. They also demonstrated that the combined use of PTX-containing nanobubbles and ultrasound caused a much higher cytotoxicity in a variety of cell lines than those without ultrasound . The useClonal evolution is one of the main processes that drives tumour progression. The emergence of heterogeneous clones within a tumour is caused by the genetic, epigenetic, and microenvironmental selective pressure that tumour cells experience during cancer progression. Clonal evolution is also one of the major causes of therapeutic resistance to anticancer agents. Understanding the evolutionary course of an individual patient’s cancer is likely to be essential in deciding their optimal treatment plan and diagnosis and subsequent points of progression or recurrence.In recent years, multi-region sequencing has allowed researchers to discover the temporal sequence of some genomic changes within a tumour, however the robust identification of repeated evolution across patients remains a challenge. Recently, Caravagna and co-authors have developed a new analysis tool, named REVOLVER (Repeated EVOLution in cancER), that utilises a machine learning approach named ‘transfer learning’ to jointly analyse multi-region sequencing data . This noDrug resistance and tumour recurrence can also be caused by clonal evolution induced by the selective pressure during therapy. Small populations of genetically distinct subclones present in a tumour can evolve under the pressure of drug treatment, resulting in the selection of the more ‘fit’ subclones. These subclones may persist after treatment and repopulate the tumour over time, leading to disease recurrence. Resistant subclones are likely to be generated in response to therapy in two different ways: (1) drug therapies induce genetic changes in a small number of cells, generating mutant subclones, which are preferentially augmented during subsequent treatment; (2) therapy resistant subclones generated during tumour progression, are present pre-treatment and continue to thrive despite further therapy. In some cases, such as metastatic breast cancer, leukaemia, non-small cell lung cancer as well as glioblastoma , it has Mathematical modelling of the evolution of drug resistance is of great clinical benefit as it could identify resistant subclones within a population that have resistance against certain therapies and therefore inform different clinical decisions. Evolutionary herding is the use of drugs that select for specific phenotypes, this can present differential therapy courses depending on each patient’s tumour heterogeneity. Each drug will produce ‘fitness peaks’, where cell populations unaffected by that drug will cluster, and ‘fitness valleys’ where affected cell populations will cluster. Combination therapy that selects for overlapping fitness peaks will select for multi-drug resistant cell populations. The idea behind evolutionary herding is for one drug to select for a cell population that falls into one fitness peak that forms a fitness valley under the influence of a second drug—thus leading to the elimination of the cancer cell population ,142.IDH1 or IDH2 mutations combined with the loss of both 1p/19q chromosomal arms. It is a relatively slow growing tumour that mostly affects younger patients, has a longer median survival time and has a better response to alkylating agents in comparison to the other diffuse gliomas [Oligodendroglioma is characterised by either  gliomas . Despite gliomas . Data frThe use of computational modelling for drug design can provide more precise insight into the structure and chemistry required from a new compound. This can help to eliminate time spent in the lab optimising hundreds of potential compounds. Virtual drug screens can be conducted using quantitative structure-activity relationships (QSAR) and ligand-based virtual screens (LBVS) .QSAR models have been previously utilised in the design and virtual testing of new EGFR inhibitors, which could provide future treatments for EGFR-amplified glioma. The study conducted by Zhao et al. employed 2D-QSAR to first ascertain whether or not a series of potential drugs would be classed as EGFR inhibitors . The 2D-DNA methylation occurs as a covalent transfer of a methyl group onto one of the DNA nucleotide bases, either as a targeted modification via DNA methyltransferases or as an effect of DNA damaging agents . MethylaGliomas exhibit several oncogenic pathways involved in tumour onset, progression and therapeutic resistance, and like other tumours their initiation and progression is associated with epigenetic abnormalities . Hence tIDH mutations occurs most commonly at position R132 and the most common mutation in glioma results in a change to R132H. The result of this mutation is neomorphic enzyme activity that instead of producing α-ketoglutarate produces 2-hydroxyglutarate (2-HG) [e (2-HG) . 2-HG ace (2-HG) .IDH mutant brain tumours have focused on understanding the pathways and mechanisms involved in IDH-related gliomagenesis, and in particular the role of the epigenetic modifications and effects on IDH mutant gliomas. A review by Park and Turcan [Multiple studies conducted on d Turcan , discussmut cancers all showed hypermethylation when compared to their IDHWT counterparts. IDHmut glioma also demonstrate the highest amount of genome hypermethylation in comparison to the other cancer types tested, with 19% of CpG sites methylated compared to 2–3% for the other cancers [WT and IDHmut gliomas (~4200 genes) in comparison to the other cancers tested (~135–420 genes). IDHmut gliomas were also enriched for sets of genes involved in the tissue development pathway as well as the immune response, which may explain why IDHmut gliomas are often poorly differentiated. The effect of IDH mutations in glioma methylation changes may be influenced by the differentiation state of the cell that the mutation occurs within. Introducing IDH1R132H into both differentiated human astrocytes and neural progenitor cells showed that undifferentiated NPCs were more susceptible to hypermethylation than differentiated astrocytes [mut may not produce reproducible gains across all IDHmut cancer types.Mapping of CpG sites across multiple cancer types including glioma, melanoma, cholangiocarcinoma, and acute myeloid leukaemia revealed that IDH cancers . These ttrocytes . AlthougWT primary GBMs. De Souza et al. proposed that a small population of cells within IDHmut primary gliomas may already have a G-CIMP low phenotype and thus contribute to the tumour’s recurrence as the more aggressive and therapy resistant phenotype [IDH mutations in glioma produces a unique methylation pattern termed Glioma CpG Island Methylator Phenotype (G-CIMP) . LGGs cahenotype .The methylation shift data presented  indicate6-mA). The N6-mA modification has been demonstrated in bacteria and mammals but was only first identified in the human genome very recently [6-mA specific antibody the existence of the modification in primary human glioblastoma