Despite advances in the development of tumor treatments, mortality from cancer continues to increase. Nanotechnology is expected to provide an innovative anti-cancer therapy, to combat challenges such as multidrug resistance and tumor recurrence. Nevertheless, tumors can greatly rely on autophagy as an alternative source for metabolites, and which desensitizes cancer cells to therapeutic stress, hindering the success of any current treatment paradigm. Autophagy is a conserved process by which cells turn over their own constituents to maintain cellular homeostasis. The multistep autophagic pathway provides potentially druggable targets to inhibit pro-survival autophagy under various therapeutic stimuli. In this review, we focus on autophagy inhibition based on functional nanoplatforms, which may be a potential strategy to increase therapeutic sensitivity in combinational cancer therapies, including chemotherapy, radiotherapy, phototherapy, sonodynamic therapy, and immunotherapy.  Cancer, a leading cause of morbidity and mortality, is a disease that is still an impossible burden on humans, a conclusion that can be derived from its diagnosis, management, and care ,2. ClassAutophagy ,11,12 isThe multistep autophagic pathway provides potentially druggable targets to inhibit pro-survival autophagy under various therapeutic stimuli ,31. ClasIn this review, we summarize works over the past 2–3 years which focus on autophagy inhibition involvement in cancer therapy in the context of nano-platforms as illustrated in The induction of autophagy in cancer cells occurred after long-term chemotherapy or in response to other therapeutic strategies ,40. AutoClinical chemotherapy is markedly hampered by multidrug resistance (MDR). MDR is inevitable following prolonged exposure to chemotherapeutic agents. Increasingly, recent studies have demonstrated that autophagy triggered by chemo-drugs may be involved in MDR, which facilitates the resistance of cancer cells to epirubicin, paclitaxel (PTX), or cisplatin. A prevailing approach to combat these thorny issues is to develop a nanotechnology-based co-delivery system incorporating a combination of autophagy inhibitors with chemotherapeutic agents. Autophagy inhibitors  such as 3O4@Ag, which has the characteristic of radiation enhancement by decreasing the cytoprotective autophagy of cells—was reported by Zhang [Clinically, radiotherapy (RT) is one of the conventional approaches for cancer therapy. A prominent challenge for RT remains that radioresistant hallmarks enable cancer cells to escape the radiation-caused cell damages, resulting in the disappointing efficacy of RT. After exposure to radiation, tumor cells activate the self-repair mechanism to elicit radioresistance. Mounting studies  have shoby Zhang  et al. Mby Zhang  et al. uPhototherapy has drawn increased attention in the field of cancer therapy due to its non-invasive and targeted cell-killing effects, which is ascribed to the inducing of hyperthermia  and production of highly toxic reactive oxygen species (ROS) (photodynamic therapy) with light stimulation. It is known that cytoprotective autophagy is imperatively induced in cancer cells as a result of the above-mentioned adverse cellular stress and might participate in resistance to phototherapy. Nevertheless, the therapeutic efficacy of PTT is still restricted by the indiscriminate heating. An ideal solution to overcome this limitation is chemo-PTT, which combines chemotherapy with photothermal therapy. However, there still remains giant challenges in the pro-survival autophagy elicited by chemotherapeutics and hyperthermia. For this question, we can attain an answer in Zhang’s proof of concept study , where tGold-based PTT  interact+/Cl− across the lysosomal membrane resulted in autophagy inhibition via increasing the lysosomal pH. Excellently, it can make up for the loss of PCN-based PDT efficiency and significantly enhance the efficacy of PDT. What’s more, SQU is involved in the dysregulation of chloride influx to deploy homeostatic perturbation therapy (HPT). Such a novel ideal strategy of ion transport nano-system provides a new direction for clinical cancer treatment research.PDT has emerged as an efficient therapeutic regimen that exerts a cytotoxic effect via ROS. Indeed, evidence arising from experiments involving the relationship between autophagy and PDT has indicated that pro-survival autophagy induced by PDT attenuates the efficacy of PDT as a cancer treatment. Wan’s group  designedSimilarly, ultrasound-based sonodynamic therapy (SDT) as an emerging alternative regimen is also one of the representative therapeutic modalities for noninvasive cancer treatment. ROS-mediated SDT has drawn widespread attention owing to its similar treatment paradigm to PDT . CompareFrom this viewpoint, Feng  developeRecently, it is highly desired to activate the host immune system against cancer, which is anticipated as a promising strategy. However, there remains unsatisfactory efficacy within current immunotherapeutic approaches such as vaccines and cytokines due to the high level of immune suppressive