Now, materials in the form of hydrogels are making headway in cancer research, where an ever-expanding tool-chest of sophisticated in vitro cell culture platforms is helping scientists to unravel the mysteries of cancer stem cells (SCs)2. These unique materials function particularly well at the interface with complex cellular systems3, thereby providing new ex vivo pathways to control cellular fate or guide cellular function. Sophisticated hydrogels that mimic properties of biological tissues have opened up new potential for biomedical engineering applications4, namely in the burgeoning field of SC therapeutics5 and cancer research6. Biological or synthetic hydrogels are now routinely produced with design features that can actively manipulate multiple aspects of SC signaling pathways leading to enhanced morphogenesis, proliferation, and differentiation9. Many of these systems take advantage of biological or bio-mimetic motifs that are incorporated into the hydrogel backbone, to alter response based on known cellular interactions with native extracellular matrix (ECM) molecules. These interactions are often combined with mechanical and biophysical features that resemble those of the SC niche, to enhance differentiation pathways towards myogenesis, chondrogenesis, neurogenesis, and more11. Now, writing in Nature Biomedical Engineering, Tanaka and colleagues1 use completely synthetic double network (DN) hydrogels to rapidly reprogram differentiated cancer cells into cancer SCs (CSCs), to identify biomarkers for targeted cancer therapy.Biomedical hydrogels, which are hydrophilic polymer-based biomaterials, are transforming the way in which materials impact the medical field1 use their DN hydrogel comprising poly(2-acrylamido-2-methylpropane sulfonic acid) and poly as a tough yet soft substrate on which differentiated cancer cells are cultivated with the aim of reprogramming these cells to their scarcer CSC progenitors. Relying solely on the ability to control the physical properties of their DN hydrogel, they reconstructed key mechanical features of the niche microenvironment that causes induction of stemness in six fully differentiated cancer cell lines. Critically, they showed that this rapid and robust reprogramming was obtained when using primary tumor cells from a human brain cancer glioblastoma patient. They unequivocally demonstrated the usefulness of this technique in terms of deriving a potentially unlimited supply of primary human CSCs for biomarker discovery research.CSCs are an elusive cell that constitute a very small fraction of the overall cell population of a tumor mass. Despite their minute numbers, these cells are capable of leading to the recurrence of cancer vis-à-vis a circulating subpopulation derived from their progeny. Identifying these cells in patients is currently not possible, because no definitive set of biomarkers exists for them. Although targeting of this subpopulation of cells could potentially eradicate cancer recurrence, the development of such therapies has been hampered by the limited availability of primary CSCs with which to study their genotype. Tanaka and colleagues12, and/or soluble biochemical agonists. A variety of techniques have emerged to produce ever larger quantities of pluripotent SCs for therapeutic application in translational medicine, including regenerative medicine and cell therapy. Far fewer strategies exist for producing CSC from primary tumor cells. This is likely owning to the complex differential spatiotemporal activation of tyrosine kinase required for this transformation, which appears to be activated by the DN hydrogel’s interactions with cellular surface proteins including the integrin family of cell adhesion molecules, but not E-cadherin or β-catenin. The DN hydrogels were not functionalized with bioactive motifs, suggesting that nonspecific spheroid formation was primarily responsible for the biophysical induction of the cells. Nevertheless, a unique combination of physical properties obtained only with the DN hydrogel, and not with any of the respective single-network hydrogels or plastic dish controls, resulted in a robust reprogramming of the cell spheroids expressing all three stemness markers, namely sox2, Oct3/4, and Nanog.The natural processes leading to SC reprogramming are complex and involve a multitude of extrinsic and intrinsic signaling in the niche. Until now, the main paradigm involved in the artificially induced SC reprogramming involved the use of transcription factors, with their stability regulation1 use their DN hydrogel, with its unique set of biophysical properties, as a catalyst to initiate brain cancer cell reprogramming, likely through the production of osteopontin (OPN) and the tyrosine kinase signaling pathways. The OPN causes a signaling feedback loop in these cultures, whereby brain cancer cells are induced to express key proteins related to stemness, such as SHH and GLI1, which in turn act on the OPN-CD44 signaling axis that enhances the stemness in the culture. This positive feedback loop causes a very rapid induction of CSCs on DN hydrogels  of the DN gel were not directly described, and further study is required”


During the last decade, studies have raised awareness of the crucial role that the right ventricle plays in various clinical settings, including diseases primarily linked to the left ventricle. The assessment of right ventricular performance with conventional echocardiography is challenging. Novel echocardiographic techniques improve the functional assessment of the right ventricle and they show good correlation with the gold standard represented by cardiac magnetic resonance. This review summarizes the traditional and innovative echocardiographic techniques used in the functional assessment of the right ventricle, focusing on the role of right ventricular dysfunction in heart failure with reduced ejection fraction and providing a perspective on recent evidence from literature. Heart failure (HF) remains a rising public health concern, with an estimated prevalence of almost 38 million individuals worldwide ,2. The tMost of the previous research demonstrated the high prognostic value of left ventricular (LV) dysfunction , while tIn the era of multi-modality imaging, cardiac magnetic resonance (CMR) imaging remains the gold standard for RV quantification, despite the technical innovations in the field of echocardiography ,11,12,13However, the high cost and low availability of CMR hamper its unrestricted use on all patients with HF. By comparison, echocardiography is a bedside, widely available tool, and novel echocardiographic techniques such as myocardial strain imaging and three-dimensional (3D) echocardiography allow an accurate RV quantification that has been validated against (CMR) ,19.The aim of this review is to summarize the role of both conventional and novel echocardiographic parameters of RV function in patients with HF and reduced EF (HFrEF), while focusing on recent research.The echocardiographic assessment of the RV faces several challenges: (1) the complex RV geometry; (2) its position behind the sternum; (3) the cumbersome endocardial tracing due to prominent trabeculations; and (4) its dependence on hemodynamic load and the RV–LV interdependence. In fact, some authors suggest that up to 20–40% of RV stroke volume results from the contraction of the LV . The RV There is no ideal echocardiographic parameter for RV quantification , hence aMost of these parameters  assess only the longitudinal RV function, while others (such as FAC) account for both the longitudinal and radial components of the RV contraction. However, most of the RV functional parameters neglect the contribution of the outflow tract contraction, potentially leading to an underestimation or overestimation of the global RV systolic performance . This liTAPSE is a highly reproducible, easy obtainable parameter  of RV loLoad-dependency means that TAPSE will change with different loading conditions without actual changes in myocardial contractility. TAPSE decreases with increased pulmonary vascular resistance , but it ≤ 14 mm predict unfavorable outcomes in patients with HF, irrespective of its ischemic or non-ischemic etiology [Ghio et al. showed that TAPSE ≤ 14 mm is an independent predictor of death or emergency cardiac transplantation in patients with congestive HF . Similaretiology .The systolic velocity of the tricuspid lateral annulus is measured in the apical four-chamber view by placing the tissue Doppler marker on the lateral tricuspid annulus ,30. SimiStudies found that decreased TDI systolic velocity of the tricuspid annulus is an independent predictor of either cardiac death ,40 or caThe RV myocardial performance index, also known as the right Tei index, is a measure of both systolic and diastolic RV function. It is a unitless parameter, calculated by dividing the total isovolumic time (isovolumic contraction plus isovolumic relaxation) by the ejection time (ET) . SystoliThe prognostic value of the pulsed-Doppler-derived RV MPI was assessed in a cohort of HFrEF patients, who were prospectively followed for 5 years for a combined endpoint of cardiac death and readmissions for HF. The authors found that an RV MPI > 0.38 was an independent predictor of adverse outcomes . In a stFAC is a 2D measure of RV systolic function obtained from the RV-focused apical four chamber view by manually tracing the endocardial border of the RV in end-diastole and end-systole. It is calculated as: (end-diastolic area − end-systolic area)/end-diastolic area × 100% . The RV-Zornoff et al. found that RV FAC is an independent predictor of total mortality, cardiovascular mortality, and development of HF in patients with LV systolic dysfunction following a myocardial infarction (MI), with each 5% decrease in FAC being associated with a 16% increase in odds of cardiovascular mortality . SimilarA small retrospective study found that RV FAC < 26.7% is predictive of death or LV assist device implantation in patients with DCM, providing better prognostic value than TAPSE and S’ wave velocity . SimilarMyocardial acceleration during isovolumic contraction is usually obtained using TDI at the lateral tricuspid annulus in the apical four-chamber view. It is calculated as the peak myocardial velocity during isovolumic contraction divided by the time needed to reach this velocity . While iSpeckle-tracking echocardiography is a non-invasive, innovative technique that analyzes the segmental myocardial deformation along different planes through the displacement of speckles . OriginaSTE assesses the deformation of myocardial speckles in two dimensions along the myocardial wall direction, thus being less confounded by the motion of the heart  and relaMartin et al. analyzed which of the RV strain parameters was a better predictor of hospitalizations for HF in patients with left heart disease. They showed that the RV global longitudinal strain independently predicts readmissions, providing additional prognostic information to that obtained by TAPSE . SimilarCarluccio et al. proved the superiority of RV strain over TAPSE, by following 200 patients with HFrEF but preserved TAPSE (>16 mm) for a composite endpoint of death and HF rehospitalization. The authors found that the RVFW longitudinal strain was an independent predictor of adverse outcome, with a cutoff value for endpoint prediction of −15.3% . In a reSeveral studies discovered independent prognostic roles for both global RV strain and RVFW strain in HFrEF. Cameli et al. found that in patients with advanced systolic HF referred for cardiac transplantation, both global and free-wall RV strain are independent predictors of an adverse outcomes , with stronger predictive power than other conventional parameters, including parameters of LV function . Another3D echocardiography overcomes the geometric assumptions used in 2D echocardiography. As such, it is particularly useful for the evaluation of the RV, which—due to its complex anatomy—cannot be comprehensively assessed with 2D measurements only. 3D echocardiography integrates both the longitudinal and radial components of RV contraction  and, unlIn a population-based cohort study that enrolled 1004 elderly people, Nochioka et al. used 2D and 3D echocardiography to analyze the prevalence and prognostic role of RV dysfunction in HF. Among patients with no HF at baseline, 3D RVEF proved to be an independent predictor of death or incident HF: each 5% decrease in 3D RVEF was associated with a 20% increase in the hazard of death or hospitalization for HF, independent of LVEF .Magunia et al. found that 3D RVEF is an independent predictor of post-operative RV failure in LVAD recipients , which iA recent retrospective study of Surkova et al. evaluated the relative importance of different combinations of reduced and preserved 3D LVEF and 3D RVEF in predicting mortality in patients with different cardiac diseases. Reduced 3D RVEF, but not LVEF, was a strong and independent predictor of both all-cause mortality and cardiovascular mortality . Moreove3D STE is a novel imaging technique that evaluates myocardial deformation using 3D full-volume data sets. It is currently limited to research and not available for routine use, but it appears to be a promising tool for the assessment of the complex RV myocardial motion . A studyThe interaction between the RV and the pulmonary circulation unit is reflected in the RV–pulmonary artery coupling (RVPAC), which is usually assessed with right heart catheterization. This parameter reflects the adaptation of the RV to afterload, and it is calculated from invasive pressure–volume loops as the ratio of RV end-systolic elastance to pulmonary artery elastance . SeveralAs innovative echocardiographic techniques become part of the comprehensive assessment of RV performance, some researchers used 2D RV longitudinal strain or 3D RVEF for the non-invasive estimation of RVPAC, which was calculated as either RV strain/PASP ratio ,90 or asFractional shortening of the RVOT (RVOT-FS) is an index of RV performance that is obtained using M-mode echocardiography in the parasternal short axis window at the level of the aortic root. It is calculated as the percentage change in RVOT diameter at end-systole compared to end-diastole . SeveralThe above-mentioned studies evaluating the prognostic role of RV dysfunction in HF are summarized in Artificial intelligence (AI) techniques, such as machine learning (ML) and deep learning, can improve the diagnostic accuracy of echocardiography, by providing fully automated image analysis and thus potentially reducing human error . So far,The RV plays a crucial role in various clinical settings. RV dysfunction is a strong independent predictor of mortality and adverse outcomes not only in diseases of the right heart or pulmonary vascular bed but also in diseases primarily involving the LV. In the particular setting of DCM, RV FAC appears to be a better outcome predictor than other conventional RV parameters, while RVFW strain has a higher prognostic value than global RV strain. There is no perfect single parameter that comprehensively evaluates RV performance. Integrating novel techniques in the RV echocardiographic assessment allows a better evaluation and an enhanced risk stratification for patients with HF, thus improving therapeutic strategies and potentially leading to an improved outcome.