as well as in validated patient-derived glioblastoma cancer stem cell models was proven [6-mA modification and when this enzyme is inhibited, tumour formation decreases, along with the self-renewing capacity of the glioma stem cells. Knockdown of ALKBH1 led to a downregulation of genes involved in the hypoxia response indicating that targeting of ALKBH1 could have clinical benefit. Like the N6-mA modification itself, ALKBH1 was highly expressed in human glioblastoma when compared to the healthy brain tissue indicating that therapeutic targeting of this enzyme could more specifically target the glioma cells over the healthy cells. The presence of high ALKBH1 levels was correlated to higher glioma grades as well as poorer survival [Whilst the methylation of cytosine bases is most commonly associated with epigenetics, another form of DNA modification exists, the methylation of adenine bases at the secondary amine  or computer tomography (CT) scans. All these techniques however have their limitations; overall the diagnostic process requires time, a lot of expensive equipment and stress and in case of tissue biopsies, it could also confer a risk of brain swelling and haemorrhages to the patient. Liquid biopsies in gliomas are a minimally invasive technique, which have great potential in all aspects of glioma management. From detection to monitoring tumour response to therapies and early recurrence. Potential biomarkers may be identified to measure disease progression. Importantly, liquid biopsies can be obtained regularly over time and can provide up to date information about the molecular heterogeneity, and longitudinal genomic evolution of the tumour from diagnosis through progression/recurrence.Many different biological materials can be sampled from liquid biopsy of human body fluids including: circulating tumour cells (CTCs); circulating cell-free DNA (cfDNA) which in cancer patients contains circulating tumourDNA (ctDNA); circulating cell-free tumour RNA (ctRNA) containing mRNAs and mainly small RNAs; extracellular vesicles (EVs); proteins, metabolites, and tumour-educated platelets . In gliWT patients having more cfDNA than IDHmut, which they explained as being down to the more aggressive nature of IDHWT tumours and their associated increase in BBB breakdown. Noushmehr H. et al., found that there was a genome wide change in methylation status of glioma patients compared to healthy tissues. This led them to test whether other CpG methylation sites could be detected in cfDNA. They found that not only was there a detectable serum methylome profile but that it was distinct from that of other neoplasms. The analysis of cfDNA methylome also showed that it was possible to decipher the IDH status of the tumour from the serum methylome. A simple blood test capable of identifying a glioma over other neoplasms and also being able to identify the IDH status may provide a non-intrusive way by which patients can be stratified early on in the treatment process without having to undergo invasive biopsies or time-consuming scans.Recently Noushmehr H. et al., provide evidence of how a simple blood biopsy, capable of detecting cell-free DNA (cfDNA), is important for patient stratification . They obRecently, increasing interest in developing tests from cerebrospinal fluid (CSF) has been developed, since this fluid is produced in the choroid plexus of the brain and would have direct access to any waste products such as cfDNA . The useGlioma surgery has been revolutionised over the last decade, due to the combination of significant advances in brain tumour imaging, intraoperative technologies and neurosurgical techniques. The philosophy of treatment, that safe, maximal glioma resection improves symptom management, quality of life, and both progression free and overall survival has also become less nihilistic. Currently, the relationship between the extent of resection (EOR) of both LGG and HGG, and clinical outcomes remain incompletely understood. There is no validated metric to quantify EOR and randomized clinical trials are impractical, which hinders achievement of level-one evidence for EOR. However, a growing body of clinical data supports the prognostic importance of gross total resection in LGG and HGG , and is Numerous intraoperative tools have been developed and evaluated, aiming to enhance the neurosurgeon’s ability to identify tumour boundaries and augment resection, while simultaneously preserving eloquent brain function.Fluorescence guided surgery allows real-time intraoperative identification of residual tumour. The best studied intraoperative fluorescence imaging technology is 5-aminolevulinic acid (5-ALA), which is used in HGG . Oral adCortical functional organization varies considerably between patients, tumour mass effect may distort anatomic relationships and cortical plasticity may result in reorganization of neural networks. Intraoperative neurophysiological/electrical stimulation mapping and monitoring either during awake craniotomy or under general anaesthesia, aims to reliably and reproducibly identify and localize the cortical areas and subcortical pathways involved in language, motor, sensory, and cognitive function and preserve these functions during tumour resection . MeRecent advances in intra-operative neurosurgical imaging including intraoperative neuronavigation, intraoperative MRI (iMRI), and intraoperative ultrasound (iUS) have significantly enhanced the potential to achieve maximal and even supra-maximal glioma resection ,178. IntFollowing surgery, diffuse astrocytoma and oligodendroglioma patients are stratified into low risk and high risk. It is worth noting that a definition for high-risk and low-risk low grade gliomas is highly variable . PatientThe standard of post-operative care for high risk high-grade gliomas is based on results from three phase 3 trials (20–22) and interim result from the CATNON trial. This includes radiotherapy (60 Gray) with either six cycles of adjuvant PCV or concurrent plus adjuvant temozolamide (TMZ). The interim analysis of CATNON trial showed that non 1p19q codeleted anaplastic astrocytomas can benefit from 12 cycles of TMZ as well. The CODEL trial is currently investigating whether TMZ can replace PCV in the treatment of 1p19q codeleted oligodendrogliomas. Further, the POLCA trial is currently investigating whether radiotherapy for 1p19q codeleted oligodendrogliomas can be replaced by PCV only. For glioblastoma, radiotherapy of 60 Gray for six weeks with concurrent daily TMZ followed by six cycles of TMZ has been the standard of care since 2005 . AdditioFor glioblastoma patients older than 70 years of age the Nordic  and the Current systemic delivery of chemotherapy in glioma patients does not reach a high concentration at target within the glioma or the CNS, and leads to systemic side effects such as myelosuppression. Novel drug delivery systems aimed at improved delivery both spatially and temporally within the brain are actively being developed. These