Connexins are proteins which comprise gap junctions in cells. These junctions can directly connect neighboring cells and the cell interior with the extracellular microenvironment and thus they act as tissue integrators. Alterations in connexin regulation can lead to unfavorable shifts in the tissue adhesive context thus eradicating the constraints of the normal tissue microenvironment, triggering (or enhancing) cell motility. This review tries to examine the role of connexins in orchestrating the tumor microenvironment and hence their role in malignancy.Today’s research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression. Modern cancer research is focused on the identification of the mechanisms of cancer initiation, promotion and progression to further develop efficient treatment strategies. Accumulating experimental data testify to the tremendous role of the tissue microenvironment in tumor development and the formation of the unique tumor microenvironment (TME) ,2,3. TMEConnexins (Cxs) are proteins that form gap junctions (GJ) in cells of chordate organisms by assembling connexin monomers into hexameric hemichannels (connexins), that can function both autonomously, or undergo coupling to form a full-fledged channel that directly connects adjacent cells. To date 21 connexin isoforms have been discovered in humans . The mosGenes of connexins are shown to have one or multiple 5′UTR (untranslated region) exons which are separated from the exon containing the coding sequence by an intron of variable size. In some connexin genes, coding sequence may be interrupted by an additional intron. This allows a great variety of alternatively spliced connexin mRNA variants . The traThe mutations in connexin gene family, both germline and somatic, have been established to be associated with multiple developmental abnormalities and syndromes. Nevertheless, the link between connexin mutations and cancer is still not clear. Several studies have revealed the association of certain type of tumors with mutated forms of connexins. Thus, the rate of mutated GJA1 gene (coding Cx43) is increased in colon adenocarcinomas , stomachConnexins are tetraspan proteins, possessing four transmembrane domains, two extracellular loops and one intracellular loop; and intracellular N- and C-terminal tails . The traThe extracellular loops are the most conserved regions of the connexin molecule, and are responsible for connexins docking. They possess consensus patterns with three characteristic cysteines in each loop . These cConnexin oligomerization can occur by two routes, determined by a signature motif in a region of the cytoplasmic loop transition to TM3. The first route is characteristic of alpha-connexins: they are stabilized in a monomeric state by quality-control proteins and their further oligomerization takes place in the trans-Golgi network. This is determined by a conserved arginine or lysine residue in the cytoplasmic loop–TM3 transition region . The sec2+, K+, bicarbonate), secondary messengers , reactive oxygen species (ROS), small molecules , peptides and microRNAs [Connexin channels mediate the passage of substances with molecular weights not exceeding 1.2 kDa: ions , underpConnexin junctions directly connect cells in the TME, so connexin localization in the cell is an important issue as it determines cell behavior . The locConnexin channels usually assemble at highly organized gap junction plaques and are associated with cholesterol and sphingolipids in lipid rafts . BesidesIn sum, the direct interaction and mutual regulation of different junctional proteins, their contribution to cell polarity and participation in motility, when disrupted can constitute dysplasia-promoting conditions in tissues. This can lead to tissue malignization and in well-established cancer helps to adapt to variable conditions within the tumor and participate in tumor heterogeneity. A plethora of controversial evidence on the role of different types of intercellular junctions and cell–ECM contacts in cancer have conceived the view that each of them is a double-edged sword ,55,56,57Cx43 often localizes to the tip areas of thin cell surface protrusions, tunneling nanotubes (TNTs). TNTs possess an actin core, their length reaches several cell diameters, and the diameter does not exceed 1 µm. TNTs are common in aggressive tumors at advanced stages, they directly connect distant cells within the TME and thus are involved in their coordination, development of resistance of normal and malignant cells to necrosis and apoptosis, act as a means to overcome hypoxia and mediate resistance to therapeutics by rapid drug efflux ,59,60,61In MCF-7 breast cancer cells, TNTs were reported to be an important means of spheroid formation in low-adhesive conditions in vitro. At its early stages, TNTs enable physical adhesion in suspended cells, establishing Cx43 contacts with further E-cadherin expression . Similar2+ waves [Taking into account that Cx43 positively regulates TNT formation, one can assume that it mediates not just the opportunistic connection of two initially juxtaposed cells but also the active seeking of ‘partner cells’. Cx43, located on the tips of interpericyte TNTs, regulates the coupling of different capillary systems within the brain niche by the controlled propagation of Ca2+ waves . Pericyt2+ waves . In cancExosomes are extracellular lipid bilayer vesicles with an average size of 100 nm produced by most eukaryotic cells and carry bioactive cargo of various nature . Tumor-derived exosomes are one of the major contributors to the TME and long-distance cancer signaling ,70. InteConnexins have been indicated to localize in exosomes, and associate  with exosomal characteristic proteins, tetraspanins . ExosomaConnexins may determine the mechanism of exosome loading with RNA and DNA. Using bioinformatic analysis, it has been suggested that Cx43 and Cx26 possess RNA- and DNA-binding motifs, potentially important for the transmission of genetic information between cells through extracellular vesicles ,76. Cx43The role of connexins in exosomal pro-tumorigenic properties is still to be elucidated, but now one can assume that it is mostly determined by the rapid cargo release into recipient cells which can either enhance the malignant potential of cancer cells or precondition a metastatic niche. Interesting speculation about the role of exosomal connexins in cancer signaling was recently suggested by Shimaoka and colleagues: Connexin channels may only release a small portion of the exosomal contents into recipient cells and this event is the decisive point in determining whether a cell will uptake the whole exosome contents by endocytosis or not .Connexin functioning as a hemichannel has a separate contribution to the TME and the development of cancer. Hemichannels can participate in the uptake of beneficial molecules  or provide the uptake of glutamate, which promotes cell survival under nutrient deficient conditions . One of At the same time, under cancer treatment connexin hemichannels enhanced anti-tumor efficacy due to the activation of ATP-mediated pro-apoptotic signaling in nanoparticle-treated glioblastoma  and PDT-Presumably, connexin hemichannels possess tumor-promoting properties in already well-developed TMEs and participate in tumor expansion. On the contrary, their anti-tumor properties can be performed in premetastatic niches where host cells try to combat the invasion. Connexin channels can also be localized on intracellular compartments, namely on mitochondria. Full-fledged versions of connexins are found in the mitochondria of a wide variety of cells, including cardiomyocytes, endothelium, astrocytes ,85,86, ac and the activation of caspase 3, thereby providing an anti-apoptotic effect and increasing resistance to toxins, chemotherapeutic drugs, or ionizing radiation [c release [One of the main signs of transformed cells is changes in the metabolic and apoptotic pathways, in which mitochondria occupy a central position. It is known that tumors tend to develop conditions similar to ischemia under chronic hypoxia. A possible way of reducing mitochondrial ROS production in such conditions depends on an increase in the phosphorylation of Cx43, which accelerates its translocation into mitochondria . Moreoveadiation . Additio release . Cx30 al release . Despite the assumptions made about an increase in cell resistance to hypoxia due to mitochondrial connexins, several works have suggested that Cx43 is involved in mitochondrial respiration, increasing oxygen consumption. The authors suggested that an increase in the expression of mitochondrial connexin shifts cellular metabolism from anaerobic glycolysis towards oxidative phosphorylation which is associated with an increased sensitivity to oxidative stress ,94 opposConnexin re-localization to the intracellular space is not necessarily aimed towards mitochondria, but it is often caused by disturbances in proper connexin trafficking due to defects in the connexin itself, impairment of other participants of the trafficking processes ,97,98, dIntracellularly localized connexins perform atypical functions. The C-tail moiety of Cx43 acts as a direct promoter of the mesenchymal marker N-cadherin . UbiquitIt is important to note that the direct triggers of the re-localization of connexins are, to a considerable extent, caused by features of the TME, such as hypoxia , changesThe coordinated actions of TME elements is one of the putative factors which determine tumor progression. Here, we intend to focus on the coordinative features of connexins in an awry tissue context, from the perspective of the functional and morphological aspects of the organization of the cells in the tumor tissue.Connexin channels are direct bridges between neighboring cells, thus the presence of functional gap junctional plaques allows us to consider a network of such cells as a functional “syncytium”, somewhat similar to cardiac tissue . The for2+ influx to the cancer cells enhancing their malignant potential [Connexins coordinate the bone metastatic niche and lead to successful metastasis establishment due to the interaction of malignant cells with the osteoclast syncytium. In the osteoblast-conditioned microenvironment, membrane-localized Cx43 mediates tumor cell chemotaxis via its non-channel functions. At the leading edge of migrating cells, Cx43’s C-tail interacts with Rac1 and contractin, thus sustaining cell migration towards the osteogenic metastatic niche . The dirotential . By inteotential . Taken totential ,83. PresIn the brain microenvironment, connexins appear to be the instrument that cancer cells use to turn astrocytes to allies. The connexin C-tail-mediated inhibition of Src kinase down-regulates β-catenin, triggering the preferential differentiation of neural progenitor cells towards astrocytes (against neurons). While the establishment of connexin GJs between normal neural cells and tumor cells can attenuate cell proliferation due to miR-124-3p transfer , astrocyConnexins heavily contribute to successful brain metastasis. Cx43 GJ communication mediates the extravasation of cancer