networks involved in cancer . A couplAlbeit, except for the classical autophagy inhibitors such as CQ and 3-MA, delivery occurs via nano-carries for autophagy inhibition, and current studies are being conducted to integrate RNA interference with nanodrugs as a method that could be identified as a more temperate strategy to mitigate the low loading efficiency of small molecule inhibitors. Intrinsically, some nanomaterials such as iron oxide nanoparticles were reported to induce autophagy dysfunction by blocking autophagy flux, and intriguingly, an anion transport SQU-mediated nano-system exhibited the capacity to alkalize lysosome, as is the case with CQ. Above all, it will be of great importance to design novel functional nanotechnological platforms based on autophagy inhibition to increase the sensitivity of therapeutic strategies for tumor. Despite the seemingly promising nanodrugs in the context of tumor treatment, there are expansive, open issues to cope with. To date, it is unknown about the side effects of autophagy inhibition for use in combinatorial therapy based on nanotechnological platforms. Accordingly, it is a giant challenge and mission for both scientists and clinicians, to successfully translate preclinical knowledge of the nanoparticle-mediated autophagy inhibition strategy for tumor treatment into a clinical environment.


Brain function relies on efficient communications between distinct brain systems. The pathology of major depressive disorder (MDD) damages functional brain networks, resulting in cognitive impairment. Here, we reviewed the associations between brain functional connectome changes and MDD pathogenesis. We also highlighted the utility of brain functional connectome for differentiating MDD from other similar psychiatric disorders, predicting recurrence and suicide attempts in MDD, and evaluating treatment responses. Converging evidence has now linked aberrant brain functional network organization in MDD to the dysregulation of neurotransmitter signaling and neuroplasticity, providing insights into the neurobiological mechanisms of the disease and antidepressant efficacy. Widespread connectome dysfunctions in MDD patients include multiple, large-scale brain networks as well as local disturbances in brain circuits associated with negative and positive valence systems and cognitive functions. Although the clinical utility of the brain functional connectome remains to be realized, recent findings provide further promise that research in this area may lead to improved diagnosis, treatments, and clinical outcomes of MDD. Major depressive disorder (MDD) is a common psychiatric disorder characterized by significant persistent sadness and emotional changes . The WorIn contrast to other fields of medicine, current diagnosis in MDD primarily relies on subjective symptoms and observable signs in combination with the Hamilton Depression Rating Scale (HAM-D) scores and Diagnostic and Statistical Manual of Mental Disorders (DSM-V) criteria . DiffereThe standard treatment for MDD includes antidepressants, electroconvulsive therapy (ECT), repeated transcranial magnetic stimulation (rTMS), acupuncture, and cognitive behavioral therapy (CBT). Responses to any given therapy for MDD are often patient-specific. For 30–40% of patients, no effective treatment is found after more than one year of testing different antidepressants using standard care and a trial-and-error process . MoreoveMagnetic resonance imaging (MRI) is becoming an important non-invasive tool to aid in the diagnosis and treatment of MDD due to its high resolution for soft tissue . In partIn this review, we summarized our current knowledge (2000–2021) of the brain functional connectome for MDD. We manually searched electronic databases PubMed and Embase for English language articles published from January 1, 2000, to February 20, 2021. We used the following search terms in combination with the terms “depression”, “MRI” and “functional connectivity”: “bipolar disorder”, “schizophrenia”, “recurrence”, “suicide”, “antidepressant”, “cognitive behavioral therapy”, “electroconvulsive therapy”, “repetitive transcranial magnetic stimulation”, and “acupuncture”. The articles were selected based on the quality of the study, and large randomized controlled trials (RCTs), high-quality observational studies, and reviews were preferred. The case reports and articles in non-peer-reviewed journals were excluded.We first described the workflow of the fMRI-based brain functional connectome and then compared the distinct patterns of the brain functional connectome between MDD and other similar clinical conditions. The later sections focused on the application of the brain functional connectome in predicting MDD recurrence and suicide risk. We also discussed the roles of the brain functional connectome in evaluating the mechanisms of various treatment approaches for MDD, including antidepressants, CBT, ECT, rTMS, and acupuncture. Finally, the conclusion and perspectives on fMRI-based brain functional connectome were presented.fMRI can map the brain functional connectome to