including injectable nanoparticles, implantable controlled-release polymer systems, convection enhanced delivery using a catheter-based approach to an agent directly into the brain tumour or parenchyma and using positive pressure infusion and BBB disruption by pulsed ultra-sound (reviewed in ).Carmustine (BCNU) loaded wafers are FDA- approved and are placed along the surface of the glioma surgical resection cavity to deliver local chemotherapy for the following days to weeks. The hypothesis was based on the fact that local delivery of a chemotherapeutic agent will enable bypassing of the blood–brain barrier. Brem et al., has showed that usage of BCNU showed improvement in median survival (31 vs. 23 weeks compared to the placebo ). While MGMT promoter and with no baseline corticosteroid use. This suggests a potential clinical benefit from a therapy with immune checkpoint inhibitors for this subgroup of patients [In the past, the brain has been considered an immune-privileged organ, meaning the effects or at least the full effects of the immune system are not expected to occur. This has been based on the fact that no lymphatics exist and the blood–brain-barrier (BBB) prevents influx of immune regulators and cells. Though it has been increasingly recognised that this might be not entirely true. Importantly, it is appreciated that brain tumours are actively evading and suppress the immune system, by minimising expression of major histocompatibility complex (MHC) proteins, reduced cell activation (T-cell in particular), and expressing pro-apoptotic factors. Understanding the immune evading mechanisms has been an emerging and exciting new research field. Three main methods for overcoming immunosuppression have been in the forefront of research: (i) cytokine therapy, (ii) active immunotherapy, and (iii) passive immunotherapy. Cytokine therapy has mainly been focused on activating the immune system, hoping it will fight the tumour. Active immunotherapy has primarily been focused on priming the immune response against known tumour antigens by vaccination and adoptive T-cell therapies . Passivepatients . FurtherAnti-angiogenic therapies have also failed the initial expectations to enhance survival and quality of life of GBM patients. Only one agent, the anti-VEGF antibody bevacizumab, has shown some efficacy in controlled clinical trials so far. This efficacy is, however, limited to improve progression-free survival (PFS) but not the overall survival in patients affected by primary and recurrent glioblastoma . TargetiEGFR-amplified GBMs have shown limited efficacy. The INTELLANCE-2 is a randomised controlled phase II trial investigating, the efficacy and safety of depatuxizumab mafodotin  in patients with recurrent EGFR-amplified glioblastoma. Depatux-M is an antibody-drug conjugate, that targets cancer cells by linking the toxin monomethyl auristatin F (MMAF) with an antibody directed against the epidermal growth factor receptor (EGFR) or mutant EGFRvIII. In this trial the efficacy of Depatux-M was studied in combination with TMZ or as a single agent, versus TMZ or lomustine alone (NCT02343406). Data obtained from the study showed a clinical benefit of Depatux-M in combination with TMZ in recurrent EGFR-amplified glioblastoma, suggesting a possible role for the use of Depatux-M in combination with TMZ, especially in patients relapsing well after the end of first-line adjuvant TMZ treatment [For instance recent studies designed to target the EGFR pathway in patients with reatment . HoweverIDHs, huge efforts have been spent pursuing the identification of novel direct inhibitors of the mutant IDH. AGI-5198 has been reported as the first novel, synthetic, direct enzyme inhibitor of the IDH mutant enzyme [IDH mutant gliomas, impairing xenograft progression in vivo [https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm569482.htm). AG-221 was tested in a clinical trial for gliomas and other IDH mutant tumours in 2014, showing inhibitory effects for these tumours. However, appropriate dosing was an issue. Also, a number of clinical trials are underway, currently evaluating the efficacy and safety profile of AG-120 and AG-881 (NCT03343197). Since the discovery that IDH1 R132H is the most common mutation in ‘diffuse’ gliomas, more clinical trials have emerged . Recently, Agios Pharmaceuticals has conducted a multicentre clinical study on recurrent LGG with IDH mutation using an AG-120 and AG-881 (NCT03343197). The primary outcome was to compare the D-2-HG concentrations in surgically removed tumours, which were treated versus not treated with the drug inhibitors. Clinical safety, dosage, tolerance, and pharmacokinetics will also be studied. This safety and feasibility trial will provide appropriate dosing for future studies. It is worth noting that the new inhibitors exhibit a good CSF-plasma ratio [Since a huge percentage of low grade gliomas and secondary glioblastomas bear mutations in t enzyme . This dr in vivo . As repo in vivo  . Initially, a spontaneous immune response to the IDH mutation has been documented . The reswww.birmingham.ac.uk/brainmatrix) is a national platform study striving to optimise this strategy. The aim of BRAIN MATRIX is to test the hypothesis that comprehensive whole-genome sequencing and epigenomic profiling of gliomas is feasible in a timely manner in the UK, and that the results improve the stratification of patients for next generation (targeted) therapies, to ultimately improve clinical outcomes and quality of life. Furthermore, optimised fresh tumour sampling pipelines can be used to provide good quality tissue in order to create autologous vaccines and potential immunotherapies. Advanced trial techniques, such as adaptive designs, can make clinical trials more flexible by using results accumulating in the trial to modify the trial’s course in accordance with pre-specified rules. These techniques can shorten trials duration, obtain more precise conclusions however are more complex and have had success in other cancers notably lung cancer .The ability to deliver precision/stratified medicine represents a challenge in glioma patients. Future treatments will benefit from ‘fresh tumour tissue’ resected at surgery that is surplus to diagnostic requirement. The tissue must be suitable for whole genome analysis, epigenomics, transcriptomics, methylomics, and proteomics to allow identification of specific tumour targets, drug sensitivities, and immune modulatory factors. The British feasibility study of molecular stratification and targeted therapy to optimize the clinical management of patients with glioma by enhancing clinical outcomes, reducing avoidable toxicity, improving management of post-operative residual and recurrent disease and improving survivorship (The Tessa Jowell BRAIN MATRIX trial Future advancements in glioma treatments require a more integrated approach and close collaboration between patients, clinicians, scientists, and clinical trial units in order to incorporate