cells to the brain parenchyma via a transcellular way. This process is initiated by the interaction of cancer cells with endothelium. The extravasated tumor cells engage Cx43 contacts with astrocytes in favor of the formation of metastasis and then form Cx43 contacts with each other, thus establishing a metastatic node . The preAn important aspect of the integrative functions of connexins in the TME is their contribution to the communication of tumor cells and cells of the immune system. Connexins participate in immunological synapses  and execImmune cells can also participate in the microenvironmental adaptation of the tumor. It has been suggested that tumor-associated macrophages (TAMs) may begin to act as intermediaries of nutrients between the tumor and vessels, creating an extensive cellular network integrated by Cx43 channels. The resulting rise in tumor metabolism not only increases its adaptability, but also leads to higher aggressiveness . The connexin-mediated cooperation of stromal cells with immune cells can contribute to tumor progression and manifests as a failed regeneration process. In the case of the giant-cell tumor of bone, Cx43 participates in the fusion of osteoclasts and monocytes. This abnormal cellular cooperation is most likely associated with an attempt by monocytes to regenerate the cancer-associated damage to bone tissues, which instead leads to an increase in the size of the tumor .Connexin contacts are established between various stromal cells, characteristic of certain metastatic niches or immune cells. Fibroblasts are a vital tissue component which substantially participates in the TME. The participation of connexins in integrating fibroblasts into the TME is tumor suppressive in the case of the early tumor and pro-tumorigenic in the advanced stage. Normal fibroblasts of the skin inhibit keratinocyte colony formation by the establishment of Cx43 GJ intercellular communications. When normal fibroblasts are exposed to sublethal doses of hydrogen peroxide  they lose Cx43 expression and thus GJ intercellular communications, which leads to the activation of keratinocyte colony formation. When the transformed fibroblasts start to prevail in the tissue microenvironment this is the turning point towards tissue malignization . InteresCells of squamous cell carcinoma down-regulate homologous GJ intercellular communications mediated by Cx43 between fibroblasts in vitro in a paracrine fashion with the involvement of calcium signaling . In the Initial connexin contacts between tumor cells and non-fibroblast cells can induce profibrotic properties in them, the most prominent of which is the enhanced expression of type I collagen. This was indicated for stellate cells in hepatocellular  and pancApparently in the TME as long as connexins are predominantly used by non-tumor cells to successfully retrain tumor cells rather than by tumor cells to subjugate normal cells, they thus act as tumor suppressors. When normal stromal communication is lost, tumor cells multiply and start to prevail in the microenvironment and thus connexins become a means of tumor progression. Early studies in connexin-deficient mice revealed their increased susceptibility to carcinogens ,140,141.H. pylori can affect connexins 26, 32, 37, 43 by triggering various signaling pathways  or through epigenetic action on DNA , thus regulating the expression levels of these molecules, governing their localization or creating their polymorphism [Shifts in overall connexin abundance, but predominantly its loss, is associated with tissue malignization under the action of various transforming factors, such as metabolic disorders, inflammation, bacterial infection, etc. Thus, non-alcoholic hepatosteatosis can be accompanied by the down-regulated expression of Cx32 which eventually causes liver fibrosis and is followed by hepatocellular carcinoma . In the morphism ,147. The improper membrane localization of connexins can also become a tumor-initiating event. Infection with human papillomavirus 16 causes mutation in the human homolog of Drosophila discs large (hDlg)-binding motif of the Cx43 C-tail, which results in the disruption of proper Cx43 and hDlg trafficking to the membrane so Cx43 remains in the cytoplasm . Another mechanism is the initiation of endometrial cancer in obese patients which can be due to the disruption of Cx43 GJ intercellular communications by hypermethylation of the Cx43 promoter in normal endometrial epithelium mediated by the microenvironment conditioned by adipose stromal cells which massively release plasminogen activator inhibitor 1 .Clonorchis sinensis metabolites and eventual cancer development is accompanied by the simultaneous decrease in the expression of Cx32 and up-regulation of Cx43 and Cx26. Cx43 in this case acts as a tumor promoter as its inhibition leads to a proliferation decrease [Considering not only the levels of individual connexins, but also taking into account their interplay with each other, relative changes in their levels are also important. For example, the turning point for the transformation of bile duct epithelium by decrease . Similardecrease . This evdecrease ,153.Another way to alter intercellular communication and hence the tissue context is the stable expression of connexins which are normally only transiently expressed in a tissue when necessary for precise functional needs. For example, in normal breast development Cx32 is expressed only during lactation precisely at the interfaces of luminal cells  but thisA rare example of non-channel connexin-dependent carcinogenesis is supposed to be associated with de novo expression of connexins, initially foreign to the tissue. For example, expression of Cx46 is not detected in normal breast epithelium and is detected in early breast cancer. De novo expression of Cx46 in breast epithelium was phenomenologically demonstrated to protect cells from hypoxia using xenograft models in vivo. The authors assumed that it may be a cancer-initiating event .Summarizing the represented data we may consider that the tumor-initiating factor, or, to be more precise, a complex of conditions in the site of tumor initiation, forces perturbations in the functioning of connexins, namely, decreasing in their amount, re-localization, shifting the ratio of connexins typical to the tissue, establishing stable expression of certain connexins which are transiently expressed in the tissue, or the de novo expression of connexins which are normally foreign to it. It is necessary to admit that these data are mainly phenomenological, and a mechanistic explanation of how the switch in the connexin profile promotes malignant transformation is a matter for future research. Still some scattered pieces of this picture can be seen already. The loss of connexins characteristic to the tissue triggers the up-regulation of oncogenes, such as c-myc ,158,159,Hypoxia is a biological condition characterized by insufficient oxygenation of tissues which, in the case of malignant tumors, is caused by rapid cell proliferation ,163. TheHypoxic conditions are, on the one hand, a circumstance that is destructive to tumor cells, which cells must cope with, and on the other hand, a powerful selection factor for their collective adaptation with the subsequent development of progression mechanisms. Connexins participate in both of these aspects. Overcoming hypoxia on a large scale is realized in tumors by promoting angiogenesis mainly by establishing the proliferative and migrative phenotype of endothelial cells. This can be realized via different types of cell–cell interactions, i.e., between tumor cells, tumor cells and endotheliocytes and between stromal cells, including vascular cells, and is mostly provided by functional GJ intercellular communications. For example, transcriptional suppression of Cx43 and Cx26 in MDA-MB-231 breast cancer cells led to the down-regulation of GJ intercellular communications between cancer cells and cancer cells-and-endotheliocytes, accompanied by reduced migrative and invasive properties, as shown by real-time cell analysis . These rOne of the characteristics of a severely hypoxic microenvironment that should be handled is acidosis, and functional GJ intercellular communications mediated by connexins allows its management. Thus, in spheroids of pancreatic cancer, it has been shown that connexin channels between hypoxic and normoxic cells allow the rapid distribution of bicarbonate ions to neutralize acidification in hypoxic areas . More thThus, connexins participate in hypoxia resistance by providing a trans-cellular path for oxygen and nutrients in arranged cells which jut into the depths of the tissue from the perivascular space  or by prmiRNAs are one of the most potent factors of the TME and they are transmitted between both malignant and stromal cells through connexin-based gap junctions or via extracellular vesicles (exosomes). Thus, Cx43-based channels have been reported to transmit miRNA-145 from microvascular endothelial cells to colon cancer cells leading to the inhibition of angiogenesis . A similA considerable amount of data has been gathered on the pro-tumorigenic role of miRNA transfer by connexin channels. The miR-5096 derived from glioma cells possesses a pro-invasive effect when transferred to astrocytes , and a pIn the case of the exosome-mediated transmission, connexin channels have been reported to recruit miRNAs to exosomes as they possess RNA-binding motifs in their structure . The miRSummarizing these data, the trend towards tumor progression or suppression may be determined by the type of miRNA which is being transferred and the direction of transfer . This can probably be determined by the presence, quantity, affinity, and conformational availability of RNA-binding motifs in various connexins.Cancer stemness is an important factor of cancer progression, which determines cancer self-renewal, dormancy, and resistance to treatment . ConnexiConnexins can reduce the stemness of cancer cells or provide assistance in cancer treatment aimed at resistant stem cells. Thus, ectopic expression of Cx43 in lung cancer cells reduces the abundance of cancer stem cells, as was shown by a reduction in tumor sphere formation and stemness markers in transfected cells . StemnesConnexins can also support the stemness features of cancer cells. The most prominent stemness feature supported by them is self-renewal which was established in the case of intracellular localization of connexins. For example, it has been shown that Cx26, Cx32 and Cx46 can form alternative signaling with the pluripotency transcription factor NANOG, up-regulate CD133 or markers of stemness Oct4 and Sox2, which leads to an increased cancer abundance of stem cells and the acquisition of an invasive tumor phenotype . The funConnexins can also be used by cancer stem cells as a means of the realization of their aggressiveness. Thus, Cx43 was reported to mediate breast cancer immune escape by establishing communications between cancer stem cells and mesenchymal stem cells which results in a preferential Treg response against T-helper 17 cells . AdditioThe