characterize the information transmission between brain regions . The bloFirst, whole-brain resting state fMRI is performed, and T1-weighted and T2-weighted images are obtained using the corresponding MRI sequences. fMRI data were also obtained by collecting BOLD signals of the whole brain. fMRI data are then preprocessed to minimize distortion, blurring, and temporal artifacts. To standardize the MRI data collection and preprocessing, in 2016, HCP published a detailed protocol for imaging data preprocessing (HCP-style paradigm) . Either Although several theories have been proposed for MDD pathogenesis, the serotonin hypothesis is still the most prevailing molecular mechanism of depression. This theory postulates that serotonin deficiency in the synaptic cleft is involved in the etiology of MDD, which is supported by the clinical efficacy of many current antidepressants such as serotonin selective reuptake inhibitors (SSRIs) . Seroton5-HTT is a protein encoded by the SLC6A4 gene in humans. Although neuronal serotonergic cells project to virtually every brain area, 5-HTT availability is not equally distributed throughout the brain. Recent studies based on RNA sequencing and positron emission tomography (PET) imaging showed that the SLC6A4 gene is highly expressed in subcortical limbic regions of the human brain, specifically the hippocampus, amygdala, and anterior cingulate cortex (ACC), which are the network hubs of the human functional connectome related to emotional processing, cognitive ability, and memory , 26. As Besides the monoamine hypothesis, it has been suggested that impaired neuroplasticity may be causally associated with the development and course of depression. The neuroplasticity hypothesis of depression is supported by the evidence of decreased concentration of brain-derived neurotrophic factor (BDNF) in both animal models of depression and patients with depression. BDNF in the brain is a common neurotrophin that play important roles in synaptic plasticity and neuronal survival through binding TrkB receptors in postsynaptic neurons . Upon biAmong different brain regions, BDNF exhibits its highest expression in the hippocampus, amygdala, olfactory bulb, and cerebellum. The hemizygosity of BDNF modifies the availability of BDNF in the synaptic cleft. Similar to serotonin signaling, the single nucleotide polymorphism (rs6265) of BDNF, also known as Val66Met, had been demonstrated to reduce not only the hippocampal volume but also brain functional connectome crucial for language, attention, and emotions , 33. ForThe misdiagnosis rate of BD patients is extremely high due to the similarities in the clinical presentation of depressive episodes present in both unipolar and bipolar depression and the higher prevalence of unipolar depression compared with BD , MisdiagThe frontoparietal network (FPN), also known as the central executive network (CEN), is involved in working memory and sustained attention. Compared to unipolar patients, BD patients showed significantly increased FC within the FPN, primarily in the left ventrolateral and dorsolateral prefrontal cortex (vlPFC and dlPFC) . The hipIn contrast, compared to unipolar MDD patients, BD patients had decreased FC in the DMN, primarily in the bilateral precuneus (PCUN) and hippocampi . High FCIn term of the biomarkers in other domains for differential diagnosis of BD and MDD, much of the interest was directed at neurotransmitters and their associated receptors as well as neurotrophic factors in brain regions such as ACC, right prefrontal lobe, and orbital frontal cortex (OFC). The most consistent results across several studies were the increased N-Acetylaspartic acid (NAA) and glutamate (Glu) levels in ACC and right prefrontal lobe in bipolar depression in contrast to unipolar , 51 Fig. The choBox 1 Three large-scale brain functional networksMDD is one of the most commonly misdiagnosed psychiatric disorders, and there is a high degree of overlap between the symptoms of MDD and SZ. A recent study reported that nearly 55% of patients with MDD in Ethiopia were more likely to be misdiagnosed as having SZ . DiffereIn terms of large-scale networks, previous studies have identified distinct dysconnectivity patterns within the triple network framework, that includes the DMN, SN, and FPN. MDD patients were found to have stronger FC between the SN and DMN than SZ patients . The mosRegarding various brain regions, FC differences were also observed in the frontal lobe and temporal lobe between SZ and MDD patients. The frontal lobe is involved in controlling important cognitive skills in humans, such as emotional expression and memory. FC within the prefrontal cortex (PFC) of the frontal lobe was significantly reduced in schizophrenia patients compared with MDD patients . Wei andA high proportion of patients with remitted MDD will experience recurring episodes, and clinical symptoms tend to be more severe in recurrent episodes than in previous episodes , 63. RecSuicide ideation and behavior are closely related to psychopathology, especially depression . It is dHypoconnectivity has been