A growing body of evidence has suggested that their dysregulated expression is involved in the pathogenesis of diverse diseases, including diabetes mellitus type 2 (DM2). Early onset of this chronic and complex metabolic disorder is frequently undiagnosed, leading to the development of severe diabetic complications. Notably, DM2 prevalence is rising globally and an increasing number of articles demonstrate that DM2 susceptibility, development, and progression differ between males and females. Therefore, this paper discusses the role of microRNAs as a source of novel diagnostic biomarkers for DM2 and aims to underline the importance of sex disparity in biomarkers research. Taking into account an urgent need for the development of sex-specific diagnostic strategies in DM2, recent results have shown that circulating miRNAs are promising candidates for sex-biased biomarkers. International Diabetes Federation (IDF) projects that the worldwide prevalence of diabetes among adults (20–79 years old) will systematically rise to finally reach about 700 million people by 2045  may serve as a valuable source of prognostic, diagnostic, and even predictive biomarkers for many human diseases , the human genome encodes more than 2500 miRNAs , may be one of the most significant targets of miRNAs involved in glucose metabolism, insulin signaling, and blood coagulation. In the cells, protein kinase A is engaged in proliferation and cell growth , enables prediction of the DM2 development with high accuracy.Considering the importance of early detection of DM2, continuous efforts are being made to find miRNAs that enable distinguishing the pre-diabetes state from the early onset of disease or identify individuals with a high risk of DM2 development  in males with early diagnosed DM2  incomplete X-chromosome inactivation leading to biallelic expression of miRNA, and in addition, (2) regulation of miRNA transcription and processing by estrogens (Florijn et al. Circulating miRNAs are promising candidates as biomarkers for screening and diagnosis of early onset, progression, and complications of DM2 or even for the selection of individually tailored medications and therapy. However, some studies have reported discrepancies in the analyzed expression level of circulating miRNA and their utility as biomarkers in different populations (Bhatia et al.


Finally, the adverse effects of adenosine signaling may potentiate a deadly outcome in the form of SUDEP by suppressing breathing and arousal in the postictal period. Evidence from animal models suggests that excessive postictal adenosine signaling contributes to the pathophysiology of SUDEP. The goal of this review is to discuss the beneficial, harmful, and potentially deadly roles that adenosine plays in the context of epilepsy and to identify crucial gaps in knowledge where further investigation is necessary. By better understanding adenosine dynamics, we may gain insights into the treatment of epilepsy and the prevention of SUDEP.Adenosine is an inhibitory modulator of neuronal excitability. Neuronal activity results in increased adenosine release, thereby constraining excessive excitation. The exceptionally high neuronal activity of a seizure results in a surge in extracellular adenosine to concentrations many-fold higher than would be observed under normal conditions. In this review, we discuss the multifarious effects of adenosine signaling in the context of epilepsy, with emphasis on sudden unexpected death in epilepsy (SUDEP). We describe and categorize the beneficial, detrimental, and potentially deadly aspects of adenosine signaling. The good or beneficial characteristics of adenosine signaling in the context of seizures include: (1) its direct effect on seizure termination and the prevention of status epilepticus; (2) the vasodilatory effect of adenosine, potentially counteracting postictal vasoconstriction; (3) its neuroprotective effects under hypoxic conditions; and (4) its disease modifying antiepileptogenic effect. The bad or detrimental effects of adenosine signaling include: (1) its capacity to suppress breathing and contribute to peri-ictal respiratory dysfunction; (2) its contribution to postictal generalized EEG suppression (PGES); (3) the prolonged increase in extracellular adenosine following spreading depolarization waves may contribute to postictal neuronal dysfunction; (4) the excitatory effects of A The purine ribonucleoside adenosine is found ubiquitously in living tissues. In the central nervous system, adenosine is an essential inhibitory modulator of neuronal excitability . Neurona2A receptor activation may have proconvulsant effects ; thus, the tone of ambient adenosine is maintained by an astrocyte-based adenosine-cycle . It is n1 receptors, which are coupled to Gi/o proteins  experience seizures and seizure-induced respiratory disruption following exposure to a high intensity broadband acoustic stimulus . SeizureIn summary, the influence of adenosine signaling in the context of epilepsy is nuanced and cannot be indiscriminately categorized as either beneficial or harmful . InsuffiThe adenosine hypothesis of SUDEP is largely predicated on the assumption that seizure-induced adenosine surging occurs in the brainstem, where adenosine is known to suppress breathing . SeizureAs discussed in the main body of this review, there are a number of mechanisms responsible for activity-dependent adenosine release. The relative contributions of these mechanisms appear to be regionally dependent. The mechanisms of activity dependent changes in adenosine signaling have primarily been studied using spontaneously occurring or electrically evoked neuronal activity. Insights gained regarding the mechanisms of activity dependent adenosine release in the context of evoked or naturally occurring neural activity may, or may not, be generalizable to seizures. Future investigations should examine the mechanisms underlying seizure-induced adenosine release and elucidate the spread of adenosine wave to the brainstem. Spreading depolarization waves result in an increase in extracellular adenosine which prolongs neuronal dysfunction , 2017; hAnatomically, where adenosine acts to potentiate SUDEP is unknown. Adenosine suppresses breathing in a variety of brainstem sites ; howeverAdenosine and breathing” subsection, adenosine modulates the signaling of a number of other neurotransmitter systems which are relevant to epilepsy and seizure-induced death. Presynaptic A1 receptor activation inhibits the synaptic release of glutamate in brain areas notable for seizure activity, such as the hippocampus (2A receptor activation facilitates hippocampal and striatal glutamate release ostensibly by diminishing the inhibitory influence of A1 receptor activation (1 receptor antagonism increases glutamate release (2A receptor antagonism decreases it (In addition to the inhibitory effect of adenosine on serotonergic neurotransmission, which is discussed in more detail in the “pocampus . In conttivation . Corresp release , whereaseases it .1 receptor activation and increased by A2A receptor activation (2A receptor mediated excitation of GABA releasing neurons in the respiratory brainstem has been used to explain the observation that both A1 and A2A receptor agonists suppress breathing, despite their divergent effects on neuronal excitability (Similar to glutamate, GABA release is decreased by Ativation . A2A rectability .Like serotonin, the monoaminergic transmitter norepinephrine may be protective against seizure-induced death. The norepinephrine reuptake inhibitor, atomoxetine, reduces the likelihood of seizure-induced death following maximal electroshock and audiogenic seizures . AdenosiArousal promoting cholinergic structures in the basal forebrain and brainstem are inhibited by adenosine . Seizure–/–Kcna1 mice, which lack voltage-gated Kv1.1 channels, experience spontaneous seizures and undergo seizure-induced death at approximately postnatal day 50 (Scn1a gene which encodes the voltage-gated sodium channel Nav1.1. Mice with similar mutations display phenotypes comparable to those seen in Dravet syndrome, including seizure-induced death which might prove to be an effective tool for SUDEP investigations (Most of the experimentation pertinent to the role of adenosine in seizure-induced death has been conducted in acute seizure models, often in seizure-naïve animals. Epileptogenesis and the occurrence of repeated seizures alters the brain in ways that might be meaningful to SUDEP; for instance, an altered expression/function of ADK and adenosine receptors has been noted during epileptogenesis . Therefol day 50 . Dravet l day 50 . Dravet igations .A growing body of evidence implicates adenosine signaling in a variety of adverse seizure outcomes such as respiratory suppression, PGES, and SUDEP , 3; howeSudden unexpected death in epilepsy typically occurs during sleep . Given tBy improving our understanding of periictal adenosine dynamics and developing novel strategies for influencing adenosinergic signaling we may gain insights into how seizures and their most tragic sequelae can be prevented. Therapeutically, adenosine augmentation strategies are some of the most effective strategies for seizure control , howeverAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The development of a biomimetic neuronal network from neural cells is a big challenge for researchers. Recent advances in nanotechnology, on the other hand, have enabled unprecedented tools and techniques for guiding and directing neural stem cell proliferation and differentiation in vitro to construct an in vivo-like neuronal network. Nanotechnology allows control over neural stem cells by means of scaffolds that guide neurons to reform synaptic networks in suitable directions in 3D architecture, surface modification/nanopatterning to decide cell fate and stimulate/record signals from neurons to find out the relationships between neuronal circuit connectivity and their pathophysiological functions. Overall, nanotechnology-mediated methods facilitate precise physiochemical controls essential to develop tools appropriate for applications in neuroscience. This review emphasizes the newest applications of nanotechnology for examining central nervous system (CNS) roles and, therefore, provides an insight into how these technologies can be tested in vitro before being used in preclinical and clinical research and their potential role in regenerative medicine and tissue engineering. Understanding the fundamental biology of neural tissue growth and synapse formation assists to define overall functions performed by these tissues . Owing tOver the last few years, nanotechnology has been emerging very fast for its applications in biological and biomedical sciences. This nanotechnology is generally defined as the manipulation of functional structures that have at least one dimension size from 1 nm to 100 nm . HoweverMany efforts have been made to uncover the underlying molecular and cellular mechanisms associated with the formation of neuronal circuits. Scientists are working on developing biomimetic and biocompatible support to control NSCs’ growth and differentiation into ordered neuronal networks ,7,8. TheFor the modulation of the cell morphology by means of nanotechnology, the synthetic nanosized materials offer inimitable surface properties that can influence cell behavior to establish a neuronal network . The impBy designing the nanoscale topography on the cell culture substrate (growth surface), the specific role of physical guidance in the formation of neuronal circuits can be understood . The abi2+ imaging techniques   was seen in nanotechnology applications. The micrometric cavities present in the porous PDMS scaffolds were exposed to the Multi-Walled Carbon Nanotubes (MWCNTs). This permits the fabrication of scaffolds with holes layered by an irregular MWCNT carpet of around 100 nm thickness. The resulting 2D and 3D scaffolds with MWCNT boost synaptogenesis to form a synaptic network of hippocampal cells and have been confirmed using immunofluorescence staining, confocal microscopy, and Cachniques  6]..2+ imagiSemiconductors, at a nanoscale level similar to nanowires, also have been used for their potential incorporation within electronic circuits for stimulating and recording cellular activities. Recently, for the first time, Indium Phosphide (InP) nanowire scaffolds mesh of 200 × 200 μm (100–800 nm dia and height around 2 μm) was used to support neuronal development and the formation of a well-connected neuronal network. InP nanowire is a direct band gap semiconductor unlike gallium phosphide (GaP) and silicon (Si) and provides a superior optoelectronic interface to stimulate neurons ,23. The a) is significant in neuronal network establishment and information processing, as found using fluorescent multi-calcium imaging and computer simulation methods. According to a study, neural cells seeded for 11 days onto corrugated surfaces (Sa > 22 nm) show small-world attributes, which enhanced exchanged information by 4-fold as compared to neural cells on a flat surface (Sa < 10 nm), which show uniform distribution over the surface with no clustering effects ..33].2, the greatest length of neurite was increased on average by almost 36% higher when compared to non-irradiated cells. The reason behind this phenomenon was hypothesized that the transient heat produced by excitation of localized surface plasmon resonance in NRs might generate extra Reactive Oxygen Species (ROS). This increased level of ROS can increase the cell metabolic activity by activating the transcriptional factors . Ch. Ch35].  