translational research focusing on precision medicine and adaptive clinical trials. The use of single cell RNA-seq methods and a wider range of in vitro and in vivo models will allow the molecular events propagating glioma to be further elucidated. The role of the tumour microenvironment as well as that of normal brain cells requires further attention in order to understand how these brain-specific components influence the development, progression, and recurrence of glioma so that more comprehensive therapies can be developed. Aiding this, in silico drug design and testing allows us to quickly understand if a certain drug will pass the blood–brain barrier, a major hurdle in the development of systemically administered drugs, and whether a new drug will provide any therapeutic benefit. Our understanding of the molecular events during glioma onset and progression have greatly improved in recent years, sadly the clinical aspects of patient stratification care have not matched this. Greater co-operation between clinicians, scientists and clinical trials will, in the future, lead to more therapeutic options to combat this invariably lethal disease.


In this commentary we discuss new findings presented by Shang et al.regarding the role of macrophage-derived glutamine in skeletal muscle repair.Loss-of-function of glutamate dehydrogenase in macrophages led to anupregulation of glutamine synthesis which sustained glutamine levels in muscletissue and facilitated satellite cell proliferation and differentiation. Macrophages are cells of the innate immune system that play a critical role inthe regulation of inflammatory responses. Tissue resident macrophages contribute toorgan development and homeostasis, whereas monocyte-derived macrophages are recruitedupon tissue injury and coordinate tissue inflammation and repair. Up until recently, theinflammatory vs. reparative capacity of macrophages was often referred to as M1 or M2activation states. However, it is now recognized that this terminology fails to describethe true diversity and plasticity of macrophage subsets in vivo [invivo setting the availability and utilization of glutamine by macrophagesin homeostasis and disease is less well understood.Over the past decade it has been appreciated that macrophage cellular metabolismoften dictates cell activation and effector functions. As an example, inflammatorymacrophages are biased towards glycolytic metabolism whereas macrophages with reparativephenotypes tend to rely upon mitochondrial fatty acid oxidation ,4. HowevGlud1),GLUD1 KO, had improved resolution of tissue damage and earlier restoration of functionalcapacity compared to wild type (WT) mice. This occurred as a consequence of enhancedproliferation of muscle satellite cells. Thus, perturbing macrophage glutaminemetabolism enhanced muscle repair and regeneration.Skeletal muscle injury occurs in cases of trauma, muscular dystrophy, drugtoxicity and aging. Macrophages contribute to skeletal muscle regeneration via severalmechanisms including the release of cytokines that promote repair, such as IL-6 andTGFβ, and growth factors, including IGF-1, that can stimulate expansion of themuscle stem cells . In an eIntriguingly, macrophage recruitment and wound healing/angiogenic capacity weresimilar between the genotypes. Therefore, to understand the mechanism of this phenotypethe authors performed metabolic phenotyping of GLUD1 KO macrophages. As GLUD1 catalyzesthe conversion of glutamate to α-ketoglutarate for entry into the tricarboxylicacid (TCA) cycle it was not surprising that KO macrophages had a ~75% reductionin glutamine oxidation capacity. Intriguingly, the authors also demonstrated thatmacrophage glutamine production increased with the loss of GLUD1 and this was associatedwith an upregulation of the enzyme glutamine synthase (GS). Macrophage-specific KO of GSin GLUD1KO mice prevented the enhanced proliferation of muscle satellite cells thatoccurred with injury. Thus, loss of GLUD1 in macrophages promoted muscle regenerationvia a GS-dependent mechanism.To understand the potential relevance of enhanced glutamine production to thecrosstalk between macrophages and skeletal muscle cells the authors used an in vitroco-culture system. Glutamine is known to be important for myoblast proliferation. WhenWT macrophages were cultured with myoblasts in glutamine rich media the growth ofmyoblasts was diminished to levels observed under glutamine-restricted conditions. Incontrast, this did not occur when myoblasts were cultured with GLUD1 KO macrophagesirrespective of the glutamine quantity added to the media. This data suggested thatunder normal conditions macrophages take up extracellular glutamine and thereby reducethe amount of this AA that is available for use by myoblasts. In line with thisobservation, glutamine concentrations decreased in the muscle interstitial fluidfollowing injury in WT mice and this drop did not occur in macrophage GLUD1 KO mice. Assuch, macrophages appear to compete with satellite cells for glutamine following muscleinjury, influencing muscle regeneration.l-glutamyl-p-nitroanilide (GPNA). Together thesefindings confirm that the salutary effects of GLUD1 deficiency in macrophages isdependent on glutamine delivery to satellite cells. Interestingly, the authors alsoconfirmed a protective effect of macrophage GLUD1 deficiency on preservation of musclemass with aging, indicating possible applications of this concept outside of acuteinjury.These findings suggested a model whereby glutamine release from GLUD1 KOmacrophages enhanced glutamine availability and fueled muscle satellite cell expansion.To explore this possibility in more detail, the authors knocked out the primary receptorinvolved in glutamine uptake, SLC1A5, in satellite cells in vitro and in vivo using aCRISPR-Cas9 approach. When satellite cell glutamine uptake was inhibited, the beneficialphenotype observed in macrophage GLUD1 KO mice was lost, confirming that glutaminereleased from macrophages was driving muscle regeneration. Similar results were observedin GLUD1 KO mice treated with the SLC1A5 inhibitorg-Despite the elegant mechanistic work performed by the authors, the questionremained as to whether this pathway could be translated into therapeutics. Therefore,the investigators treated mice with the GLUD1 inhibitor R162 after muscle injury.Inhibition of this enzyme also improved muscle regeneration and satellite cellproliferation. Moreover, in aged mice R162 treatment for one month improved muscle massand exercise capacity. Thus, pharmacologic strategies targeting GLUD1 have promise forthe treatment of acute and chronic muscle injury.The study by Shang et.al. is an exciting addition to the field ofimmunometabolism. The authors likely anticipated that disrupting glutamine oxidation inmacrophages would have a direct effect on the macrophage polarization and thereby alterthe injury response. Instead, they uncover a novel pathway whereby the release ofglutamine from macrophages into the muscle microenvironment drove regeneration andhealing. Equally as exciting, this study provides compelling evidence that this pathwaycould be exploited for therapeutic purposes .