observed between large-scale brain networks, especially those involved in executive functions, in both adult and adolescent MDD patients with suicidal ideation compared with those without suicidal attempts Fig. . For insDifferential functional connectivity in brain circuits related to cognitive functions has also been observed between MDD patients with and without suicide attempts Fig. . Fronto-Several studies also reported brain region-dependent hyperconnectivity in MDD patients with suicidal ideation. Resting-state FC between the superior frontal gyri and left precuneus was found to be positively correlated with suicidality . In addiMachine learning approaches can process high dimensional variables and assess all the brain compartments simultaneously . A recenInterestingly, previous studies also reported some protein and metabolite biomarkers for suicidal behaviors in the brain regions with functional connectome alternations and the peripheral blood –92. ThesFor a long time, drug therapy has been the first choice of treatment for depression because of its advantages, such as simplicity and strong operability . Brain cThe amygdala, located in the medial temporal lobe, is a critical brain region of emotion regulation. SSRIs including fluoxetine, sertraline, and citalopram, are among the most commonly prescribed antidepressants. It was reported that 8 weeks of SSRI treatment normalized the increase in functional connectivity between the amygdala and bilateral PFC in MDD patients . VenlafaAlthough SSRIs and SNRIs have dominated the treatment of depression, a major limitation of these antidepressants is the delayed clinical onset . KetaminTreatment responses to SSRIs and SNRIs vary greatly between patients . The fMRCBT is a type of time-limited, intensive, symptom-focused psychotherapy that aims to alleviate depressive symptoms by encouraging patients to change destructive or disturbing thought patterns that have negative influences on behavior and emotions . StudiesTo date, CBT treatment has been primarily found to be associated with amygdala resting-state connectivity Fig. . StudiesThe subcallosal cingulate (SCC) is known to be involved in top-down emotion regulation . The effElectroconvulsive therapy (ECT) is a common approach for the treatment of MDD, especially for patients with refractory or severe depression , in whomBy analyzing resting-state fMRI data from 84 subjects, Bai et al. found that ECT treatment restored the reduction in functional connectivity between dmPFC and PCC in MDD patients. Increases in FC between these two brain regions following ECT were correlated with improvements in depressive symptoms in MDD patients . AdditioDisruptions in functional interactions within the cognitive emotion regulation network (ERN) is one of the core features and underlying mechanisms of MDD . A recenAlthough ECT is generally safe, it may cause memory loss, confusion and low verbal frequency . These srTMS is a non-invasive treatment that uses specially designed magnetic coils placed near the head to electrically stimulate the brain to trigger neuronal action potentials by generating a rapidly changing strong magnetic field , 139. rTClinical improvements for patients with TRD following rTMS were found to be related to the decreased functional connectivity between the sgACC and right dlPFC and increased connectivity between the rACC and left lateral parietal cortex (llPC) Fig.  141]. I. I141]. The effectiveness and safety of acupuncture treatment, an essential therapeutic modality in complementary and alternative medicine, for depression have been confirmed in a number of studies –145. fMRThe amygdala and OFC are two key regions in the emotional regulatory nervous system. Dugan et al. found that acupuncture stimulation at BaiHui increased functional connectivity between the amygdala and the OFC and decreased FC between the amygdala and the angular gyrus in MDD patients . RepeateA small clinical trial with 46 patients also showed that acupuncture treatment resulted in an increase in FC in brain regions involved in reward processing, such as FC between the ventral striatum and mPFC, and between the amygdala and ventral prefrontal putamen (VPP) . At the The development of fMRI-based brain functional connectome techniques has enabled us to navigate the living human brain in a way that was never before possible. Through analyses of the brain functional connectome, we can compare the brain circuits between MDD and other conditions and then zoom into the differential brain circuits to explore the functions that depend on them. Studies published in the past 20 years have demonstrated that connectome dysfunctions between brain circuits associated with negative/positive valence systems, cognitive systems, and cross-cutting elements are the core features of MDD Table . The sysDespite the substantial progress that has been made in the analysis of brain functional connectome, several technical issues remain unresolved. First, brain segmentation of function connectome is still challenging due to the complexity of the human brain and the absence of a true