recent conceptual papers by J.E. Trosko discussed the hypothesis that connexins may act as key molecules which underlie cancer stem cell origin and determine the cancer stem cell type due to their tissue integration properties with the most crucial point, cell differentiation. Cancer cell stemness, in this case, is assumed to be maintained on the one hand by the sustained expression of the Oct4A oncogene which prevents the expression of connexin genes, thus making it impossible for cells to differentiate at all, or in the case of when Oct4A oncogene is absent the stemness is maintained by other oncogenes which prevent proper connexin localization and the execution of their differentiation functions ,191,192.The holistic picture of cancer origin and development is still an unsolved issue. Accumulating data indicate that tissue context is crucial in this process. In the 1990s a tissue-organization field theory (TOFT) of cancer origin and development was suggested, which postulated that cancer is a tissue disease and that it is based on the disorder of cell organization, rather than one renegade cell . So, basLoss of functioning connexin channels, or, as a worse outcome, mislocalization of connexin can lower the level of tissue orderliness. The disbalance in the abundance of connexins which are characteristic of the tissue, or stable expression of connexins which are only needed for precise functional states of the tissue , or eveConnexins are indeed found to be involved in regulation of EMT. For example, Cx43 can reverse EMT, as its overexpression leads to the elevation of epithelial markers, E-cadherin and ZO-1, and the down-regulation of mesenchymal markers, vimentin, Snail and Slug ,195. ThiThe interactions mediated by connexins between tumor cells and anti-tumor stroma result in the restriction of tumor growth and is most clearly represented by immunological synapses ,121, intDuring metastasis, connexins participate in all stages, from preconditioning the metastatic niche, establishing cell motility, enabling collective migration of tumor cells coupled with immune cells and endotheliocytes, diapedesis of the metastatic unit and homing to the metastatic site D.In order to resolve the controversy of the role of connexins in cancer and precisely in the TME, a complex approach should be executed. If we assume that intercellular junctions are one of the putative means defining a multicellular organism, we should study the dynamics of the overall junctional complex of the cells in the TME, as during tissue development the role of each type of these junctions can hardly be separated. Such attempts have been made ,202. It The importance of the TME in tumor progression and response to treatment has led to special attention being paid to (i) tissue-relevant tumor models used in drug discovery, (ii) clinical diagnostic methods allowing the assessment of the tissue organization of tumors in addition to its size and localization, and (iii) TME-targeting tumor treatment approaches. Thus, 3D in vitro models are increasingly favored over conventional monolayers ,209,210.The list of diagnostic imaging methods used by researchers and clinicians has recently been expanded with magnetic resonance imaging (MRI)-based techniques  providing information on the spatiotemporal pattern of tissue vascularity and perfusion, cellularity, extracellular pH within the tumor niche and other metabolic features ,213,214.In addition to TME assessment by imaging methods, the detailed information on the TME–connexins interplay can be obtained from cellular level analyses using sequencing and omic techniques. These approaches are becoming important instruments in TME research and clinical practice due to a growing interest in personalized medicine. Single-cell sequencing is able to delineate the heterogeneity of tumor and tumor-associated cell sub-populations, such as cancer-associated fibroblasts, responsible for the development of chemoresistance via Cx43 up-regulation ,222, or The TME and, specifically, connexins can be considered as a target for tumor treatment. To date several approaches utilizing connexins as potential therapeutic targets are being developed. Firstly, the restoration of GJICs either by combating with conditions which block it (by inhibiting the enzymes which produce blocking substances) or the restoration of the expression of connexins . In meta


The complexity of brain functions is supported by the heterogeneity of brain tissue and millisecond-scale information processing. Understanding how complex neural circuits control animal behavior requires the precise manipulation of specific neuronal subtypes at high spatiotemporal resolution. In utero electroporation, when combined with optogenetics, is a powerful method for precisely controlling the activity of specific neurons. Optogenetics allows for the control of cellular membrane potentials through light-sensitive ion channels artificially expressed in the plasma membrane of neurons. Here, we first review the basic mechanisms and characteristics of in utero electroporation. Then, we discuss recent applications of in utero electroporation combined with optogenetics to investigate the functions and characteristics of specific regions, layers, and cell types. These techniques will pave the way for further advances in understanding the complex neuronal and circuit mechanisms that underlie behavioral outputs. Optogenetics has become a powerful tool in neuroscience for understanding the complex functions of neural networks ,2. In thNeurons, like all other cells, have a plasma membrane. The plasma membrane serves as a barrier between the neuron and its surroundings, controlling the movement of substances into and out of the cell. This movement is controlled by transmembrane proteins (embedded in the plasma membrane), which act as ion channels or pumps that control the transport of specific molecules across the membrane. Electrically charged ions are one of the substances transported by ion channels and pumps. Ion channels and pumps transport specific ions and generate voltage gradients across the membrane, which are referred to as membrane potentials. In particular, a rapid influx of sodium ions generates action potentials, which contribute to neuron-to-neuron communication in the following manner. in neurons, action potentials are conducted along axons toward synaptic boutons at presynaptic axon terminals, where the electric signals are converted into chemical signals  and GABA (gamma-aminobutyric acid) in most cases). The neurotransmitters  bind to ion channel-coupled receptors on postsynaptic terminals at chemical synapses, which transform these chemical signals back into electric signals known as postsynaptic excitatory or inhibitory potentials. Collectively, the generation of ac-tion potentials and the subsequent activa-tion of postsynaptic neurons are achieved by a number of ion channels.In the field of optogenetics, researchers often genetically modify neurons to manipulate their activity by inducing their expression of light-gated ion channels or pumps known as opsins ,14,15,16Among the various methods for promoting the expression of opsins in a specific neuronal population, we focus on in utero electroporation, which allows researchers to study the diversity of the neuronal populations composing neural circuits ,25,26. TFirst, we introduce the unique features of the technique by comparing it with similar techniques. Then, we precisely illustrate the basic function and characteristics of in utero electroporation. Finally, we discuss future perspectives by showing examples of previous studies that have combined in utero electroporation with optogenetics to reveal important features of neural circuits.Neural networks comprise a myriad of cells, each receiving and sending projections to various brain regions. Thus, cellular- and projection-specific optogenetic control of neural activity is required to understand the complex network structure of neurons. Several methods, including (i) genetically modified animals a, (ii) vGenetically modified animals can be created by altering their genetic materials  with genetic engineering techniques. Conventional genetic engineering methods allow researchers to insert ‘foreign’ genes at a random location in the genome of a host animal to create transgenic animals. However, conventional genetic engineering methods do not necessarily yield the desired outcomes because foreign genes are inserted at unpredictable loci in the host animal’s genome. In this light, this approach has recently been described as ‘ham-fisted’ or clumsy .In addition to the conventional methods described above, gene editing technologies based on the zinc finger nuclease (ZFN) ,32,33,34Another method uses a viral vector with opsin genes (see the next section). There are numerous Cre lines for specific neurons in mice, but the number of opsin-expressing mouse models is limited. Moreover, the drawback of this system is that genetically modified animals for species other than mice are meager and scarce. Theoretically, one could design and create novel transgenic or knock-in animal lines for various species, but this would be a time-consuming and costly process.Using viral vectors for gene expression is an alternative to producing transgenic animals. Gene expression with viral vectors  and lentivirus) is achieved by replacing the viral genome with a custom-designed construct containing a target opsin . This teIn utero electroporation is used to induce the expression of target genes in neural precursors to control the activities of neurons. This method is less invasive and more convenient than postnatal injections of viral vectors ,54,55,56Electroporation consists of two major steps: (1) electropermeabilization of the cellular membrane and (2) electrotransfer of a DNA plasmid. Both steps in the electroporation process require a train of electric pulses to drive the DNA into target cells. The efficient transfer of plasmid DNA into the cells depends on (1) the permeability of the cell membrane and (2) the probability  of DNA transfer across the cell membrane toward the nuclear membrane. These can be controlled by optimizing the frequency and intensity of the electrical pulses. In the following section, we discuss the basic mechanisms of electroporation.In the first step of electroporation, a series of electric pulses are used to disrupt the cell membrane, resulting in the formation of temporary pores in the membrane ,70. The In the second step, the negatively charged DNA migrates toward the positive electrode ,74. The The brain comprises multiple functionally distinct regions, and proper neural circuits between these regions are required for higher brain functions. Various genetic modification techniques, such as the use of transgenic Cre lines and viral vectors, have been developed to induce the conditional expression of transgenes in specific neural populations.In utero electroporation can be used to induce gene expression in certain brain regions by targeting progenitor cells that migrate and develop into neurons in specific brain regions . This isThe bipolar in utero electroporation technique was first developed in 2001 ,81 Figua. InitiaConfinement of the transgene-expressing region is also a benefit of using in utero electroporation. As is often the case with viral vectors, simple diffusion after injection causes surrounding regions to also be transfected. However, since transfection of plasmids in in utero electroporation is also affected by the direction and intensity of the electric field, controlling these parameters will enable researchers to confine the region of transgene-expressing tissues. Although region-specific expression of transgenes is possible with double-electrode probes, the diffused electric field may cause the transfection of surrounding tissues, and an asymmetrical electric field results in an undesired transfection of the other ventricle when loading both ventricles with plasmid vectors. Undesired expression in other regions can be overcome by using triple-electrode probes to precisely regulate the direction and strength of the electric field . Using aIn utero electroporation is an effective method for revealing the function of specific cell populations by selectively inducing the expression of target genes. The opsin-expressing construct is injected into the ventricles of the embryonic brain and electroporated into adjacent cells. Since the cellular lineage of the progenitor cells lining the lateral ventricle varies depending on the embryonic day of the animal, the electroporated cells can be characterized based on the developmental stage . For exaThe same technique can be applied to promote the expression of transgenes in interneurons. Interneurons originate in the ganglionic eminence and migrate to the cerebral cortex b. BorrelMoreover, changing the time of electroporation enables the transfection of not only neural progenitor cells but also astrocytes. For instance, when rat embryos were electroporated at E18, transfection of astrocytes in the neocortex was observed .In utero electroporation allows target genes to be expressed in the region- and cell type-specific manners. Including opsin-coding genes in the vector plasmids allows the expression of opsins in specific neurons. This technique enables optical control of neuronal activity ,89 and iIn neural circuits, information is exchanged between different neurons through local and long-distance connections. One way to elucidate the complexity of this neural circuitry is to express opsins in cell- and region-specific manners and manipulate these opsins with optical stimulation. By taking advantage of the cell type- and region-specificity of in utero electroporation, a variety of connections in neural circuits have been scrutinized.Combining whole-cell recording of synaptic currents with the photostimulation of electroporated channelrhodopsin-2 (ChR2)-positive neurons has allowed mapping of the circuits between presynaptic and postsynaptic neurons. This technique, known as ChR2-assisted circuit mapping (CRACM) ,93, can In a similar manner, Adesnik et al. analyzed horizontal projections in cortical domains . In uterBitzenhofer et al. used in utero electroporation to promote ChR2 expression in pyramidal neurons in layers II/III or layers V/VI. They photoactivated ChR2-transfected pyramidal cells with blue light and observed the resulting changes in network oscillations . They shIn utero electroporation has also been used to analyze network plasticity in the neocortex. Lourenço et al. found that layer V pyramidal neurons in the mouse barrel cortex modulate information processing in the cortex via perisomatic inhibitory synaptic plasticity . In uterLayer specificity in the neocortex has also been used to identify the source of spontaneous low-frequency oscillatory dynamics in the mammalian cortex . BeltramWide patterning of brain regions occurs in the early days of embryonic development. Therefore, electroporation into embryos performed as early as at E9.5 allows researchers to study the development of region-wide interactions from early embryonic stages.Ahlbeck et al. applied in utero electroporation to study the interaction between the hippocampus and neocortex in neonatal mice . They spThe development of extracellular oscillations in the cerebral cortex has been studied by exploiting the advantages of in utero electroporation, allowing neuronal populations to be manipulated in a layer-specific manner. For example, Bitzenhofer et al. analyzed the origin of gamma oscillations in the developing mouse brain ; gamma oIn addition, in utero electroporation can be used to study neuropsychiatric pathologies that emerge during development. Bitzenhofer et al. analyzed the effect of disturbed neuronal activity during various developmental stages by transiently increasing the network activity of layer II/III pyramidal neurons in developing mouse brains . In thisTo reveal a causal relationship between the activation of specific groups of neurons and animal behavior, either excitation or inhibition of the target population of neurons in a specific region can be used. To accomplish this, electrical microstimulation via locally placed electrodes is widely used ,102,103.Since in utero electroporation can be used to confine the expression of opsins to specific layers of the cortex, it is possible to control the number and type of neurons excited by photostimulation with optogenetics. Therefore, in utero electroporation is a useful method for analyzing the number of neurons required for perception .As described above, in utero electroporation is advantageous in terms of the region-, layer-, and cell type-specific manipulation of neurons. To further dissect the establishment and functions of neuronal networks, it is essential to understand the connectivity and interactions between multiple subsets of neuronal populations. A recent method called dual in utero electroporation serves as an effective tool to target multiple populations of neurons within the same circuit in a spatially and temporally specific manner ,105,106.In utero electroporation enables researchers to transfect DNA into a minor subpopulation of neurons ,112,113.The drawback of standard in utero electroporation is that electroporated transgenes remain episomal and thus are lost during cell division, suggesting that transgenes are not expressed in proliferating cells, such as cells in a neural lineage. However, combining the binary piggyBac transposon system with in utero electroporation enables the stable expression of transgenes in cells in the neural lineage, such as radial glia ,121,122.In this light, the combination of a variety of in utero electroporation-based techniques  with optogenetics will open new avenues for the theoretical and experimental investigations to answer fundamental questions in neuroscience.


Multiparametric MRI has been extensively studied in prostate imaging, showing diagnostic benefits with high-quality evidence. In 2019, the PI-RADS Steering Committee confirmed that multiparametric MRI helps to distinguish patients with clinically insignificant cancer from those with life-threatening cancer, and the Committee suggested multiparametric MRI routine adoption in clinical practice  mapping for the differential diagnosis of gliomas between a series of genome types. The OEF mapping was calculated with an integration of both quantitative susceptibility mapping (QSM) and quantitative blood oxygen level-dependent magnitude (qBOLD). As a biomarker predominantly reflecting the hypoxia status, the OEF mappings achieved the AUCs of 0.828, 0.784, and 0.764 for the detection of IDH1 mutation in low-grade gliomas, MGMT promoter methylation in glioblastomas, and RTK aberration in the general cohort, respectively . With a cohort of 91 patients with pathologically confirmed gliomas, these results suggest the potential of OEF mapping in gliomas differential diagnosis and treatment guidance.On cancerous neuro disease, Bao et al., Kisel et al., and Liu et al. focused on the application of multiparametric MRI in neurodegenerative diseases. When compared to the age-matched healthy controls, the remitting-relapsing multiple sclerosis (RRMS) patients demonstrated significantly different patterns of fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity in a range of brain areas, as revealed by Bao et al. using diffusion tensor imaging, suggesting demyelination and axonal injury in RRMS patients even in some normal-appearing white matter regions. Kisel et al. reviewed the value of Macromolecular Proton Fraction (MPF) measurement, using magnetization transfer (MT) MRI, in the observation of the de- and re-myelination process. This work demonstrated the animal studies that formed the basis of MPF mapping theory, the correlation of MPF maps with histological studies, and the recent translation of this technique at clinical setups, suggesting the potential application of MPF mapping in a range of neurodegenerative diseases including MS by providing additional pathological information in a non-invasive way. Meanwhile, Liu et al. applied chemical exchange saturation transfer (CEST) MRI to Alzheimer's disease (AD), revealing higher glutamate concentration in AD mice than in wild type of the same age. With a Resnet neural network, they were able to achieve an accuracy of 75.6% in the classification of glutamate distribution images of AD mice and the wild type. Their result implies a potential solution to AD diagnosis comprising both novel MRI techniques such as CEST and the recent development in artificial intelligence (AI).On the other side of the disease spectrum, With incremental clinical values revealed and validated by histological studies and with integrated development in both acquisition technology and AI-based image interpretation, the studies in this Research Topic have shed light on the potential clinical impacts of multiparametric MRI. However, before being translated into clinical practice, several questions mentioned above remain unanswered. A particularly important concern is the repeatability and reproducibility of multiparametric MRI, which is a legacy problem that has for a long time hampered the clinical translation of quantitative MRI. Is the multiparametric MRI-based diagnosis model more prone to the across-site and across-vendor signal variations, even assuming the same imaging sequence and protocol, or is it more robust than traditional quantitative MRI because of the integration of different measured parameters? In addition, the standardization of acquisition sequence and protocols is another major challenge for the routine use of multiparametric MRI. Nevertheless, any progress in multiparametric MRI toward clinical translation will be plausible to the modern healthcare system due to its remarkable cost-benefit and reduced operational risk when compared to the traditional biopsy-based tissue examination.JW wrote the manuscript. All authors critically reviewed and approved the manuscript. All authors contributed to the article and approved the submitted version.JW is an employee of Philips Healthcare. The remaining 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.