While each route of therapeutic drug delivery has its own advantages and limitations, oral delivery is often favored because it offers convenient painless administration, sustained delivery, prolonged shelf life, and often lower manufacturing cost. Its limitations include mucus and epithelial cell barriers in the gastrointestinal (GI) tract that can block access of larger molecules including Therapeutic protein or peptide-based drugs (TPPs), resulting in reduced bioavailability. This review describes these barriers and discusses different strategies used to modify TPPs to enhance their oral bioavailability and/or to increase their absorption. Some seek to stabilize the TTPs to prevent their degradation by proteolytic enzymes in the GI tract by administering them together with protease inhibitors, while others modify TPPs with mucoadhesive polymers like polyethylene glycol (PEG) to allow them to interact with the mucus layer, thereby delaying their clearance. The further barrier provided by the epithelial cell membrane can be overcome by the addition of a cell-penetrating peptide (CPP) and the use of a carrier molecule such as a liposome, microsphere, or nanosphere to transport the TPP-CPP chimera. Enteric coatings have also been used to help TPPs reach the small intestine. Key efficacious TPP formulations that have been approved for clinical use will be discussed. Currently, small molecule drugs  dominate the pharmaceutical drug market. However, recent advances in recombinant DNA technology and solid-phase synthesis of peptides and proteins have enabled large-scale production of therapeutic peptides and proteins (TPPs) . The sucFrom a clinical perspective, TPPs are inherently more specific to their target sites than small molecule drugs, resulting in reduced interference with the patient’s normal physiology and consequently in fewer off-target effects.Another advantage of using proteins that are produced naturally in the body for therapeutic applications is that they tend to be less immunogenic, producing fewer side effects due to inadvertent stimulation of the immune system. From a financial standpoint, median total pre-market development times are shorter for biologics 10.6 years) than for small molecule drugs (12.6 years), as estimated by the Merck Index. In 2009, the US Congress passed the Biologics Price Competition and Innovation Act (BPCIA) which gave newly approved biologics 12 years of guaranteed exclusivity . Thus, t years thThe most commonly utilized routes of administration for TPPs are intravenous (I.V), intraperitoneal (I.P.), and intramuscular (I.M.) injections. However, oral administration is the route most preferred by patients as it avoids the pain and discomfort associated with injections. Furthermore, oral medications are less expensive to produce than medications that must be isotonic and sterile to use for injection and cheaper to administer since they require no specialized equipment or trained professionals . HoweverOrally administered TPPs follow the same path as ingested food in their passage through the G.I. tract , before being absorbed into the bloodstream from the small intestine . TherefoProteolytic enzymes promote the digestion of peptides and proteins into their constituent amino acids. This is accomplished primarily by pepsin, trypsin, chymotrypsin, and elastase enzymes produced by the pancreas and by the mucosal cells that line the gut ,13,14. PSeveral factors affect the proteolysis of proteins in general, including molecular weight, hydrophilicity, the potential for ionic or hydrogen bond formation, the number of enzyme susceptible groups present, and low structural flexibility. For example, peptides that lack a cyclic structure and small peptides fewer than 12 amino acids in length  are less readily digested by proteases than proteins or larger peptides containing more than 12 amino acids  and having more complex structures . The amiThe low bioavailability of orally administered TPPs is linked to their reduced ability to cross the epithelial lining in the small intestine and its protective mucus layer. Appropriate knowledge of the structure of intestinal epithelium and absorption mechanisms at intestinal mucosa can help in engineering delivery vehicles for TPP to overcome this barrier B. The inThe use of mucoadhesive polymers for the delivery of drugs like TPPs relies on hydrophobic, hydrogen bonding, and Van der Waals interactions between the polymer and the mucin protein. The first successful strategy to use nanoparticles as drug delivery particles involved coating 200–500 nm (diameter) nanoparticles with a dense layer of a muco-resistant, low molecular weight polyethylene glycol (PEG) polymer . Its mucOnce through the mucus layer, the epithelial cell layer still blocks access to the bloodstream. Although mucous penetrating nanoparticles do facilitate the passage of drugs through the mucous barrier, their densely charged hydrophilic surface prevents them from entering the epithelial cells ,30. MoreHowever, entry into epithelial cells still may not ensure the absorption of the drug into the bloodstream. The apical side of the epithelial membrane contains efflux pumps that can return the drug back into the lumen of the gut, thereby contributing to multidrug resistance in humans . One suczonulae occludens) [The single-cell epithelial barrier can be crossed via three different mechanisms, namely paracellular transport, passive transcellular transport, and active carrier-mediated transcellular transport B. Paracecludens) . Howevercludens) ,35,36,37Transcellular transport can be mediated by either passive diffusion or actively by specific amino acid, peptide transporter, or endocytosis while paracellular transport takes place via passive diffusion only . The traMost TPPs including vaccines are administered to children using a needle and syringe. The oral route is the most convenient way of administering medicine in children as if offers a painless approach. However, oral delivery of medicines including TPPs in children remains challenging as many of the conventional strategies do not work on this age group. Children also show different responses to many active ingredients and excipients than do adult patients. Pediatric formulations need special consideration depending on the developmental age of the child. For instance, a Danish study shows that children less than 6 years of age have difficulties in swallowing solid dosage forms like tablets. Parents report that unpleasant taste and swallowing are a huge problem for solid dosage forms. Therefore, a liquid formulation with acceptable taste is preferred for this age group as they are more readily swallowed . The dosChildren’s anatomy and physiology change very rapidly with increased age, especially in the developmental ages (1–1.5 years) and