gold standard. Second, at present, most studies have not conducted in-depth optimization of the identification methods for time series characteristics of fMRI data. Third, there are discrepancies in using different algorithms to construct functional networks based on deep learning, and how to select algorithms to improve their applicability in the brain functional system remains to be studied. Furthermore, it is worth integrating multi-level templates and multi-modal images using machine learning approaches to enhance the accuracy and efficiency of large-scale brain connectivity.Given the increasing public health need, we must improve our understanding of how connectome dysfunctions alter behaviors in MDD and the associations between connectome changes and the abnormalities of molecular biomarkers. Most past studies were limited by the small sample size. Connectome atlases based on a larger number of subjects per group and high-resolution imaging techniques will provide more reliable and consistent results. Connectome analysis could include more diverse age groups, especially adolescents. Additionally, the integration of results from different connectomes, including connectomes based on proteomics, metabolomics, transcriptomics and exposomics, may prove more informative than analyses based on a single connectome , 150.In conclusion, alterations in the human brain functional connectome are central to the neurobiology of MDD and include changes in multiple, large-scale brain networks as well as local disturbances in brain circuits related to negative/positive valence systems and cognitive functions. With the growing compilation of brain connectome data, the development of high-resolution MRI imaging techniques, and the application of data-driven machine learning algorithms, we anticipate that brain connectome analyses will continue to surprise us and provide a better understanding of living healthy brains and MDD.


Functional bowel disorders such as irritable bowel syndrome (IBS) are common, multifactorial and have a major impact on the quality of life of individuals diagnosed with the condition. Heterogeneity in symptom manifestation, which includes changes in bowel habit and visceral pain sensitivity, are an indication of the complexity of the underlying pathophysiology. It is accepted that dysfunctional gut-brain communication, which incorporates efferent and afferent branches of the peripheral nervous system, circulating endocrine hormones and local paracrine and neurocrine factors, such as host and microbially-derived signaling molecules, underpins symptom manifestation. This review will focus on the potential role of hepatic bile acids in modulating gut-to-brain signaling in IBS patients. Bile acids are amphipathic molecules synthesized in the liver, which facilitate digestion and absorption of dietary lipids. They are also important bioactive signaling molecules however, binding to bile acid receptors which are expressed on many different cell types. Bile acids have potent anti-microbial actions and thereby shape intestinal bacterial profiles. In turn, bacteria with bile salt hydrolase activity initiate the critical first step in transforming primary bile acids into secondary bile acids. Individuals with IBS are reported to have altered microbial profiles and modified bile acid pools. We have assessed the evidence to support a role for bile acids in the pathophysiology underlying the manifestation of IBS symptoms. Over a hundred trillion microbial organisms, mostly bacteria, inhabit the human colon and have co-evolved with their hosts to have diverse, but primarily beneficial, functions. They scavenge additional calories by fermenting non-digestible foods, secrete vitamins and ensure normal physiological development. A plethora of studies have demonstrated that microbes have the capacity to modulate host physiological homeostasis and have been linked with cognitive disorders such as anxiety, depression, Parkinson’s disease, autism spectrum disorder and schizophrenia , 2, in avia the biliary ductal system. Comprising about half of the total solutes in bile, their amphipathic structure facilitates emulsification and subsequent digestion and absorption of dietary lipids being emptied from the stomach  and chenodeoxycholic acid (CDCA), into the duodenum  stomach . The entle acids Figure 1le acids . Bile sale acids . Given tle acids –14 and ble acids . We havevia the portal vein. The ~ 5% of bile acids which reach the colon, are either reabsorbed via passive diffusion or lost in the feces  is the most extensively studied nuclear bile acid receptor, for which CDCA is the most potent agonist , however3 and certain dietary ligands, LCA acts as endogenous ligands for VDR , also called Takeda G-protein-coupled receptor 5 (TGR5) . TGR5 isIBS is a clinically diverse disorder, with a multifactorial etiology. Global prevalence varies from ~1% to more than 45%, with between 5-10% reported for Europe, the United States and China . CharactE. coli -secreting enterochromaffin cells are one such chemosensory cell type and they are coupled to sensory