Sm-CeO2 .Electric stimulation of various cells has been widely used to treat several conditions related to musculoskeletal and neurological disorders and provided a significant impact on laboratory research. It balances the electric signal propagation by compensating for the altered electric activity of cells and improve their growth and tissue generation properties. Owing to the intrinsic electroactivity properties of nerve cells, scaffolds with conductive properties and the ability to deliver electric stimulation have gained interest for their application in neuroscience. Numerous ways of invasive and non-invasive stimulation have been reported and practiced to precisely confine the activation of specific nervous structures. The exclusive mechanical, electric and biological properties of different nanoparticles make them a potential candidate for providing a neural interface to decide neural cell fate  and neural network formation ,39.Nowadays, piezoelectric materials are widely being used in biomedical research due to their fascinating property of generating electric fields upon applying mechanical stress, called the “direct piezoelectric effect.” Many in vitro studies have shown that different cell types behave differently when incubated with piezoelectric substrates/scaffolds.Ciofani et al. (2010) reported the use of Boron Nitride Nanotubes (BNNTs)  and ultrasound to stimulate neuronal-like cells in culture. Despite the structural similarity of BNNTs with CNTs, BNNTs show superior chemical, mechanical and electrical properties. They proposed a unique way to stimulate the cells in vitro, based on the piezoelectric properties of BNNTs without the usage of electrodes in culture. BNNTs of length 200–600 nm and diameter of 50 nm, incubated with the neuronal-like PC12 cells and ultrasounds were used to deliver mechanical stress to BNNTs. This led to the polarization of nanotubes due to the piezoelectric properties of BNNTs and delivered electrical stimulus to the cells. PC12 cells stimulated with this innovative technology showed a 30% increment in neurite sprout after 9 days of treatment.2+ transients, known as Ca2+, whereas only ultrasound stimulation without BTNPs could induce Ca2+ transients of low amplitude. Furthermore, these Ca2+ waves are known to propagate intercellularly through gap junctions on adjacent neurons. These waves are important in the establishment of a neural network, especially by controlling the neurite outgrowth [As suggested by Ciofani et al., this concept model can also be implied in life science to electrically stimulate the cells when required . Similarutgrowth ,41.2+) response in murine Neuro-2A (N2A) cell line in primary mouse cortical neurons. Gold nanoparticles (AuNPs) excite the cells by transferring energy from a laser pulse to the cell membrane. By visualizing the lipid peroxidation and calcium flux in cells by fluorescent dye, they hypothesized that when AuNPs are irradiated at their plasmon resonance frequency, the release of Ca2+ from the endoplasmic reticulum is triggered [2+ acts as universal messengers and plays a crucial role in numerous signaling pathways and cellular stress response. Furthermore, Ca2+ is a source of cell stress and tracking the calcium flux of AuNPs-laser-treated cells can reveal information on different aspects of Ca2+ role and health [Moreover, neuronal stimulation mediated by laser has opened another potential field of research. In this technique, a laser pulse is used to stimulate cells instead of an electric field, which provides superior spatiotemporal resolution by avoiding the electronic crosstalk. Recently, a similar study by Johannsmeier et al. (2018) demonstrated gold nanoparticle facilitated laser stimulation to excite the cells and studied calcium , carbon nanotubes (CNTs), and graphene, have shown their potential roles in neuroscience due to their mechanical, electrical and biological properties ,47. ReseAt present, the primary aim of neuroscience is to discover the relationships between neuronal circuits and their pathophysiological functions. To do so, various technologies have been developed using micro and nanotechnology for neuron signal recording in in vitro, such as patch-clamp array for intracellular recording and substrate-integrated MEAs for extracellular recording. Both the techniques have some limitations; for instance, the patch-clamping technique permits high-precision intracellular recording but is not appropriate for the network-level investigations whereas, MEAs enable extracellular neuronal network recoding precisely and are not very sensitive for intracellular recording ,52. The seal) with the plasma membrane when they penetrate it. Robinson et al. developed vertical nanowire electrode arrays (VNEAs) from silicon-on-insulator, and each NW in the array comprises of doped silicon core encapsulated by silicon dioxide and sputter-deposited metal tip Ti/Au. The metal tip and silicon core were used for providing internal access to the cell, and the glass shell prevents current leakage and tightly seals the cell membrane. The developed VNEAs had 16 stimulation/recording pads and lied into 3 × 3 arrays of nine silicon NW . For the optimization of VNEAs, embryonic rat cortical neurons or HEK293 cells were cultured on VNEAs for few days, and approximately 50% of electrodes instinctively penetrated the plasma membrane. This caused a potential drop across the membrane, and seal resistance was developed between VNEAs, and the plasma membrane was estimated to be 100–500 MΩ [Patch-clamp electrodes and glass electrodes are known for intracellular recording by producing seal resistance  nanoribbons, which could detect the cell deflection of 1 nm when 120 mV is applied to the cell membrane. The cell line used for the experiment was rat pheochromocytoma (PC12 cells), which is similar to sympathetic neurons when treated with nerve growth factor (NGF). The measured deflections resembled the theoretical model where applied voltage causes cell depolarization and leads to alter membrane tension, which makes the cell change its radius to keep pressure constant across the membrane [Even though it has been considerably studied where electric responses by the neuronal cells are induced by applying voltage, less is known about the mechanical response of neuronal cells when excited electrically ,57. The membrane ,60. Anotmembrane . The disUndoubtedly, nanotechnology has provided numerous benefits over microtechnology for culturing neuronal cells in vitro. Moreover, the fabrication of different nanostructures and their integration with microtechnology have offered in vivo-like environments. However, there are still a few challenges that need to be addressed. The critical challenges found in the aforementioned four key areas are: (i) a lack of fundamental understanding of the mechanisms governing the formation and elimination of synaptic connections for precise neural network, (ii) difficulties in developing the platform where multiple cell lines can be appropriately programmed for controlling cell behavior and morphology, (iii) optimization of the stimulus parameters, such as type of stimulation, stimulation power and duration, avoiding inappropriate stimulations which lead to apoptosis, and (iv) biosafety issues caused by cytotoxicity and genotoxicity of nano-sized materials.In particular, nanotoxicity has emerged as one of the major concerns with using new nanomaterials for cell culture or in vivo application ,63,64,65Furthermore, there is a need to emphasize finding the accurate balance of microenvironment factors to obtain the pure population of differentiated neural cells. The sorting of differentiated populations, due to the absence of control on the differentiation process, may limit their application on larger scales. To overcome this, a specific microenvironment for neural stem cell differentiation should be discovered. Another major concern that needs to be addressed is the complexity of fabricating 3D porous scaffolds with certain nanotopographies on them. Although at a small scale, the development of nanotopographies on the biomaterial has been achieved, and these nanotopographies had shown good interaction with neural cells to establish a neural network. However, nanopatterning technologies for biomaterials on a large scale need to be achieved. Moreover, the biocompatibility and biodegradability of the nanomaterials need to be checked properly before their use in vitro and in vivo. These technologies can provide 3D neuronal cultures, which will not only offer the platforms for toxicology assays but also help in designing future biocompatible devices. Upcoming advancements in this field will develop sophisticated, more functional, and safer platforms for its application in both clinical and scientific endeavors.In summary, we have highlighted how nanotechnology can be used to guide neural stem cell growth and their differentiation in in vitro culture. Nanotechnology-based methods can be customized to regulate the neuronal cell culture for establishing an in vivo-like model for research. The scaffold developed using nano/microtechnology for stem cell applications is biomimetic and biocompatible and supports neural cell growth and differentiation. Biomaterials with some specific nanotopography have shown their ability to control cell behavior, morphology, and fate. The interaction of different nanostructures with neuronal cells can be stimulated (electric and laser) for different purposes, such as more proliferation, enhancement of neurite length, and differentiation of neural cells. The development of nanotechnology devices, such as nanoelectrode arrays, with circuit integration technology can be used to estimate intracellular membrane potentials and postsynaptic current from hundreds of connected neurons to form networks in vitro. This would be a potential step to discover the relationships between neuronal circuit connectivity and their pathophysiological functions. Furthermore, the cultured neuronal network can play a pivotal role in finding cures/answers for many neurodegenerative diseases which cannot be solved through simple in vivo cell culture models.