these changes have a direct impact on the pharmacokinetic profile of any formulations . The Int®, a marketed tablet formulation manufactured by a conventional compressing method but that incorporates taste-masking and effervescent agents [In this section, we will discuss some novel formulation approaches for pediatric oral drug delivery. Conventional tablets are slow to disintegrate and children may find them difficult to swallow. Orodispersible tablets rapidly disintegrate in the mouth when they come in contact with saliva or water, thereby easing the process of swallowing, and can be taken without water . These ft agents . Orodispt agents . OrodispOne way to improve the bioavailability of TPPs in the circulation is to increase their ability to cross the mucus and/or epithelial cell layers to reach the bloodstream . HoweverChelating agents such as ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA) modulate or perturb structural integrity of tight junctions by chelating calcium ions essential for their structural organization. This disruption increases the paracellular transport of TPPs. Calcium chelating agents can be toxic when calcium depletion induces distortion of actin filaments, adherent junctions, and cell adhesion molecules .Surfactants (detergents) destabilize proteins and lipid present at membranes and increase intercellular space for transportation of molecules like TPPs. These include anionic detergents such as Tween 20, sodium dodecyl (lauryl) sulfate , and sodium dioctyl sulfosuccinate and non-ionic detergents such as polysorbate 80 and polyoxyl 40. Anionic surfactants are more effective than non-ionic surfactants in increasing transepithelial permeability . Other sBile salts such as sodium glycocholate and sodium taurocholate represent another class of surfactants anchor at the lipid bilayer of the epithelial cell membrane and form micelles above or near the critical micellar concentration (CMC), thereby eliminating the membrane lipid components . The bilMedium-chain fatty acids (FAs) function as absorption enhancers by a mechanism that is not well understood. They are hypothesized to downregulate tricellulin and claudin-5 expression, thereby affecting the integrity of the tight junctions, and to solubilize the epithelial cell plasma membrane phospholipid bilayer reversibly . Orally Vibrio cholerae reversibly increase paracellular absorption of orally administered insulin in rabbit intestine 10-fold via enhancing the permeability through the tight junctions without toxicity issues [Other molecules can also function as PEs. Zonula occludens toxins (ZOT), produced by the bacterium y issues . ZOT only issues .The structure and function of proteins are tightly linked, particularly for enzymes that must maintain the conformation of an active site to retain enzymatic activity. Alteration of TPP structure can improve stability and membrane penetration, decrease immunogenicity, and improve resistance to proteolytic degradation. Such alterations can occur by modification of accessible amino acid side chains, by alteration of the carbohydrate moieties of glycoproteins, or by conjugating them to lipophilic molecules such as fatty acids to increase their hydrophobicity . For exaAnother approach is the introduction of protective groups that prevent enzymatic degradation of TPPs. The addition of α-lipoic acid to insulin protects it from digestion by trypsin . SimilarAn alternative to structural alteration is the co-administration of protease inhibitors like pancreatic inhibitor, soybean trypsin inhibitor, camostat mesylate, and aprotinin, which increase the bioavailability of orally administered TPPs by reducing their enzymatic digestion by trypsin or α-trypsin . ProteasOther protease inhibitors such as chymostatin, Bowman–Birk inhibitor, and aprotinin increase the bioavailability of orally administered insulin microspheres by preventing degradation by digestive enzymes like pepsin, trypsin, and α-chymotrypsin . The higTwo assays have been used to evaluate the protective effects of these inhibitors. One test for measuring luminally-secreted protease activity is to incubate the TPP delivery system with the protease inhibitor in the gastric or intestinal fluid at 37 °C and then determining the amount of residual substrate drug in the medium ,85. For Another alternative strategy for the protection of TPPs from proteases is to covalently attach them to a biocompatible polymer, leading to reduced immunogenicity, enhanced bioavailability, and enriched pharmacokinetic profile. Polymers such as PHPMA (poly(N-2-hyfroxypropyl methacrylamide)), POEGMA (polyoligo(ethylene glycol) methyl ether methacrylate), PNIPAm (poly(N-isopropylacrylamide)), PLGA (poly(lactic-co-glycolic acid)), PDEAM ); and PEG (polyethylene glycol) are used to facilitate oral delivery of TPPs. PEG exhibits several advantages over other polymers of the same molecular weight, including biocompatibility, lower toxicity, improved biological and circulation half-life, and lower cost . PEG is PEGylation also serves to decrease the immunogenicity and immunotoxicity of TPPs in vivo . Conjuga®  that adhere to the mucus layer in the G.I. tract provide an excellent platform for oral delivery of TPPs. Mucoadhesion enhances the degree of drug absorption by extending the residence time of the drug carrier and providing higher drug concentration to the absorption site  or PMA (polymethacrylic acid) hydrogels with azo-aromatic crosslinkers have been developed for targeted delivery of macromolecules to the colon. The swelling of these polymers and release of their cargo occurs only with an increase in pH and is highest in the neutral pH of the colon. However, the enzyme azoreductase, produced by microbial flora in the colon, can degrade azo aromatic linkers . AnotherIncorporating a biocompatible endogenous permeation enhancer such as spermine or spermidine improved the bioavailability of calcitonin-loaded mucoadhesive PAA nanoparticles, which. showed a significant and prolonged reduction in blood calcium levels when administered orally in rats. This polyelectrolyte nanoparticle complex was made from the interaction of the carboxylate groups of the PAA and magnesium ions or the amino acid groups of the spermine .