Despite medication-related osteonecrosis of the jaw (MRONJ) being first reported in 2003, the optimal treatment and prevention modalities for MRONJ are not clear. As a result, dentistry, oral surgery, and departments involved in the treatment of cancer and bone diseases are struggling with the management of MRONJ. Several cases of MRONJ cannot be managed by conventional treatment strategies recommended in various position papers. Therefore, studies have been conducted to investigate the efficacy of novel therapies for MRONJ. However, the optimal treatment is unknown. Several cell therapies including autologous cell transplantation have been reported for MRONJ. Although the efficacy of cell therapy for MRONJ has been demonstrated, large, statistically accurate clinical trials are lacking. We have been investigating the efficacy of MRONJ treatment using mesenchymal stromal cell (MSC) sheets since 2013 and confirmed its efficacy through various experiments, wherein MSC sheets were transplanted in model rats and beagle dogs with MRONJ-like lesions. Based on these results, we are planning to conduct a clinical trial of MRONJ therapy using periodontal ligament-derived MSC sheets. Bisphosphonates (BP) and anti-receptor activator of nuclear factor kappa-Β ligand (RANKL) antibodies (denosumab) are drugs that inhibit bone resorption by suppressing osteoclasts and are used in many patients with osteoporosis, bone metastases of cancer, and multiple myeloma. However, after the first report of BP-related osteonecrosis of the jaw in 2003 , reportsThe American Association of Oral and Maxillofacial Surgeons position paper advocates treatment according to the stage of the disease. For cases with mild symptoms (stages 1 and 2), conservative treatment such as administration of antibiotics, debridement, and mouthwash use is recommended. In cases progressing to stage 3, surgery to remove the jawbone is recommended. However, the cure rate after this treatment protocol is low. Rupel et al. reported a cure rate of 33% for stage 1 and 24% for stage 2  MRONJ. ICell therapy has been investigated as a new treatment modality for MRONJ, and eight studies have been reported on the efficacy of cell transplantation in animal models of MRONJ  Barba-R. Most ofSince 2013, we have been focusing on the effects of MSCs on wound healing, angiogenesis, bone metabolism, apoptosis, and immunomodulation to develop a new treatment modality for MRONJ using MSCs. Although intravenous administration is widely used as a method of transplantation of MSCs, most of the transplanted MSCs fail to reach the affected area, pulmonary embolization  can occuWe have investigated the efficacy of MSC sheet transplantation for the treatment of MRONJ in rats and beagle dogs . Before The usual cell harvesting method using proteolytic enzymes destroys the adhesion proteins between the cells, so the cells are scattered. Therefore, it is difficult to transplant cells efficiently without the use of scaffolds. In contrast, cell sheet engineering is a technology that allows cells to be collected in a sheet form while retaining their extracellular matrix by using temperature-responsive culture dishes . TherefoThe selection of a cell source for MSCs and the establishment of a cell bank of allogeneic (human) MSCs are essential for the clinical application of this therapy. We have established a bank of human periodontal ligament-derived MSCs (PDLMSCs) and conducted a physician-led clinical trial to investigate the safety and efficacy of human PDLMSC sheet transplantation for periodontitis. PDLMSCs have angiogenic, osteogenic, and immunomodulatory abilities comparable to those of MSCs derived from other tissues . TherefoCurrently, there is no established treatment or prevention method for MRONJ. We have demonstrated the efficacy of MSC sheet therapy for MRONJ in animal experiments. Based on the results, we are planning to conduct a clinical trial of MSC sheet therapy for MRONJ using PDLMSCs.


The agricultural sector is a foremost contributing factor in supplying food at the global scale. There are plethora of biotic as well as abiotic stressors that act as major constraints for the agricultural sector in terms of global food demand, quality, and security. Stresses affect rhizosphere and their communities, root growth, plant health, and productivity. They also alter numerous plant physiological and metabolic processes. Moreover, they impact transcriptomic and metabolomic changes, causing alteration in root exudates and affecting microbial communities. Since the evolution of hazardous pesticides and fertilizers, productivity has experienced elevation but at the cost of impeding soil fertility thereby causing environmental pollution. Therefore, it is crucial to develop sustainable and safe means for crop production. The emergence of various pieces of evidence depicting the alterations and abundance of microbes under stressed conditions proved to be beneficial and outstanding for maintaining plant legacy and stimulating their survival. Beneficial microbes offer a great potential for plant growth during stresses in an economical manner. Moreover, they promote plant growth with regulating phytohormones, nutrient acquisition, siderophore synthesis, and induce antioxidant system. Besides, acquired or induced systemic resistance also counteracts biotic stresses. The phytomicrobiome exploration is crucial to determine the growth-promoting traits, colonization, and protection of plants from adversities caused by stresses. Further, the intercommunications among rhizosphere through a direct/indirect manner facilitate growth and form complex network. The phytomicrobiome communications are essential for promoting sustainable agriculture where microbes act as ecological engineers for environment. In this review, we have reviewed our building knowledge about the role of microbes in plant defense and stress-mediated alterations within the phytomicrobiomes. We have depicted the defense biome concept that infers the design of phytomicrobiome communities and their fundamental knowledge about plant-microbe interactions for developing plant probiotics. A glaring overhaul has been observed in the 21st century in regard to climate change where environmental stresses have caused global threat toward food safety and security. The world population has been projected to be 9 billion (approx.) by 2050, and environmental disturbances cause reduced crop productivities and soil fertility thereby impeding agricultural sustainability . ConsequSince decades, enormous studies have depicted the sky-scarping diversity of microbiome co-linked to agricultural plants . MicrobiPhytomicrobiome is the most sustainable and efficient approach that is effective both in terms of productivity and quality . Harnessvia nitrogen fixation, mineral acquisition, inhibiting pathogenic organisms and modulating plant defense responses toward stresses  that enhance the beneficial traits in plants during stresses. Various biochemical and molecular pathways are adopted by microbes for maintaining growth through hormonal regulation and nutrient acquisition, thereby enhancing the plant growth regulators for resistance against adverse conditions . As depi9–1010 microbial cells with an estimated 104–106 species  and are generated due to weathering and inadequate rainfall   that enhance the immunity of plants against invaders and induce their mortality  , which degrade the cell wall and cause cell destruction of phytopathogens , which inhibit the mycelial growth of harmful fungal strains (Bacillus cereus triggers the expression of Induced Systemic Resistance (ISR), inhibiting the infection of Pseudomonas syringae in Arabidopsis  . Variousortality . Bacteriectively . A gaseos, etc.) . It is as, etc.) . Differeathogens . Various strains . They albidopsis . It is a amounts . SeveralThe plant immune system is activated during biotic stress, which triggers the roots to secrete several exudates, such as organic acids, phytohormones, alexins, etc. These exudates act as signaling components and are enhanced in response to various stresses. The complexes like polyphenols, plant hormones , AHLs (N-acyl-homoserine lactones) are also involved in plants-microbes associations under stress severity . The AHLin vitro for studying their interactions with plant microbial symbionts and pathogens, thus ultimately studying their impact on plant health. However, due to the chemical complexity of varied soil types, root exudation analysis remains an important hurdle in natural settings. A few plant compounds have been discovered in having a positive impact on the rhizosphere microbiota. These compounds, which include flavonoids, peroxidases, oxylipins, coumarins, benzoxazinoids, phenylpropanoids, aromatic chemicals, triterpenes, and mucilage, have all been shown to attract beneficial microorganisms that modulate plant defense system  has rhizosphere rich in Flavobacterium spp.  was found to deal with phosphate stress responses and regulates a set of plant immune system genes, which ultimately lead to changing composition of root microbiome  and many other jasmonate-responsive genes , behave as signaling molecules during stresses  mechanism. SAR inhibits many pathogens and leaves prolonged effects through their mobile signals with the ability to activate the transcription of defense-related genes and plant immunity  that enhance immunogenic responses in plants by identifying various chemical cues from microbes, and this immunity is called as PAMP-mediated immunity. In this process, the plants lead to callose deposits within the cell wall and ROS production with the activation of signaling and defense-related genes, such as stresses . Furtherectively . Along wimmunity . SA has immunity . During immunity .nod gene expression. Rhizobia also release lipochitooligosaccharides (LCO) that are mostly recognized by lysine-like receptors, thereby forming a symbiotic pathway to generate the nodulation . Under stresses, plants synthesize chemical cues in excessive amounts, like amino acids and sugars, when released in rhizodeposits and behave as chemo-attractants for beneficial microbes, causing enhancement in the microbial populations within rhizosphere. The mucilage secretions also consist of anti-microbial compounds that prevent the pathogen attack and establish mutual or symbiotic associations among plants and microbes . The legdulation . The comdulation . The LCOdulation . Jasmonidulation . In the dulation . LCO alsBacillus thuringenesis that responds to plant signals and promotes plant growth along with inhibiting pathogenic organisms  is favorable to plants in enhancing host disease resistance  and its application. Due to the complexity