We report an effective surface immobilization protocol for capture of Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor whose elevated concentration in cerebrospinal fluid has recently been associated with Alzheimer’s disease (AD). We employ the proposed surface functionalization scheme to design, fabricate, and assess a biochemical sensing platform based on plasmonic interferometry that is able to detect physiological concentrations of TREM2 in solution. These findings open up opportunities for label-free biosensing of TREM2 in its soluble form in various bodily fluids as an early indicator of the onset of clinical dementia in AD. We also show that plasmonic interferometry can be a powerful tool to monitor and optimize surface immobilization schemes, which could be applied to develop other relevant antibody tests. Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that affects more than five million Americans and approximately 50 million people worldwide . AD causAD is traditionally diagnosed by monitoring subjects’ behavioral changes because it is challenging to diagnose more definitively without an invasive examination of the brain. This inexact method, which has a misdiagnosis rate of up to 45%, contributes to the untenably high attrition rate of drug candidates by creating an incomplete understanding of disease etiology and, as such, a lack of robust and valid biomarkers on the causal path of the disease .Most cases of AD have a complex, highly polygenic architecture . A numbeTREM2 may be found in its soluble form in cerebrospinal fluid (CSF) and other bodily fluids, such as saliva . CSF conSeveral optics- and nanostructure-based alternatives to conventional biosensing methods have been developed, including sensing based on magnetic nanoparticles , carbon he metal . When thhe metal ,44,46,47he metal ,42,43,46he metal ,42,44,46Compared to more conventional surface plasmon resonance (SPR) sensing platforms, sensors based on plasmonic interferometry (PI) retain high sensitivity and low detection limits whilst providing several advantages, including: (1) broadband (as opposed to single wavelength) operation that allows for spectroscopic capabilities and detection of refractive index changes at multiple wavelengths of interest, simultaneously ,46; (2) ame time ,39,42.Here, we employ plasmonic interferometry to develop and assess a surface functionalization protocol designed to detect TREM2 in solution. Specifically, we (i) monitor each step of the proposed functionalization protocol using the intensity change in the transmitted spectra of plasmonic interferometers and (ii) employ this surface functionalization scheme to detect biological levels of TREM2 in solution.The proposed biosensing chip comprises four arrays of nominally identical GSG plasmonic interferometers that were designed and fabricated with asymmetrical arm lengths  for a duration of 2.5 h, followed by rinsing with phosphate buffer solution (PBS) and DI water. The sulfo-NHS-biotin solution was prepared in 0.01 M PBS  immediately before using. On an amino-terminated gold surface, sulfo-NHS-biotin will covalently attach to the amino groups via ester linkage, as shown in In order to saturate the sulfo-NHS-biotin binding sites on the APTES-functionalized chip and to form a monolayer of streptavidins and antibodies on the surface, the chip was optically characterized after every hour of chemical treatment until no significant spectral peak shifts were observed in the optical transmission spectra through the GSG interferometers; the concentration ratio of sulfo-NHS-biotin and streptavidin was optimized to achieve a high biotinylated antibody covering rate. Subsequently, the chip was incubated with 0.1 mg/mL biotinylated TREM2 antibody PBS for 2.5 h, as shown in After each functionalization step, the sensor chip was rinsed with DI water to remove unbound molecules/proteins and thoroughly dried under a stream of nitrogen gas to remove any residual water droplets/molecules that might affect the final wavelength shift measurement. This washing and drying protocol was enforced throughout the experiments to make sure that actual protein-protein binding events were measured, which allowed us to better validate the proposed surface functionalization and sensing methods. After surface rinsing and drying, the transmitted intensity of each plasmonic interferometer on the sensor chip was measured. To perform the optical characterization, a plasmonic interferometer sensor chip containing functionalized plasmonic interferometers was placed on the controllable moving stage of a Nikon Eclipse Ti Series inverted microscope. The collimated white light beam from a xenon arc lamp coupled with an optical lens system was focused onto the plasmonic interferometer sensor chip, transmitted through the slit, and collected by an objective lens . The collected light was further dispersed by a monochromator and detected by a CCD camera. Transmitted spectra of the single slit accompanying each plasmonic interferometer were taken as well for spectral normalization.kth GSG interferometer and Each solid line in As shown in The sensing chip was functionalized and stored in a refrigerator at 4  results . Figure For sensing experiments, various concentrations of TREM2 in buffer solution were obtained by diluting a stock solution of TREM2 ) in 0.5% BSA PBS . BSA serves as a protein stabilizer to maintain the integrity of TREM2 at low concentration.TREM2 binding experiments were performed by directly dispensing 40 To understand the kinetic interaction of the binding between TREM2 and its antibody, we first consider an equilibrium model for the chemical reaction ,54,55,56complex] is the molar concentration of antibody-antigen complex, [Ab] is the molar concentration of unoccupied antibodies, [Ag] is the molar concentration of antigen in the solution, The time-dependent rate equation that governs this reaction can be expressed by:In addition, if we assume that the total number of antibodies immobilized at the sensor surface is fixed and the concentration of antigens is constant due to the large volume (or continuous dispensing) of the sample solution onto the surface, the following expression holds true at all times:quations  and 7)  (7)[Ab]0quations  and takiEquation  implies  factors ,58. Morea, which represents the total wavelength shift at equilibrium, increased with the concentration of antigens. These experimental findings are consistent with the simple model described by Equation  by using three of the four sensing spots of the chip, separately, and investigated the concentration dependence of the binding kinetics. Note that the chip was first regenerated to bare gold by 15 min of RCA1 cleaning, then functionalized and blocked again with BSA PBS before TREM2 detection. After regeneration, we confirmed that the normalized transmission spectra went back to the initial spectra, well within the confidence intervals reported in Interestingly, the binding reaction data corresponding to the higher concentration (8.1 ng/ml TREM2) displayed a kinetic curve that has a second slow rising stage, which could be fit with a double-exponential function:  surface ,59. The  surface . In addi surface ,62, espeAlthough a more complex model may be needed to fully describe all of the possible microscopic phenomena underlying the actual reaction kinetics, to first approximation the data presented in We demonstrate a biophotonic sensing platform to monitor the chemical reactions associated with surface functionalization of a metal in time series through wavelength shifts in the transmission spectra of plasmonic interferometers. This work helps us devise and assess a functionalization protocol for detecting TREM2, a biomarker associated with the development of AD and other neurodegenerative diseases, in solution. We devise a chemical equilibrium model which we fit with the reported data. The fitting parameters from the data reinforce the theoretical model and are consistent between experiments, suggesting that the experimental process is repeatable. The chemical equilibrium model allows us to generate calibration curves for the reported data which are able to differentiate between different concentrations of TREM2. The results reported here open up the possibility to employ the proposed sensing platform based on plasmonic interferometry for the detection of physiological concentrations of TREM2 in CSF or other bodily fluids. In the future, plasmonic interferometry may be a promising method to test functionalization protocols for deployment on other scalable platforms, such as colloidal nanoparticles, which are widely used in biological testing, to help validate the importance of TREM2 to the development pathway of AD. Additionally, plasmonic interferometry may be adapted to other surface functionalization protocols for different antibody-antigen pairs to enable development of a wide array of testing schemes for a variety of clinically relevant biomarkers.