®), Eudragit L100®, or Eudragit S100®, covered by an adhesive compound. The middle layer is comprised of a cellulose membrane containing the drug of interest, either alone or coupled with substances like citric acid and a non-ionic detergent, polyoxyethylated castor oil derivative (HCP-60®). This unique system was initially formulated for oral delivery of recombinant human G-CSF in dogs and resulted in a slight (1.3-fold) increase in total white blood cells [The gastrointestinal mucoadhesive patch system (GI-MAPS) was developed for oral administration of TPPs, including granulocyte colony-stimulating factor (G-CSF). GI-MAPS is comprised of four distinct layers, each serving different functions, and contained in an enteric coating. The base layer contains a water-insoluble ethylcellulose polymer that protects the protein cargo from proteolytic enzymes. The surface layer is comprised of an enteric pH-sensitive polymer like hydroxypropylmethylcellulose phthalate  protein, model amphipathic peptide (MAP), synthetic transportan peptide, and the nnapedia ,118. Fornnapedia . CPPs hannapedia .Some delivery strategies seek to overcome issues of low bioavailability by avoiding the gut entirely. One such strategy involves the delivery of drugs into the bloodstream in the oral cavity, which avoids their degradation by the gastric acid and proteolytic enzymes present in the GI tract but introduces new challenges in delivering drugs in therapeutically relevant quantities by this route. A greater understanding of the anatomy and physiology of the oral cavity can help in designing suitable delivery strategies that overcome low bioavailability issues.The oral cavity consists of several parts, including the lips, alveolar mucosa, hard and soft palate, cheeks, gingiva, and tongue. Over sixty percent of the total surface area of the oral lining consists of the lining mucosa. This is comprised of stratified squamous epithelial cell layers, separated by a basement membrane from the underlying lamina propria and submucosa, both of which contain blood vessels. Absorbed drugs traverse through the small capillaries of the lamina propria to the lingual, facial, and retro-mandibular veins that connect with the jugular vein or systemic circulation . The bucSeveral strategies have been applied to develop effective vehicles for improved oral delivery of TPPs. These are reviewed briefly here.Enteric coatings such as pH-sensitive polymers have been used to protect TPPs from proteolysis in the gut and thereby facilitate their delivery to the absorption site in the small intestine. This strategy has been used for the oral delivery of protease-sensitive insulin to the small intestine . InsulinLipid-based carrier systems have been widely studied for oral delivery of proteins, but they are inefficient in encapsulation of hydrophilic peptides and proteins, are instable in the GI tract, and exhibit poor permeation through the mucosa to reach the epithelial layer. However, these challenges can be countered significantly by modifying the liposome surface with a mucoadhesive polymer like chitosan and protease inhibitors like aprotinin or the Bowman–Birk inhibitor. Orally delivered achitosan-aprotinin conjugates showed a 15-fold increased AUC (area under curve) compared to calcitonin when administered as a single agent . AnotherSulfolobus acidocaldari) is superior to that by traditional liposomes in lowering blood glucose levels.Liposomes can be chemically modified to improve its delivery characteristics. Liposomes can be surface modified to deliver their cargo to a specific delivery site. For example, liposomes made from di-stearoyl phosphatidylcholine, phosphatidylserine, and cholesterol were selectively directed to the Peyer’s patches of the lower ileum of the small intestine . ModifyiVibrio cholerae whole-cell oral microsphere vaccine relies on the formation of poly(lactic-co-glycolic acid) copolymer (PLGA) microspheres containing a whole cell lysate from V. cholerae [Polymeric microspheres that range in size from 1 to 1000 μm can also facilitate controlled oral delivery of peptides and proteins ,141. Polcholerae . Japanescholerae . Anothercholerae . Some prcholerae .Insulin loaded microspheres were fabricated from blends of PEG with poly(L-lactide) (PLA) homopolymer and poly(DL-lactide co-glycolide) (PLG) copolymers by water-in-oil solvent extraction method. These particles exhibited controlled release of insulin over 4 weeks, although only the PLA/PEG particles and their insulin cargo were stable over the 4 weeks .Biologically erodible microsphere formulations made with polyanhydride copolymers of fumaric and sebacic acid (poly(FA:SA)) demonstrated better adhesive properties than microspheres made with other polymers and were able to penetrate through the intestinal epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer’s patches . Stable Microparticles or nanoparticles (10–200 nm) penetrate through the mucus pores, cross the epithelial membrane, and reach the bloodstream. However, larger nanoparticles (200 nm–500 nm) that are surface-modified with PEG also effectively diffusing through the mucus layers . NanoparThere is much interest in the pharmaceutical industry in exploring methods of oral delivery for TPPs. For example, in Tarsa Therapeutics’ ORACLE trial, salmon calcitonin was delivered orally to osteoporotic postmenopausal women for 48 weeks, resulting in a 1.5% increase in the bone density together with a decrease in serum cartilage breakdown markers when compared with control. This study demonstrated that oral delivery of proteins can be efficacious . Clinica® (Emisphere) is a delivery agent that uses sodium N-(8-(2-hydroxybenzoyl) amino caprylate) (SNAC) as a carrier molecule for attaching drugs through weak non-covalent association. The integrity of the drug remains unchanged when conjugated with the carrier molecule and can be delivered across the epithelial membrane. SNAC enhances insulin absorption 10-fold transcellularly without significantly damaging the tight junctions. It also helps to maintain TPP stability in the GI tract by protecting the coupled TPP/SNAC protein from proteolytic enzymes [Eligen enzymes ,151. A p enzymes . In a ph enzymes .CLEC is a novel platform for the production of protein microcrystals cross-linked with glutaraldehyde, which serves to stabilize protein structure and reduce proteolytic degradation. This approach has been used successfully for lipases, esterase, and the protein hormone calcitonin . CommercThe size and surface area of nanoparticles makes them appropriate vehicles for site-specific delivery of protein cargo. They are nonimmunogenic, biodegradable, easy to modify, easy to metabolize, and stable during storage . The BioOral-Lyn is used commercially for aerosol delivery of insulin delivery. Excipients used for formulating Oral-Lyn are nonchlorofluorocarbon-based propellent and stabilizing agents to protect the aerosolized protein formulation from denaturation. Insulin is converted into its micelle form with a size greater than 7 µm, which is too large to enter the lungs but can reach the bloodstream via the buccal route of oral delivery. Oral-Lyn aerosols are delivered with the RapidMist device also used for the delivery of asthma medications. Clinical data shows that Oral-Lyn in its Phase III has been used for treating Diabetes Mellitus (NCT00668850). Generex has been marketing the same product in India under the name of Oral Recosulin since its launch on 20 November 2008 in a collaboration with Shreya Life Sciences ,163.Hydrophilic proteins and peptides are difficult to deliver through the oral route. Nobex and Biocon together are trying to address this limitation on a commercial scale by introducing a lipophilic moiety