in the mechanism of interactions among soil, plants, and environment, there was difficulty in persisting this phytomicrobiome study because only limited communications have been studied under naive conditions. Many industries showed interest in the use of microbial products that contain specific microbes and carrier-dependent inoculant strains. The implication of emerging technology “Microbiome on a Chip” was effective in studying the multitrophic interplay (microbiome plants) through the combinations of microbial colonies, the response of the host, and several environmental incentives . AdvanceThe enhancement in the production of goods was achieved by modulating the beneficial microflora of the soil at the blooming phase of seeds. This technology introduced harmless microbial strains and protected the plant biome from other competitive strains that further enhanced the survival and colonization of inoculated microbial strains. The chain of multiple harmless strains of microbes has more compatibility as compared to single-strain designed experiments because the networking and the grouping of beneficial strains with the existing microflora of soil favor the host. There were various systematic approaches to isolating microbial strains that are associated with phyllo- and rhizosphere .in situ engineering of the microbiome for the profit in the agriculture and food sector. In this technology, the crops probiotics formulation was composed of the engineered microbiome that further manipulated the microflora (soil and plants) and its activity during its application  that engineered their own microbiome and enhanced the interactions with other beneficial microbiomes. It was reported that the miRNA was associated with maintaining the structural property of the microbiome of the rhizosphere. Therefore, these interactions are defined as a key to allocating the miRNA and engineering the beneficial microbiome from the target to beneficiary soil for desirable products. Different experiments were conducted with engineered soil or plant-enhanced microbes to manage the ecological engineering of the microbiome of plants and soil in an artificial selection system . This ar species .Food security and sustainable agri-products will be a considerable challenge in the coming years for the growing population. The stressed conditions reduce the productivity and availability of food for the growing population that creates rigidity in sustainable farming. Therefore, for enhancing the sustainability and productivity of soil, the phytomicrobiome is being used as an imperative biological toolkit to help and improve agricultural soil flora and fertility. It is a forgoing resource that counteracts various stresses by enhancing the synthesis of phytohormones, enzymes, nutrients, etc. This toolkit has a solution of boosting plant resistance against stresses by supplementing beneficial microbial stress-tolerant strains in soil or plants. The interface of the plant-soil microbe will promote productivity and growth by enhancing the production of hormones, siderophores, and the antioxidative defense system. Another technique, i.e., the application of plant growth-promoting rhizobacteria will also support the development of internal and external resistance or tolerance in plants under stress conditions. The plant growth-promoting bacteria may be used as priming agents, in the form of biopesticides and biofertilizers or as a direct supplement. It is revealed that the rhizo-microflora play an important role in plant growth and health, and the plant itself manages the composition of microbiomes. This mechanism of selection and regulation of microbial strains by plants is mostly favored for their maturation that appears as a natural-picking pressure whenever plants need microbial help.via lab and field experiments. This approach of understanding plant immunity, physiology, genetics, and biome under stress conditions needs to be explored more for improving productivity and ecological sustainability. Moreover, the biological explanation of the mechanism of microbiome shifts induced by stress would also allow the secretion of chemicals in biomes against stress is still unclear. The interplay between plants and microbes is a complex mechanism that needs a thorough investigation as it is still in its initial stage of experiments. Scientific policy organizers and associates need to make a toolkit to identify the plant growth-promoting microbes that are stress tolerant, and, also, the effective strains should be verified to overcome the harmful impact of the stressed environment. Proper research and study will achieve the process of selection and engineering of phytomicrobiome to make them stress resistant, resulting in enhanced production and yield of crops. Plant-microbe interactions and biotic stress-induced alterations have been mostly shown in plants, but the mechanism associated with metabolic pathways of microbes is still yet to be explored. Research should be focused on the characterization and effect of the microbiome on the gene expression in plants, resulting in altering the plant functioning both at proteomics and genomics levels. The CRISPR-Cas9-based forward genetic screen is a comprehensive approach to explain the interactions of plant and microbiome that will be beneficial in the future. To expand the knowledge of plant-soil-microbes interaction, information at the genomic level and engineering of beneficial microbial factories should be explored.The microbes act as ecological engineers to cope with environmental stresses. Still, stupendous research will be needed in the field of sustainable agriculture approaches by knowing the durability and survival, commercial design, inconsistency, and host relevance in extensive conditions to meet full utilization of these approaches and provide food security demands in the future. Also, there are other challenges in interpreting plant or soil physiological data and multi-omics with appropriate validation KK, RB, SK, ASh, PAh, PAl, and TA designed the outline and revised and finalized the draft. All authors contributed to the article and approved the submitted version.


Climate change (CC) is currently responsible for global weather extremes. These weather extremes could contribute to changes in the pattern of health problems. The purpose of this review is to discuss the role of CC on remapping of hepatic diseases and the mechanisms of re-mapping.CC was found to have a major influence on the distribution and severity of hepatic diseases, such as outbreaks of vector-borne, water or food-borne, parasitic diseases, re-emerging of disappeared diseases, or emerging of new forms of infectious agents. Migration of infected people from endemic areas due to the CC disasters results in rapid dissemination of infectious diseases that leads to outbreaks or endemicity of diseases in new areas. CC could cause increasing chemical emissions, or change in its biodegradability, or restriction in its dispersion, such as PM, PAHs, heavy metals, mycotoxins, and aquatic toxins. Increase in the concentrations of these chemicals may have significant impacts in changing the health map of hepatic toxicity and liver cancer.The current review confirms the role of CC in changing the pattern of several liver health problems and remapping of these problems in several regions of the world. This review could be of high importance to the health decision-makers as an early alarm and prediction of hepatic health problems with the projected CC. Human activities have proved to cause increased greenhouse gases that warm the atmosphere, land, and ocean. Widespread and rapid changes in the air, soil, ocean, biosphere, and cryosphere have occurred over the past few decades . ClimateA lot of recently published evidence indicate that CC has hazardous impacts on the liver health in several aspects. CC may lead to increase in the communicable and non-communicable liver diseases. Global warming has been reported to have an impact on the pathogen selection and resistance, host ecology and immunity, as well as vector ecological niches and capacity, with more potential influence on vector-borne and zoonotic diseases . CC, dueAdditionally, most of the non-communicable liver diseases could be attributed to the interaction of CC with several factors that affect development, concentration, and dispersion of air pollutants. This interaction could affect the burden of diseases and mortality associated with these pollutants. Global warming increases the risk of development of poor air quality due to the chemical reactions as a result of either biodegradability of some of these chemicals or increases the concentrations of the emissions due to non-biodegradable of others. CC could also lead to restriction of the dispersion of pollutants due to atmospheric stability . TherefoFurthermore, desertification and climate change expand the global area of dry land  and incrGlobal warming and the change in the pattern of precipitation will increase the frequency of hepatotoxic cyanobacterial blooms. In addition, CC induces the conditions suitable for the growth of aflatoxigenic producing fungi and mycotoxin production which are carcinogenic to the liver.This review aimed to discuss the mechanisms of CC. It also informs about the impact of changes in the pattern of temperature, precipitation, and increased extreme events by increasing the frequency of global liver diseases and remapping of some hepatic health problems, including communicable and non-communicable liver health problems.A literature search was done by authors, which focused on data from peer-reviewed published articles, which contained the keywords of CC and its effects on remapping of the hepatic diseases vulnerable to CC, through PubMed, Google, and Medline searches. Information from non-peer-reviewed sources, such as editorials, letters, websites, and the sources with non-English language, were excluded.Search: (("Climate Change"[Mesh]) OR ) AND  OR ("emerging disease") OR ("re-emerging disease") OR ("communicable disease") OR ("non-communicable disease") OR ("infectious disease") OR ("endemic disease") OR ("epidemic disease")).A PubMed MeSH search was done using the below keywords, to find the peer-reviewed published articles which discussed the relation between CC and health map of communicable and non-communicable diseases without limitation of time:Search: ) AND  AND 2016/01/01:2021/12/31 [Date - Publication] AND ("liver disease" [All Fields] OR "liver*" [All Fields] OR ("hepatic" [All Fields] OR "hepatics" [All Fields]) OR ("gastroenterology" [MeSH Terms] OR "gastroenterology" [All Fields] OR "hepatology" [All Fields]) OR "hepato*" [All Fields] OR ("hepatitis" [MeSH Terms] OR "hepatitis" [All Fields] OR "hepatitides" [All Fields] OR "hepatitis a" [MeSH Terms] OR "hepatitis a" [All Fields]) OR "hepat*" [All Fields])The second step was filtration to include the peer-reviewed published articles that focused on the impacts of CC on liver during without limitation in time as seen below:The relationship of CC with liver health problems due to exposure to environmental toxins and pollutants did not appear in the previous search; therefore, the below PubMed MeSH search was done, which revealed only one article published in the last 5 years. For that, additional Google and Medline manual searches were done.Search: (("Climate Change"[Mesh]) OR ) AND  Filters: Free full text,For the effect of mycotoxins on the liver, PubMed MeSH search was done, and then filtration of the results to detect the articles discussing the relationship of CC, mycotoxins, and liver:Google and Medline searches were done using the keywords of this review focusing on pollutants and toxins vulnerable to various parameters of CC, such as temperature, precipitation, and salinity, which have impacts on the liver. Through manual selection, English peer-reviewed published articles during the last 5 years were included. Articles that did not include effects of any toxins or pollutants sensitive to CC on human liver were excluded.The result of the first PubMed MeSH search was 7166 peer-reviewed published articles about climate change and human health during the period between 1976 and 2022. After specifying peer-reviewed published articles that focused on the impacts of CC on liver, 33 peer-reviewed published articles were found during the period between 1991 and 2021.The second PubMed MeSH search to select the peer-reviewed published articles discussing the relationship of CC on liver health problems caused by exposure to environmental toxins or pollutants revealed that only one article was published in the last 5 years. PubMed MeSH search for the effect of mycotoxins on the liver resulted into 31 articles, and after filtration only five peer-reviewed published articles were detected discussing the role of CC on mycotoxins affecting the liver.The results of Google and Medline searches and the manual selection of peer-reviewed published articles focusing on the relationships between the various parameters of CC, such as temperature, precipitation salinity, the different toxins and pollutants, and human hepatic health problems, resulted in 10 peer-reviewed published articles.This review discusses the role of CC on the mapping of some common communicable and non-communicable liver diseases and highlights the importance of considering CC as risk factor for remapping of hepatic diseases and the impacts of CC on the different hepatotoxic or hepato-carcinogenic causative agents mentioned in Table Vectors transmitting parasitic or viral diseases are extremely sensitive to CC, as heat waves, extreme weather events, and salinity have direct effects on breeding rates and sites, survival rates and activities of these vectors, and indirect effects on the pathogens such as shortness or elongation of the incubation period inside the vectors. Therefore, CC will change the geographic distribution, intensity of transmission, and seasonality of VBDs . In addiAedes, Culex, and Anopheles spp. Aedes aegypti is a mosquito vector notorious for spreading different viruses causing hepatic complications such as yellow fever, dengue, Rift Valley hemorrhagic fever, chikungunya (CHIKV), and Zika.Extreme weather events are favorite for mosquito breeding and cause rapid proliferation of several species of mosquitoes, including Aedes aegypti and Aedes albopictus. After infection with CHIKV, the virus replicates in many organs including liver and persists in it. Meanwhile, the main reservoir for persistent CHIKV infection is macrophages. CHIKV could also be affected by people and vector movements, as CHIKV outbreaks spread from Indian Ocean to Europe to be registered in Italy [CHIKV, as an example, is transmitted through the bite of the infected mosquitoes, mostly in Italy  and autoin Italy .Malaria transmission is nonlinear as a response to CC. When maximum temperature is close to the upper limit for vector and pathogen, this leads to reduction of transmission, while daily temperature near the minimum boundary increases the transmission. Infection of the liver cells can cause organ congestion, sinusoidal blockage, and cellular inflammation . Moreove5 and 0.399/105 [Ambient temperature, precipitation, floods, and humidity may have a significant role in distribution of intermediate hosts and pathogens that cause digestive diseases. In China, severe flood events due to CC were found to be associated with increase in the incidence of hepatitis A viral infections (HAV). Based on the data between 2005 and 2010 and flood event scenarios, the incidence of HAV infections was projected to be increased for the year 2030 to be between 0.382/10.399/105 . Moreove.399/105 .Pseudamphistomum truncatum is a newly emerging zoonotic liver trematode affecting Baltic grey seals, with the roach (Rutilusrutilus) as a paratenic host. Accidentally, human may ingest fish with metacercariae, which develop to adults in the bile ducts. This accidental ingestion is of potential risk for humans and will be associated with chronic inflammation, fibrosis, and liver failure [ failure . Global  failure .Fasciola hepatica is a real example of the impact of CC on the change in the distribution and the magnitude of parasitic infestations. Fasciola hepatica is mainly a sheep liver-fluke but could be transmitted to human if they ingest infested liver. Wet and mild warm climate is essential for the parasitic free-living stages and the intermediate snail host to grow on grassland and needs approximately 3 months for parasite stages to develop from the eggs and for the release of the infective metacercariae [ercariae . With glercariae .The main classes of toxicants of clinical significance discussed in this review are the chemicals that could be affected by CC and have toxic impacts on the liver or/and gastrointestinal tract (GIT), including particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs), and heavy metals, in addition to the mycotoxins including aflatoxins and aquatic toxins such as cyanobacteria. The increase in the concentrations of these chemicals may have significant impacts in changing of the health map of some non-communicable hepatic diseases, such as hepatotoxicity or cancers. Biomass combustion associated with heating and cooking needs is a major source of household air pollution, such as PM, PAHs, and different gases .Mostly, PM occurs naturally in the environment as desert dust, forest fire, sea salt, and sulfates from volcanoes; however, PM concentrations were projected to increase with CC in the atmosphere as a result of chemical reactions between the different pollutants due to the global warming, which could cause mutations and cancers of liver and lungs . Artific2.5 and 85% for PM10 [2.5 air pollution was associated with adverse effects on liver enzymes [During the 3-day Saharan episode in Texas in 2008, the total dust contribution increased to 64% for PM enzymes  and shor enzymes .2.5-induced oxidative stress in the liver [Moreover, aflatoxigenic producing fungi may by carried on PM, which increases hepatic cancers . Hepatothe liver . Howeverhe liver , due to he liver .PAHs are found in coal and in tar sediments and produced from incomplete combustion of organic matter as in the cases of forest fires, incineration, and engines. Climate change may increase the emission of toxic environmental chemicals from biomass combustion associated with heating and cooking needs . The amoHeavy metals, such as lead, chromium, arsenic, mercury, nickel, and cadmium, may cause hepatotoxicity, in addition to ultrastructural changes in the hepatocytes, that is induced by oxidative stress reactions . Some stCadmium (Cd), another heavy metal, has been associated with hepatocarcinogenic potential via multiple mechanisms as shown in animal studies . This maRepeated occurrence of extreme events as flooding due to climate change leads to inundation of contaminated land with heavy metals which will be transported in floodwater reaching the freshwater and marine environment and finally to human beings . AlternaGlobal warming and acidification in sea water may increase the methylation rates of heavy metals, such as mercury in tuna and tuna-like species . MoreoveFreshwater harmful cyanobacterial blooms (CyanoHABs) are recognized to produce a wide range of toxins and bioactive compounds, which are secondary metabolites. The most commonly occurring cyanobacterial toxins are the microcystin and nodularin which are hepatotoxic . These tClimate change plays a significant role in the development of CyanoHABs in fresh water, due to the alteration of temperature and light of the ecosystem that could support the growth of CyanoHAB species. Global warming and the change in the pattern of precipitation will increase the frequency of CyanoHAB, which increases the magnitude and the duration of blooms. Furthermore, high atmospheric temperature leads to increase in the stratification and lowers the viscosity of seawater which is favorable to CyanoHAB growth . Thus, t2 by dense CyanoHAB creates a concentration gradient across the air-water interface [Therefore, due to CC, there is a projection that the frequency and duration of cyanobacteria bloom exposure will be increasing in the Baltic Sea within the coming years, as wellnterface , which wAspergillus, Penicillium, and [Mycotoxins are the secondary metabolites produced by certain fungal species including ium, and . Mycotoxium, and . There iium, and .Aspergillus species and infect crops including, e.g., peanut, cotton-seed, maize, nuts, cereals, spices, and dried fruits. AFs are highly liver carcinogenic and can also cause acute toxicity or even be fatal for both livestock and humans if ingested in sufficient amounts; the availability of mathematical models as risk assessment tools for prediction of the possibility of A. flavus infection and levels of AFs contaminations in maize within CC highlights the agricultural practices used to prevent or mitigate the deleterious consequences of AF contaminations [Among all mycotoxins, the biggest attention has been given to aflatoxins (AFs), namely AFB1, because it is a well-known human carcinogen, as CC is considered to be an important predictor for the increase of AFs’ risk of crops’ contamination and improvement of predictive modeling, extension to the impact of CC on fungi and mycotoxin co-occurrence, both in crops and their value chains, up to consumers . AFs areinations .In equatorial and sub-equatorial developing countries, traditional agriculture is usually carried out under optimum conditions and suitable climate for fungal growth and aflatoxin production. This situation is further worsened due to CC, which produces conditions increasingly suitable for the growth of aflatoxigenic producing fungi and mycotoxin production . TropicaAmanita, Lepiota, and Galerina [Mushrooms are important forest fungi in many ecosystems, being highly vulnerable to climate change . AmatoxiGalerina . Alday eGalerina .Africa is vulnerable to CC due to weak adaptive capacity, high dependence on ecosystem for livestock, and less developed agricultural systems. Expectations of changes in temperature, rainfall patterns, safe water, and man-made ecological changes of irrigation schemes will lead to increase the burden of schistosomiasis, as well as malaria in endemic areas in Africa. CC impacts also the infection control scenario of zoonotic infections including fascioliasis and the expansion of vectors or reservoirs of infection such as visceral leishmaniasis and the other parasitic liver diseases in Africa such as human echinococcosis and amebic liver abscess .The current review discusses the role of CC in changing the distribution of some forms of hepatic health problems, which will lead to change in the global health maps. The objective of this review was to make a focus on the importance of planning health strategies for surveillance and prediction of epidemics that could be associated with the climate disasters, which seem to occur more frequently with CC.All the above factors led to redrawing of the maps of liver diseases globally. CC in the form of changes in rainfall patterns, global temperature, humidity, and tropical dusty storms may have a major influence on the distribution, features, and severity of communicable and non-communicable liver diseases. Migration of infected people from endemic infection areas due to the CC disasters to other areas is an important way for the rapid dissemination of infectious diseases. Health infrastructures and population density will affect the incidence and prevalence of the controllable infectious diseases. Proper healthcare programs and infrastructures can prevent the development of outbreaks or endemicity of the disease in the new areas.Proper expectation and designed protective strategies will play a significant role in minimizing the social and economic burdens of the emerging or re-emerging hepatic health problem globally.Thus, early warning surveillance, monitoring, and mitigation strategies are essential tools to be implemented, besides the sustained mapping and watching of infectious diseases vulnerable to CC to guide the healthcare decision-makers to design health policies according to the risk factors and the indicators for control or elimination programs. Moreover, CC impacts liver by exposure to environmental pollutants sensitive to global warming. CC could also affect the geographical distribution of many infectious diseases, as well as hepatic toxicity and carcinogenicity. Different strategies to improve air quality are needed to decrease these hepatic health problems resulting from exposure to environmental pollutants. These strategies require an estimation of the cost-benefits gained from proposed mitigation programs. Public awareness with the scientific approaches must be considered as an essential target solution to decrease this threat and propose sustainable solutions.In conclusion, this review can help policymakers and stockholders in suggesting proper strategies according to the expectations and predicted scenarios of hepatic health impacts due to CC. It can also be used in planning strategies for CC adaptation and mitigation. For the gastroenterologists, this review will develop new vision in the diagnosis and treatment of the hepatological problems. Clinicians should consider CC as a risk factor for several liver diseases and put this into consideration during the management of patients. They should also consider the re-mapping of liver diseases to improve the diagnosis of their patients. For example, considering migration of infected patients from their endemic regions due to the CC would lead to emerging diseases in some areas which will expand the scope of diagnosis of liver diseases. Further researches on CC by hepatologists would be necessary to understand the different mechanisms of inducing liver diseases by CC. This will benefit vulnerable communities to minimize CC risks. Therefore, the review could be of high importance to the health decision-makers as an early alarm for the prediction of hepatic health problems related to CC.


In the Anthropocene the consequences of land-use transformation on ecosystem services are of growing concern, particularly in fragile areas of mountain agriculture that often represent high nature-value farmland. This study uses a decadal repeated survey approach to analyse the effects of modernisation on oasis systems in the Jabal Al Akhdar region of northern Oman. This rugged mountain region at the north-eastern tip of the Arabian Peninsula experiences a growing influx of regional and international tourists since the opening of a modern highway 15 years ago. In 2007, at the onset of transformation processes, a survey was conducted with all households (HH) located in three major settlements along the 1000-m-altitude gradient of the Wadi Muaydin watershed. The survey was repeated in 2018, including all remaining HH of the three settlements. This longitudinal approach allowed studying the consequences of social-ecological transformation processes on crop and livestock husbandry, agricultural labour use, product marketing, and perception of the region’s future by its local residents. Though the village inhabitants are aging and declining in numbers, they still adhere to agriculture, largely because of tradition and identity. Fallowing and abandoning farmland increased over the investigated time span but was paralleled by increased application of agrochemicals and animal manure on fields, purchase of roughage and concentrate feeds for small ruminants, concentration on cash crop and meat production for sale, and increased employment of migrant workers. These indicators of modernisation of oasis agriculture are accompanied by predominantly pessimistic views on future prospects of oasis farming. Commonly perceived problems are shortage of irrigation water and profound societal change. Against these challenges, value chain generation and direct marketing opportunities for local agricultural produce are seen as prerequisites to keep the high nature-value farmland of these mountain oases alive. In the Hajar Mountains of northern Oman, oases settlements are very particular social-ecological systems –3 that hvia livestock manure . Yet, it was in any case far higher than the minimum wages -for agricultural labour- in the workers’ home countries, namely Bangladesh, northern India, and Pakistan . Increased recurrence to migrant labour is also reported from intensifying oasis systems in Algeria, whereby workers in the Adrar region mainly migrate within the country [Across the study period, small ruminant herding on mountain pastures remained an important part of animal-keeping routines. In line with the insights on arable farming, this is a clear indicator that also in livestock farming, innovations and traditional management aspects are combined to ensure the health of the overall system and the individual animal, by allowing for daily exercise outside enclosures and access to a wide variety of fodder plants on the rangelands . At the  country . Replaci country , 58. Acr country . In the Agricultural product processing was of little importance in both survey years, and the major products, vinegar and rose water, were of simple nature. The share of HH engaged in product sales slightly decreased from 2007 to 2018, with an increasing importance of farm-gate sales and a decreasing importance of sales to shops in Sayh Qatanah and at the Nizwa market. Government-promoted olive tree cultivation, fruit pressing, and oil marketing  has not In both study years, farmers strongly expressed the wish to maintain their agricultural activities as part of family tradition and personal and cultural identity, an observation also reported for the inhabitants of the low mountain range region of Transylvania, Romania . For thesarooj mortar [Campanula akhdarensis A.G. Mill. & Whitc., of which the worldwide largest population had been found in S, but has completely disappeared during the last years ([In view of the above, it can be maintained that until today, central elements of sustainable oasis agriculture, namely use of crop rotations, herding livestock on mountain ranges, and close crop-livestock integration with its central component of organic manure application to terraced farmland, are still in place. Even though these practices are increasingly executed by hired foreign workforce, the high amounts of manure applied, the perpetuated quality herding of animals, the relatively low absolute extent of abandoned and fallowed surfaces, the increasing focus on cash crop cultivation in parallel to a still high agro-biodiversity point to the farmers’ dedication to maintain their farming system while adapting it to current socio-economic conditions and possibilities. Hybridization of old and modern management approaches such as use of “non-halal” treated wastewater for crop irrigation, and the renovation of terrace walls and ancient irrigation channels with cement rather than traditional j mortar  indicatej mortar . While wj mortar , 62, thet years (; A. Patzt years (, 64, 65.t years (, 23, evet years (, 33. Thot years ( could beLike other agricultural systems in mountainous or island regions, oases settlements in the northern Hajar Mountains of Oman are undergoing major land-use transformations typical for many high-nature value farmlands worldwide. As elsewhere, their cultivation increasingly relies on culturally foreign, largely unskilled, migrant labour. While some of the traditional agricultural practices that secured the oases’ long-term persistence over centuries are still in use, political support is needed to foster their continued existence as social-ecological model systems whose sustainability has stood the test of time. To support local farmers in their endeavors to continue practicing mixed irrigated agriculture on the steep slopes of Jabal Akhdar as part of their identity, the establishment and use of profitable value chains for primary and processed agricultural products in combination with sustainable forms of ecotourism seem an option worthwhile to pursue.