Despite advances in diagnosis, imaging methods, and medical and surgical interventions, prosthetic valve endocarditis (PVE) remains an extremely serious and potentially fatal complication of heart valve surgery. Characteristic changes of PVE are more difficult to detect by transthoracic echocardiography (TTE) than those involving the native valve. We reviewed advances in transesophageal echocardiography (TEE) in the diagnosis of PVE. Three-dimensional (3D) TEE is becoming an increasingly available imaging method combined with two-dimensional TEE. It contributes to faster and more accurate diagnosis of PVE, assessment of PVE-related complications, monitoring effectiveness of antibiotic treatment, and determining optimal time for surgery, sometimes even before or without previous TTE. In this article, we present advances in the treatment of patients with mitral PVE due to 3D TEE application. Prosthetic valve endocarditis (PVE) remains an extremely serious and potentially fatal complication of heart valve surgery, associated with a mortality risk of about 20–30% ,2. Its iStaphylococcus aureus, less commonly by coagulase-negative Staphylococcus species) and late  [According to the time of infection occurrence since valve replacement surgery and specific microbiology, PVE can be classified into two types: early  . The timacteria) .More than two-thirds of PVE occurs during the first year after valve replacement ,5. MechaTreatment of PVE is directed at eradication of the infective agent with different antibiotic protocols, effective in about 50% of patients ,5. CardiAccording to current guidelines, echocardiography is considered the basic diagnostic method of PVE ,6. HowevIn this review, we present advances in the diagnosis of mitral PVE patients due to 3D TEE application.Two-dimensional TTE sensitivity and specificity for diagnosis of PVE are low due to limitations caused by acoustic shading, body habitus, and possible concomitant pulmonary pathology ,5,6,7,8.According to recent reports, 3D TEE provides clear, rapid, and detailed anatomical visualization of mechanical and bioprosthetic mitral valves in real time ensuring better assessment of mitral PVE-related complications and significant improvement in monitoring and preoperative surgical planning ,12,13,14oscillating intracardiac mass in accordance with vegetation,abscess formation,presence of paravalvular regurgitation/leakage or dehiscence of prosthetic valve.The ‘Duke criteria’, introduced in 1994 and modified in 2000, formalized the role of echocardiography in the diagnostic strategy of IE ,16. EchoEchocardiographers should keep in mind that the pathophysiology and echocardiographic presentation of PVE have a certain different specificity in relation to the type of prosthesis and the time elapsed since valve replacement. In mechanical PVE, infection usually begins early after replacement from the prosthetic ring suture line or from a nonbacterial thrombotic lesion near the recirculation flow area or prosthetic ring, leading to complications associated with a worse prognosis than late PVE requiring valve replacement ,3,5. BecWith TTE, it is more difficult to detect vegetation on PV than that involving native valves, because the highly echogenic suture ring and PV support structures can prevent detection of vegetation within the valve apparatus or its shadow ,7. ThereHowever, vegetative growth can occur as thickening and irregularity of the normally smooth contour of the suture ring  or in thAccording to the European Society of Cardiology guidelines, there are three main criteria for indicating emergent surgery to prevent embolism on the left PVE: PV with vegetation >10 mm after a previous embolic episode, very large isolated vegetation (>30 mm), and isolated vegetation with main diameter >15 mm and no other indication for surgery [Because the risk of embolism in patients with IE is related to the size, morphology, and mobility of vegetation, proper assessment of vegetation and monitoring of their size during antibiotic therapy have important prognostic implications ,11. TherIt is obvious that by providing better images in comparison to 2D TEE, 3D TEE allows detailed anatomical visualization of PV and better characterization of vegetation allowing their differentiation from non-PVE changes such as thrombi, pannus, prosthetic sutures, mitral subvalvular tissue remnants d,e, and Perivalvular complications are particularly common in early PVE because the annular ring is usually the primary site of infection ,4. The sPerivalvular abscess is more often associated with PVE of the aortic valve than with mitral PVE and is thought to be present when a specific area of reduced echo density is found on the echocardiogram or when echolucent cavities are found within the valvular ring or myocardial structures surrounding the valvular infection . In caseAlthough 2D TEE is a useful diagnostic tool in detection of paravalvular complications (sensitivity 90% and specificity 90%), 3D TEE provides anatomical imaging that can better identify location, number, shape, and size of damage ,13. ReceDiagnosis of PVE is challenging and includes identification of the infective pathogen and detection of cardiac lesions and noncardiac localization of the disease. Evidence of intracardiac material involvement is a major diagnostic criterion, with TEE representing the first-line imaging method . Higher Three-dimensional TEE has several limitations that include poor visualization of anterior cardiac structures, such as aortic and tricuspid valves, while mitral valve as a posterior structure due to reduced distance from TEE probe can be visualized significantly better; suboptimal images due to poor ECG triggering in patients with arrhythmias; reduced spatial and temporal resolution with narrow angled acquisitions; and tissue dropout . These lTwo-dimensional TEE detects signs of mitral PVE with excellent sensitivity and specificity. However, the higher quality of 3D compared to 2D images allows a more realistic anatomical view and a delineation of valvular vegetation and abscesses, perforations and paravalvular leaks, dehiscence, and localization of infection with regard to valve anatomy and its relationship with surrounding cardiac structures. Those providing additional information important for fast and accurate diagnosis and timely therapeutic decisions. Finally, the ability of 3D TEE to accurately display mitral PV from a left atrial perspective identical to that seen by the surgeon during surgery (surgeon’s view) allows better communication within the IE team.Conclusively, 3D TEE imaging improves visualization in the assessment of mitral PVE in terms of prognosis and selection of the best therapeutic options; as is the least costly and most affordable method.


Long non-coding RNAs (lncRNAs) exceed 200 nucleotides in length are considered to be involved in both developmental processes and various diseases. Here, we focus on lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), which was one of the most important lncRNAs in proliferation, apoptosis, and migration. MALAT1 plays a regulatory role in liver diseases, including hepatic fibrosis, liver regeneration, liver cancer, and fatty liver diseases. In the current review, we summarize the latest literature about the function roles of MALAT1 in liver disorders. Probing the regulatory mechanism and cross talk of MALAT1 with other signaling pathways of pathological processes would improve the prognosis, diagnosis of liver diseases, and offer a promising candidate target for therapeutic interventions. Seventy-five percent of the human genome generates transcripts, yet only about two percent of the genome encodes proteins. The majority of transcripts, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), do not encode proteins . Small nTo date, over 50,000 human lncRNAs have been identified . One of MALAT1 is preferentially associated with transcriptionally active genes in the nucleus . MALAT1 MicroRNAs bind to the 3'UTR of their target genes and negatively regulate gene expression through depressing translation or promoting mRNA attenuation. This strategy was proposed initially by Poliseno to explain how mRNAs communicate with lncRNAs through miRNA response elements (MREs) as “language” . In the MALAT1 binds to chromatin of activated transcribing genes, transcription factors and transcriptional co-activators and regulates their expression at the transcriptional level to promote cell proliferation and inhibit cell apoptosis. MALAT1 was also triggered to upregulate transcriptional activators of proteosome genes .Hepatic fibrosis is characterized by chronic abnormal hyperplasia and accumulation of large amounts of extracellular matrix (ECM) [including smooth muscle actin (SMA) and type I collagen], and the release of proinflammatory and profibrotic factors. Fibrosis can result from a variety of chronic liver diseases such as viral hepatitis, alcoholism, drug abuse, metabolic syndrome, genetic metabolic diseases, and autoimmune hepatitis . The regThe normal liver has a strong regenerative ability and can maintain the original liver volume by inducing mitosis of its own cells, or tissue repair through rapid division and proliferation from endogenous liver stem cells  to mature liver cells . BecauseLiver regeneration is initiated immediately after liver resection, and upregulation of hepatocyte growth factor (HGF) induces the expression of MALAT1, damaging the stability of the p-catenin degradation complex . The resThe expression of MALAT1 is significantly increased in HCC tissues and cell lines, which promotes HCC proliferation and metastasis and inhibits HCC cell apoptosis by acting as an oncogene –49. SileMALAT1 plays a role in HCC cell proliferation and apoptosis by multiple pathways. Upregulation of MALAT1 promotes cancer cell proliferation, and its downregulation promotes cancer cell death and autophagy. Malakar et al.  found thHCC invasion and metastasis are aggravated via signaling pathways and MALAT1. YAP1 in vascular endothelial cells reduces vascular proliferation . ExosomeMALAT1 also promotes glycolysis in HCC. Aerobic glycolysis (Warburg effect) is a marker of tumor cells' ability to escape apoptosis to promote proliferation and migration. It could provide material basis for tumor cells and create an acidic microenvironment , 60. MalCancer stem cells (CSCs) are characterized by self-renewal and differentiation and are considered the seeds of tumor genesis, development, and metastasis. MALAT1 promotes HCC stem cell properties. The higher the proportion of CSCs, the more aggressive the tumor . He et aMALAT1 significantly inhibits palmitic acid-induced lipid accumulation and increases expression of SREBP-1c, an important regulator of cholesterol and fatty acid synthesis in abnormal lipid metabolism and fatty liver disease , 70. MALLncRNA is a hot research topic in the field of liver disease in recent years. As a member of the lncRNA family, MALAT1 is a multi-functional lncRNA and an important regulator in hepatic fibrosis, liver regeneration, cancer, and fatty liver diseases. The molecular mechanisms mediated by one lncRNA would be complicated. Compared to most lncRNAs, MALAT1 is expressed at relatively high level in almost all human tissues in a variety of regulating pathways, thus makes it intricate to be targeted by simply silencing or overexpressing in pathological conditions. Further challenge to therapeutically measures would include small molecules specifically designed to intervene the gene-protein interaction for MALAT1 functions . A deepeJLu and KX structured the text and content. JLu and JG wrote and edited the manuscript. JLi generated the figures. XM reviewed the literature. All authors contributed to the article and approved the submitted version.This research was funded by the Independent Project Fund of the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the National Key Research and Development Program of China (No. 2016YFC1101404/3), and Zhejiang Basic Public Welfare Research Program of China (No. LQ20H030012).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.


Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a heritable renal disease that results in end-stage kidney disease, due to the uncontrolled bilateral growth of cysts throughout the kidneys. While it is known that a mutation within a PKD-causing gene is required for the development of ADPKD, the underlying mechanism(s) causing cystogenesis and progression of the disease are not well understood. Limited therapeutic options are currently available to slow the rate of cystic growth. Epigenetic modifications, including DNA methylation, are known to be altered in neoplasia, and several FDA-approved therapeutics target these disease-specific changes. As there are many similarities between ADPKD and neoplasia, we (and others) have postulated that ADPKD kidneys contain alterations to their epigenetic landscape that could be exploited for future therapeutic discovery. Here we summarise the current understanding of epigenetic changes that are associated with ADPKD, with a particular focus on the burgeoning field of ADPKD-specific alterations in DNA methylation.  Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common heritable renal disease in humans. Characterised by the development of large, fluid-filled cysts in both kidneys, this disease has an estimated prevalence of three cases per 10,000 , to patiAs a consequence of cyst growth, renal function is impaired. It is assumed that either the destruction of the renal parenchyma , the fibADPKD is associated with significant morbidity, and patients have a reduced life expectancy ,13 when PKD1 is the most prevalent disease-causing gene for ADPKD, with approximately 85% of patients having mutations within this locus. Mutations in PKD2 account for most of the remaining 15% of patients [GANAB [DNAJB11 [de novo mutations in PKD1 [ADPKD is genetically heterogeneous—a germline mutation within one of a handful of PKD-causing genes is a prerequisite for patients to develop the disease. patients . More res [GANAB  and DNAJ[DNAJB11 , which a in PKD1 .PKD1 or PKD2; mutations are highly variable, are spread across each gene, and are typically unique within families. A database of disease-causing mutations within PKD1 and PKD2 has been established (pkdb.mayo.edu (accessed on 30 October 2021)) [There is no mutation ‘hot-spot’ within PKD1 or PKD2 mutations can explain the initiation of cyst growth over time, and several studies have identified somatic “second hit” mutations in isolated cysts [While these germline mutations correlate with the development of ADPKD, they are still insufficient to fully explain the process of cystogenesis. Although each kidney’s approximately one million nephrons all carry the germline PKD-causing mutation, less than 1% of these will develop cysts . Varioused cysts ,27,28,29ed cysts . The neted cysts .‘neoplasia in disguise’ [The uncontrolled growth of cysts that occurs in ADPKD has clear parallels with cancer. Cysts are effectively benign neoplasms , and theisguise’ . Featureisguise’ ,34, and isguise’ .An underexplored parallel between these two disease states includes changes in epigenetic modifications, which can result in large scale gene expression changes. Exploring epigenetic alterations in ADPKD may not only shed light on the process of cystogenesis and cyst growth, but could lead to new therapeutic options being available to slow the course of this disease.In addition to germline or somatic mutations, relatively stable alterations in gene expression can occur through epigenetic mechanisms. These are molecular changes that occur to the DNA molecule without changing the genetic sequence. While epigenetic mechanisms are essential for normal cellular function/development , these pDuring development, epigenetic mechanisms play a role in cellular growth and differentiation, and as cells mature, these epigenetic modifications change to suit the role of the cell. Once the cell differentiates, epigenetic modifications are specific for each cell type. These modifications (and any subsequent disease-causing epigenetic changes) can be inherited through progressive cell lineages.Two key forms of epigenetic regulation are histone modification (such as acetylation and methylation of histone proteins) and DNA methylation. These mechanisms act to modify the chromatin state and accessibility of the DNA to transcription factors . An assoBCL2 and the oncogene MYC [MicroRNAs (miRNAs) are a class of non-coding RNAs that are approximately 20 nucleotides long. These sequences function as post-translational regulators to alter protein expression by destabilising mRNA translation . It has gene MYC .Previous research in renal development has confirmed that miRNAs are required for embryonic development of the kidney, and several miRNAs have been implicated in glomerular and tubular diseases . ScreeniInhibition of the miR-17~92 cluster has been demonstrated to reduce cyst proliferation in ADPKD mice . FurtherPKD1 gene itself houses several miRNA genes  was performed in five human ADPKD kidney samples and three non-ADPKD samples [PKD1, 2.6 kb from the transcription start site; see While miRNAs that are associated with ADPKD are located at disparate sites throughout the genome, the enes see . To deteIn the nucleus, DNA is wound around histone protein octamers, which allow for the tight packaging and organisation of the DNA molecule within the nucleus. The N- and C-terminal portions of the histone proteins (histone ‘tails’), play a central role in this process, guided by a range of chemical modifications. These post-translational modifications determine how closely the histones are packaged together, in turn facilitating how freely DNA can be transcribed.There are three main modifications to the histone tails: phosphorylation, methylation and acetylation . There iPKD1 promoter, an effect which has shown to be attenuated following HDAC inhibitor treatment [PKD1-dependent fluid stress-sensing in renal epithelial cells, in which the PKD1 protein product, polycystin, facilitates calcium influx into the cell and subsequent protein kinase C phosphorylation of HDAC5 [Pkd1 cells and activates a number of different factors associated with cyst growth, such as EGFR and EGF-induced β-catenin nuclear localisation. Inhibition of HDAC6 prevents the release of Ca2+ from the endoplasmic reticulum, consequentially attenuating cyst growth through cAMP signalling [Pkd1 mutant cells. Treatment with a specific SIRT1 inhibitor decreased proliferation and increased apoptosis of cystic epithelial cells [A number of studies suggest that HDACs are regulators of PKD genes and/or the signalling pathways that are involved in cystogenesis. HDACs mediate p53-induced repression of the reatment . HDAC5 hof HDAC5 . HDAC6 ignalling . Additioal cells .Pkd1 mutant and wildtype cells [Numerous studies have identified the activity of HDACs as a driver of neoplasia, due to the aberrant expression of HDACs in tumours , and thepe cells .DNA methylation is a stable and somatically heritable epigenetic mark which plays a regulatory role in shaping the phenotype of a cell, and has been heavily implicated in human disease ,67. DNA Methylation within a gene promoter has historically been associated with repression of the gene, by reducing the ability of transcription factors to bind . One mecMethylation of CpGs within the gene body is not as well understood as that of the promoter, but typically results in sustained or increased expression of a gene ,72. A vaThe pathology of several diseases, most notably cancers, has been linked to the dysregulation of DNA methylation both globally and locally . As suchPKD1 gene body (exon 43) and demonstrated hypermethylation in ADPKD samples, negatively correlating this with the expression of the gene. Demethylation of an immortalised cell line (WT 9–12) resulted in increased PKD1 mRNA expression, and treatment of the cyst-forming cell line MDCK with a DNMT inhibitor repressed the growth of cysts [A 2014 paper by Woo et al.  was the of cysts .MUPCDH (mucin-like protocadherin) gene to be hypermethylated in its promoter, and this was associated with significant repression of MUPCDH gene expression in ADPKD samples. The authors suggested that the methylation status of the MUPCDH promoter could be used as a novel epigenetic biomarker and a therapeutic target in ADPKD.It was theorised by Woo et al.  that if PKD1 gene body was hypermethylated in ADPKD samples from the RRBS study, but this was associated with an increase rather than a decrease in PKD1 mRNA expression [PKD1 promoter hypermethylation inversely correlated with gene expression in patient blood, further indicating a role of DNA methylation in PKD1 expression.In contrast to Woo et al. ., Bowdenpression . A laterpression  demonstrDNA methylation plays a key role in gene expression modulation during disease progression, as shown in the development of aggressive metastatic tumours . In the PKD1  studies. A global analysis using dot blot and immunochemistry methods reported that global levels of 5-hydroxymethylcytosine were reduced in mouse kidneys affected by ischemia reperfusion injury (IRI). However, the level of DNA methylation (5-methylcytosine) was reported to be globally unchanged in ischemia reperfusion . A subseCell lines generated from cystic tissue have also been used to study DNA methylation in ADPKD . HoweverIn contrast to that which was observed with immortalised cell lines, Kenter et al.  generateAnother variable among these DNA methylation data sets is the source material. Patient tissue is typically from ESKD patients at around age 50, at which point a multitude of environmental factors are likely to have impacted each patient’s methylome . Additionally, each study discussed has been generated from patient data within a defined population, which could also contribute to these differences.ADPKD is a disease that could be characterised as cysts arising from different nephron segments. Thus, a “cyst” sample taken from tissue is very likely to represent different nephron segments and therefore could be heterogenous. Large levels of variation in DNA methylation have been observed between cysts taken from a single patient . This suEpigenetics has increasingly become an area of interest for researchers working on ADPKD, as the identification of pathways associated with cystogenesis becomes increasingly complex. New data that can shed light on additional mechanisms of disease gives us hope that additional therapeutic strategies are possible for this relatively common genetic disorder.PKD1 gene itself, provides evidence of an epigenetic influence in this disease process, yet there is no clear direction for targeting treatments at this stage. However, the fact that both HDAC and DNMT inhibitors have been identified in a recent high-throughput screening platform for polycystic kidney disease drug discovery [While novel experimental approaches have characterised the epigenome of ADPKD kidneys, more work needs to be done to establish a consensus of DNA methylation changes in ADPKD. Conflicting data on the methylation status of the genome, as well as within the iscovery  suggestsA solution to help disentangle DNA methylation variables may be to perform multiple methodologies on biological replicates, however this does not solve the larger issue of DNA methylation methodologies each containing their own internal biases. Some standardisation of the DNA methylation field is required to develop synchrony between research groups, to more efficiently assist the analyses of patient material and to provide a clear path forward.


Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications. Branched chain amino acids (BCAAs), valine , leucine (Leu) and isoleucine (Ile), are essential amino acids that cannot be synthesized by animals, but only from bacteria, plants and fungi and must be obtained from diet sources. These amino acids have branched hydrophobic side chains and play a primary role in protein structure . All thrThe first step in BCAA catabolism is a reversible transamination process in which the BCAAs are converted to their branched-chain α-keto acids (BCKAs), 2-ketoisocaproate (KIC), 2-keto-3-methylvalerate (KMV), 2-ketoisovalerate (KIV) from leucine, isoleucine and valine, respectively A. This r2 and NADH. By this process, the carbon skeletons of KIC, KMV and KIV are converted to isovaleryl-CoA, 2-methylbutyryl-CoA and isobutyryl-CoA, respectively (2β2) decarboxylase (E1) that catalyzes the oxidative decarboxylation of the BCKAs, a dihydrolipoyltransacylase (E2) to transfer the acyl groups to CoA and a FAD-dependent dihydrolipoyl dehydrogenase (E3) to transfer the released electrons to NAD+ [+ and of acyl-CoA/CoA-SH as well as ATP [The second step in BCAA catabolism is the irreversible oxidative decarboxylation of the α-ketoacids to their respective branched-chain acyl-CoA esters, COectively A. This r to NAD+   by the enzyme 2-ketoisocaproate dioxygenase (KIC dioxygenase)  Figure B. BCAAs and especially leucine induce a number of metabolic and signaling functions, particularly via the activation of the “so called” mechanistic Target Of Rapamycin (mTOR) signaling pathway. mTOR is a protein kinase and acts as a central regulator of many fundamental cell processes from protein synthesis to autophagy and glucose homeostasis, while impaired mTOR signaling has been involved in the progression of pathological conditions such as cancer and diabetes as well as in aging . mTOR isIncreased intracellular levels of amino acids and especially leucine, promote the activation of mTORC1 through the active complex of Rag guanosine triphosphatases (GTPases) consisting of GTP-bound RagA/B and GDP-bound RagC/D  Figure . The binMoreover, leucyl-tRNA synthetase (LRS) functions as another leucine sensor for mTORC1 signaling. LRS is translocated to lysosomes where it acts as a GAP promoting the formation of GDP-RagC/D by the GTP hydrolysis of RagC/D, leading to mTORC1 activation . Thus, LMaple syrup urine disease (MSUD) or branched chain α-ketoacid dehydrogenase deficiency, is an autosomal recessive disorder, caused by mutations in the subunits of the BCKD complex A and occCurrent management for MSUD patients includes dietary modifications with limited protein consumption and BCAA-free medical formulas and in some cases liver transplantation to restore BCKD activity and BCAA homeostasis . DisruptBCAA homeostasis is of major importance for brain function as the accumulation of BCAAs and of toxic BCKAs is associated with severe neurologic diseases. As a consequence, a better understanding of the mechanisms that connect BCAA metabolism with the central nervous system may provide novel therapeutic approaches in MSUD.+ ratio due to enhanced fatty acid oxidation may contribute to the impaired BCAA catabolism as a consequence of the decreased supply of amino group acceptors  and the inhibitory effect of NADH and acyl-CoAs on BCKD [It has been proven repeatedly that in animal models of T1DM ,33,34 an on BCKD ,39.Increased plasma BCAAs in obesity and insulin resistance states were reported for the first time in the 1960s ,41. ThesIn the adipose tissue of obese and insulin resistant humans and animals, the expression of genes encoding the enzymes BCAT2 and BCKD, that catalyze the first two steps of BCAA catabolism as mentioned above, are significantly suppressed ,46 with In recent years, genome-wide association studies have revealed common genetic variants that correlate with blood amino acid levels. It is reported that mutations in the PPM1K gene in humans and rodents that cause defects in PP2Cm production, the only BCKD phosphatase, result in a defective activation of the BCKD complex  Figure . TherefoPrevotella copri and Bacteroides vulgatus and deprivation of genes encoding the transport system for bacterial BCAA uptake, results in increased plasma BCAAs [In insulin resistance states, dysbiosis in the human gut microbiome, e.g., gut enriched with the main drivers of the gut bacterial biosynthesis of BCAAs ma BCAAs  , an important regulator of de novo lipogenesis. ACL promotes increased formation of the lipogenic substrates, acetyl-CoA and malonyl-CoA, which result in the elevated fatty acid synthesis and dyslipidemia that contribute to insulin resistance .It has been documented that the activity of BCAAs catabolic enzymes greatly increase over the course of adipocyte differentiation ,56. BCAAIt is proposed that disturbed BCAA catabolism in adipose tissue and liver promote a shift of BCAA flow to skeletal muscle turning this tissue into the main BCAA oxidation site . SeveralIn addition, an excess of BCAA oxidation in skeletal muscle that depletes intracellular glycine  and reduFurthermore, increased BCAA oxidation in skeletal muscle results in the elevated production of 3-Hydroxyisobutyrate (3-HIB), a catabolic intermediate of valine, which acts as a paracrine factor, leads to increased endothelial fatty acid uptake, accumulation of incompletely esterified lipids in skeletal muscle and consequently lipotoxicity and impaired insulin signaling  Figure . MoreoveBCAAs have emerged as predictive markers for future risk of T2DM ,66 and pChronic hyperglycemia leads to long-term complications, such as nephropathy and renal failure that contribute to increased disability and reduced life expectancy in patients with diabetes . It is rAcross gestation, the sum of plasma BCAAs decreases while the BCKAs are found either unaltered  or decreased (2-ketoisocaproate). In human placental tissue, high activity of BCATs has been detected indicating that BCAAs are transaminated for placental nitrogen demands such as glutamate synthesis . AlthougIn experimental studies, obese and insulin resistant Zucker rats treated with thiazolidinedione (TZD) drugs showed an improvement in insulin sensitivity and in BCAA metabolic pathways in adipose tissue . More spBCAA catabolism has also been investigated in cardiovascular disease and heart failure. Higher plasma BCAAs as well as their catabolic products are associated with increased risk of cardiovascular disease ,84. BothAD is a chronic neurodegenerative disease and the predominant cause of dementia. It is characterized by extracellular beta-amyloid (Aβ) deposits and intracellular hyperphosphorylated Tau proteins . Recent Abnormal BCAA levels have also been reported in patients diagnosed with cancer. Increased plasma BCAAs were found in patients 2–5 years before the diagnosis of pancreatic ductal adenocarcinoma (PDAC) and these elevations were associated with an up to two-fold increase in the risk for the development of pancreatic cancer . Subclinwt gliomas while in IDHmut gliomas it was suppressed [mut enzymes. The oncometabolite 2-HG reduces the activity of 2-KG-dependent enzymes such as histone demethylases, leading to widespread hypermethylation of the BCAT1 promoter region and thus to the BCAT1 suppression [mut glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo, suggesting an effective role of this inhibition against IDHmut gliomas [Several studies focus on the activity of BCAT1 in cancer, as alteration in the expression of this enzyme is differentiated among the cancer types and is associated with tumor aggressiveness ,100. Trappressed . This supression ,100. Bre gliomas .wt acute myeloid leukemia, higher BCAT1 levels are associated with shorter survival and display a DNA hypermethylation phenotype similar to previously described IDH mutations. These findings suggest that BCAT1 stabilizes HIF1α protein, which is required for the maintenance of leukemia stem cells, by limiting intracellular 2-KG and regulates the epigenomic landscape [In leukemia cells, overexpression of BCAT1 decreases the intracellular 2-KG and causes DNA hypermethylation through inactivation of 2-KG-dependent enzymes and especially TET (ten-eleven translocation family of DNA demethylases) . In IDHwandscape . In chroandscape .Tumor tissues of patients with hepatocellular carcinoma (HCC), compared to adjacent non-tumor tissues, displayed a significant increase in BCAAs but a reduction in their oxidized metabolic intermediates, indicating impaired liver BCAA catabolism . MoreoveIn contrast, breast cancer displays enhanced BCAA catabolism with higher gene expression of BCAT1, BCKD and other downstream enzymes . Thus, iThese findings indicate the major role of BCAT1 in tumor proliferation and that its expression in different cancer types may possibly serve as a therapeutic target. In addition, in the presence of cachectic disorder in cancer patients, supplementation of BCAAs improves the quality of life, due to their anabolic effect, and subsequently the effectiveness of the chemotherapeutic interventions .Decreased BCAAs and elevated aromatic amino acids represent characteristic alterations in blood of cirrhotic patients along with an increased risk of hepatic encephalopathy and muscle wasting disorders . The leaIn hepatitis B, disturbed amino acid metabolism, expressed mainly as increased tyrosine and decreased BCAA levels, results in a significantly lower ratio of BCAAs to tyrosine (BTR) that is correlated with the type and stage of HBV infection . BTR canElevated plasma BCAA levels have been recorded in patients with nonalcoholic fatty liver disease (NAFLD); however, it remains unknown if this is due to increased muscular proteolysis, obesity, and/or increased insulin resistance or impaired liver and adipose tissue catabolism of these amino acids . FurtherBCAA supplementation may have beneficial effects in patients with chronic liver diseases. In cirrhotic patients, it improves the nutritional status, weakness and fatigue and reduces the incidence of hepatic encephalopathy and HCC . MoreoveFinally, in chronic renal failure, plasma BCAAs and BCKAs as well as valine levels in muscles are found significantly decreased ,119. In Recent approaches have emphasized the role of BCAAs not only as important constituents for protein synthesis but also as important regulators of whole-body metabolism. BCAA-rich diets may have a beneficial role in metabolic health, mainly due to their anabolic effect, but further investigation is needed regarding the relationship of elevated BCAAs with a wide range of pathological conditions. Apart from the well-characterized Mendelian disorders, defective BCAA catabolism is linked to the pathogenesis and progression of many prevalent diseases. A better understanding of the mechanisms underlying the role of BCAAs in these pathological conditions may be helpful and may lead to better outcomes possibly by “restoring” BCAA metabolism.