into the protein structure. The hydrophobic nature of the protein would help in increasing its ability to cross the epithelial membrane. Hexyl-insulin monoconjugate (HIM2) is a modified form of insulin where amphiphilic oligomers conjugated with a primary amine group of the Lys-29 residue found in the beta chain of human insulin . It is rTM formulation uses sugar nanoparticles coated with vitamin B12 for oral delivery of functional insulin molecules encapsulated in the matrix and can actively normalize blood glucose levels when delivered orally [Although enteric coatings are successful for the delivery of insulin, proteolysis, and absorption at intestinal enterocytes limit its commercial use . Vitamind orally ,165.AI-401 is a recombinant insulin formulation developed using oral tolerance therapy, which suppresses autoreactive T cells in type I diabetes mellitus . Phase IOrasome is a polymer-based liposome that is designed to protect insulin and human growth factor from the acidic stomach pH and release it into the small intestine. Orasome also helps in protecting the protein from low pH and bile salts ,169.® is another oral delivery technology used for enhancing protein absorption by as much as 200 times when delivered orally. This delivers oral therapeutics such as Insulin and GLP-1. GIPET-1 is currently used in tablet form of insulin with three different coatings and has been studied for its pharmacokinetics and pharmacodynamics properties towards the treatment of diabetes mellitus and is currently the subject of a Phase I clinical trial (NCT01931137) [GIPET-11931137) ,171,172.Oramed is also a carrier system used for delivering GLP-1 and Insulin. Ormade’s oral insulin is currently being investigated for both type1 and type2 diabetes. This formulation is available as ORMD-0801, which protects insulin from proteolytic degradation and enhances its oral delivery when passing the G.I. tract. It is under phase II clinical trial when used for insulin delivery and phase I trial when used for GLP-I delivery (NCT02535715) ,174.TM platform, successfully completed clinical trials and has obtained the approval of its new drug application (NDA) from the FDA [This is an enteric-coated tablet formulation that increases solubility and absorptions of peptides. This formulation has two main components: a surfactant permeation enhancer that loosens the tight junctions and potentiates paracellular transport and a multifunctional pH-lowering citric acid component that increases absorptive flux acts as a membrane wetting agent and is a calcium chelator . This st the FDA .This is an orally degradable drug composed of C-Trelin, an active analog of Thyrotropin-releasing Hormone, which functions by inhibiting glutamate activation and is one among the most successful drugs for the treatment of SCA . CurrentQRH-882260 is an orally bioavailable heptapeptide imaging agent labeled with the fluorophore cyanine 5.5 (Cy5.5) linked to the 7 aa peptide that binds specifically to the epidermal growth factor receptor, EGFR, and is used to image EGFR-expressing tumor cells. Because 97% of adenocarcinomas of the colon overexpress EGFR, this peptide provides an important imaging agent for multiple areas in the human G.I. tract . CurrentStreptococcus mutans without interfering with other strains of Streptococcus [This is a targeting antimicrobial peptide developed as an oral gel treatment of dental caries. It specifically targets the cariogenic oral pathogen tococcus . Its G2 tococcus .Oshadi Icp is a carrier system developed for oral delivery of a combination of insulin, proinsulin, and C-peptide. This formulation was developed and tested as OR14-1 . It enabRsCT is a 32-amino acid naturally-occurring calcium regulating peptide that can bind to G-protein receptors expressed on osteoclasts, leading to inhibition of nonapoptotic osteoclastic activity. rsCT is a tablet formulation of orally available calcitonin primarily used in women for preventing bone loss and treatment of low bone density for avoiding the risk of fracture. The oral form of calcitonin might have higher efficacy than compared to the nasal form of its administration based on the ORACAL trial research . A PhaseOral hPTH is a synthetic parathyroid hormone therapy for the treatment of hypoparathyroidism that targets eucalcemia and converts quiescent bone to high-turnover bone . A PhaseSemaglutide is a glucagon-like peptide 1 receptor agonist (GLP-1 RA) drug for type II diabetes Miletus that plays an important role in lowering hemoglobin A1c, but its sub-cutaneous administration has caused a loss in its therapeutic efficacy. Oral administration may prevent drug disintegration in the stomach, thereby facilitating its transcellular absorption via the gastric membrane, allowing it to reach the systemic circulation. A Phase III clinical trial is completed (NCT02906930) and the FDA approved it for treatment of type 2 diabetes ,189,190.Also known as oral anti-CD3, is already an FDA-approved drug for the treatment of patients undergoing solid organ transplantation but it has shown up to significantly toxic when administered intravenously. It is also known as Muronomab-CD3, which was the first monoclonal antibody-based therapy for clinical application . In an aForalumab is an oral formulation of anti-CD-3 antibody developed for the treatment of autoimmune and inflammatory diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). A Phase II clinical trial (NCT03291249) conducted by Novimmune was withdrawn at the request of the Ministry of Health . FurtherThe use of TPPs as drug candidates has been facilitated by the discovery of recombinant DNA technology and solid-phase protein synthesis, which allowed a large scale of synthesis of TPPs. A variety of peptides and proteins are currently in use for treating different diseases but mostly through the parenteral route, yet the oral route is the most preferred route for administration due to patient compliance and ease of administration. The therapeutic potential of orally delivered TPPs is somewhat hampered by systemic instability in the GI tract and poor absorption through intestinal epithelia. To exert their activity, TPPs must remain stable and active before reaching the systemic circulation. In the last few years, significant efforts were made to enhance the bioavailability of orally administered TPPs. Absorption enhancers, enzyme inhibitors, structural modifications, and carrier systems were developed to tackle systemic instability and increase the bioavailability of TPPs. These strategies have met with initial success but very few of them have yet reached the clinics. An ideal oral formulation must meet all the demands of patients, healthcare providers, and pharmaceutical manufacturers. Furthermore, a thorough understanding of the factors that affect stability in the GI tract and permeation across the intestinal epithelia of TPPs will be essential in developing the ideal TPP formulations for oral delivery, particularly those of children.