Metzincins are a superfamily of metalloproteinases characterized by the presence of a conserved methionine residue downstream the active site zinc . This FrRose et al. comprehensively review the current knowledge about regulatory mechanisms of ADAMTS activity. Protein levels and activity of ADAMTSs can be modulated at different levels, including transcriptional regulation, alternative splicing, proteolytic activation, endocytosis, and inhibition by tissue inhibitors of metalloproteinases (TIMPs). A deeper understanding of such regulatory mechanisms can lead to new strategies to target the pathological potential of specific ADAMTSs without affecting the physiological activity of other family members. The contribution by Jiang et al. provides updated insights on multiple layers of regulation of one of the best characterized ADAMTS family members, ADAMTS5. ADAMTS5 is an important pharmaceutical target in osteoarthritis (OA) due to its ability to cleave aggrecan, the major proteoglycan in articular cartilage . While a fine regulation of their activity ensures tissue homeostasis, several pathological conditions are associated with their aberrant activity. artilage . Since nMohamedi et al. overview this complex and multifunctional metalloproteinase on the 20th anniversary of its identification. The authors discuss functions of ADAMTS12 in inflammatory processes, arthritis, degenerative intervertebral disc, chondrogenesis, tendon degeneration, cancer, neurological and fertility disorders. Different domains of ADAMTS12 can interact with different ECM components, which in turn modify its proteolytic activity. The authors emphasize that identification of ADAMTS12-binding partners would contribute to a deeper understanding of its biological functions.ADAMTS12 is another example of how we are just starting to scratch the surface of ADAMTS complexity. Leduc et al. investigated substrates in vivo by directly comparing skin from Adamt2−/−, Adamts14−/− and Adamts2−/−/Adamts14−/− knockout mice with those of wild type littermates. Their results confirm the cleavage site preference (P1 and P1′ residues) of these proteases and provide more detail about their role in collagen fibril organization. As often in proteomics, further studies are required to assess the relevance of these intriguing proteolytic events and novel substrates.A central aspect of this Research Topic is the identification of novel metzincin substrates using state-of-the-art proteomics approach. In the last decade, mass spectrometry-based techniques have dramatically expanded our knowledge of proteases’ substrate repertoire, the degradome . The mosPedrina and Stambas, the ECM offers novel opportunities for intervention. The proteoglycan versican is a major ECM component and has been shown to be involved in the immune response to viral infection by binding chemokines that guide leukocyte extravasation. Versican also provides a barrier for leukocyte migration, as summarized in the contribution by McMahon et al. ADAMTS4 and ADAMTS5 are the two major versicanases (Adamts5 knockout mice (The current pandemic has stressed the need for better intervention strategies aiming at the prevention and treatment of infections caused by respiratory viruses. As highlighted by icanases , so it dout mice . On the out mice , suggestout mice  and thesout mice , a caveaout mice .Gellrich et al. characterized these two variants and found that, similar to wild type meprin β, the G45R variant is able to shed a number of specific substrates and promote invasion when expressed in HeLa cancer cells. On the other hand, the G89R variant showed impaired trafficking to the cell surface, reduced shedding of its target substrates and diminished cancer cell invasion. A similar mechanistic approach can be extended to elucidate the activity of other meprin β variants (Like ADAMs, Astacins can cleave membrane-tethered proteins and therefore act as sheddases . Meprin variants .Overall, the studies included in this Research Topic expand our knowledge of the role and regulation of ADAMs, ADAMTSs and Astacins in a variety of biological processes, something that would not have been possible without the powerful new -Omics tools that have been widely adopted in the last decade.


Chlamydomonas, regulating its inducible carbon concentration mechanism that is dependent on cellular machineries of transcription, protein folding, and retrograde signalling, is reviewed here.The interplay of photosynthesis with carbon acquisition and fixation in  Chlamydomonas reinhardtii has been well defined from a molecular and ultrastructural perspective. Inorganic carbon transport proteins, and strategically located carbonic anhydrases deliver CO2 within the chloroplast pyrenoid matrix where Rubisco is packaged. However, there is little understanding of the fundamental signalling and sensing processes leading to CCM induction. While external CO2 limitation has been believed to be the primary cue, the coupling between energetic supply and inorganic carbon demand through regulatory feedback from light harvesting and photorespiration signals could provide the original CCM trigger. Key questions regarding the integration of these processes are addressed in this review. We consider how the chloroplast functions as a crucible for photosynthesis, importing and integrating nuclear-encoded components from the cytoplasm, and sending retrograde signals to the nucleus to regulate CCM induction. We hypothesize that induction of the CCM is associated with retrograde signals associated with photorespiration and/or light stress. We have also examined the significance of common evolutionary pressures for origins of two co-regulated processes, namely the CCM and photorespiration, in addition to identifying genes of interest involved in transcription, protein folding, and regulatory processes which are needed to fully understand the processes leading to CCM induction.The inducible carbon concentration mechanism (CCM) in  Inorganic carbon, in the form of bicarbonate, is delivered to the chloroplast stroma using a series of membrane transporters. Saturating internal CO ambient . The avaSubstantial molecular and mechanistic advances in our understanding of the algal CCM have been recently reviewed . CCM indChlamydomonas and identify future avenues for investigation. First, we consider the control of nuclear gene expression and the need to identify transcription factors (TFs) associated with sensing the different environmental stimuli—light and CO2; second, regulation of export of translated proteins and folding within the chloroplast, and associated chaperone systems; third, formation of the pyrenoid matrix, starch sheath, and intrapyrenoidal tubule network; fourth, the role of retrograde signalling in delivering signals to alter nuclear gene expression; and, finally, we discuss the potential evolution of CCM induction from existing photorespiration regulatory mechanisms, through the master regulator CIA5.The aim of this review is to characterize the various novel aspects of molecular mechanisms leading to mechanisms of CCM induction and establishment. We explore the regulatory interplay between environmental sensing, photosynthesis, and CCM induction that is critical for 2] in the external medium have traditionally been thought to be sensed through Ci of the photosynthesizing algal cell and conveyed to the nucleus to change the expression of genes that turn on/off the CCM. Studies carried out with asynchronous cells grown under continuous light revealed [CO2]-dependent expression for >5000 genes at the transcription level  how do they bring about changes at the transcriptional level? In this section, we discuss regulatory mechanisms operating at the transcriptional level to modulate the inducible CCM in Chlamydomonas.Changes in   CCM into higher plants might require tinkering with PR processes, in addition to introducing various CCM components   , these proteins are conserved across photosynthetic organisms, including three homologues in Chlamydomonas  have emerged as important modulators of thylakoid membrane bending and plasticity. First identified in d matrix .Chlamydomonas pyrenoid   . RNA-seqgun4 mutant , bestrophin-3, three CAs , and the chaperone DNJ15. That PR, mitochondrial CAs, and PET proteins are influenced by GUN4 further strengthens the crosstalk between these processes which has been highlighted in the section- ‘CCM induction and control of gene expression’. GUN4-influenced CCM genes also include DNJ15, implying protein homeostasis regulation by retrograde signalling. Seven of the CCM genes in While the role of retrograde signalling in regulating photosynthesis is accepted, we checked if it also plays a part in modulating the CCM. We compared the 803 differentially expressed genes in the 4 mutant . This li1O2). Modified and degraded GUN4 products are hypothesized to initiate the retrograde signalling to nucleus  is a chloroplastic thylakoid membrane protein that mediates signalling, as part of acclimation to high-light and low-carbon (LC) conditions  in DNJ31, encoding a DnaJ chaperone discussed above, is also influenced by CAS. These expression features of DNJ31 make it an interesting candidate requiring its functional characterization and identification of its substrates. Two mitochondrial carbonic anhydrases  and two mitochondrial envelope proteins (CCP1 and CCP2) of unknown function  genes, and the GDC-H gene. GDC-H is in a GDC complex with two other subunits (GDC-T and GDC-P) that are encoded by genes which are part of ‘CCM clusters’. PGP1 has already been discussed above. Although the PGP genes PGP1, PGP2, and PGP3 did not show CIA5-dependent differential expression  or product (glycolate) as CCM induction signalling molecules. 2-PG was suggested as a potential signalling molecule in a pgp1 algal mutant study  in the same study. This similarity in expression profiles dependent on [CO2] changes and the presence of a common TF led us to look at the evolutionary origins of these processes. These studies provide compelling evidence that the stimulus for the CCM is likely to be determined by interactions between light intensity and PR activity when external CO2 is limiting.A recent study (Chlamydomonas) from a common ancestor for all green plants and algae is dated ~700 million years ago (mya) (4 and Crassulacean acid metabolism (CAM) plants]  (go (mya) . The end4/CAM have greater affinity for O2 (i.e. a lower specificity factor) than those lacking concentration mechanisms  to identify the signalling mechanisms which orchestrate the biophysical CCM.Major questions still remain, such as the origins and regulatory processes leading to the knotted thylakoid tubule network at the heart of the pyrenoid, the formation of associated connective minitubules, and spatial segregation between PSI and PSII. The energetic balance between CO