nerves. Catecholamines and microbially-produced short-chain fatty acids, such as butyrate and isobutyrate  can stim origins , and con origins . Abnorma origins , 97.Recent research has detected expression of TGR5 in enterochromaffin cells in the colon but not the small intestine . In micevia cellular transducers in the epithelium with precise, temporal transmission of sensory signals (Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY)-secreting L-cells are electrically excitable biosensors integrated into the epithelium. They express a plethora of receptors including receptors for GABA , short-c signals .Bile acids are noted for their antimicrobial properties which prevents small intestinal bacterial overgrowth . This maAn adaptive or allostatic response to a perceived environmental threat underpins the stress response and is initiated by release of corticotropin-releasing factor from the hypothalamus to stimulate HPA activity. Chronic activation of the HPA axis has, however, been associated with altered bowel morphology, function and visceral pain sensitivity , 118 andColonic exposure to excess bile acids, which may be due to loss of bile acid transporters in the ileum causing bile acid malabsorption , overproModification of epithelial permeability and stimulation of pro-secretory pathways involving cAMP is likely to underlie the manifestation of bile acid-evoked watery diarrhea , 129, whAlthough the modulatory effects of bile acids on absorpto-secretory function are most obvious, both primary and secondary bile acids can also modify intestinal motor function. In a patient study, modest increases in stool bile acids were noted as an underlying factor in the onset of diarrhea in individuals diagnosed with IBS-D, but who did not have bile acid malabsorption, and it was found that this was mediated by an increase in gut motility . PerfusiIn addition to altered bowel habit, individuals with IBS often present with bloating and abdominal pain indicative of visceral hypersensitivity. Recently reported findings from a rodent study have implicated Nerve Growth Factor (NGF) and transient receptor potential vanilloid channel (TRPV1) nociceptors in a signaling pathway where bile acids could modulate visceral pain signals. NGF, an important mediator in the generation and maintenance of pain, was upregulated following exposure to bile acids and this was mediated through activation of FXR. As mast cells synthesize, store and secrete NGF, the authors suggested that bile stimulated NGF release from mast cells, which, in turn, activated nociceptors and induced visceral hypersensitivity . TRPV1-eResearch into the etiology of IBS recognizes the complexity of this multifactorial and heterogenous bowel disorder. There is growing evidence to support a role for bile acids in the pathophysiology of IBS, through interactions with the microbiome and host sensory and/or immune cells or through direct actions on the peripheral and central nervous system Figure 2Mechanistically, bile acid receptors are expressed on intrinsic and extrinsic nerves, which could facilitate direct neurally-mediated changes in gut function. Moreover, when bile acids are at supraphysiological levels, they can breach the blood brain barrier to modify the HPA axis, which in turn, can modulate intestinal function and luminal microbes. However, bile acid receptors are also detected on enteroendocrine and immune cells, which could both act as signal transducing intermediaries, secreting factors which subsequently modify colonic activity either through direct actions or by modulating neural regulation of the gut. Further complexity lies in the variability of responses evoked by conjugated or unconjugated, primary or secondary bile acids. As bile acids have emerged as effectors in microbe-host signaling and can directly and indirectly modulate gut homeostasis, these bioactive molecules should not be overlooked as the pathophysiology of IBS is elucidated. Indeed, interventions to modify colonic exposure to bile acids could reveal effective therapeutic options for this functional bowel disorder.RD, QX, and DO’M prepared this review together. All authors contributed to the article and approved the submitted version.DO’M is a funded investigator in APC Microbiome Ireland, which is supported by Science Foundation Ireland [Grant SFI/12/RC/2273]. QX is supported by CSC, PR of China.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 reviewer VC declared a shared affiliation with the authors to the handling editor at time of review.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.


During the past decade, a stem cell-based hypothesis has emerged (among many others) to explain the pathogenesis of endometriosis. The initial hypothesis proposed that endometriosis arose from a single or a few specific cells with stem cell properties, including self-renewal and multi-lineage cell differentiation. The origins of the endometriosis-initiating stem cells were thought to be the bone marrow, uterine endometrium, and other tissues. Based on the implantation or metastatic theory in combination with the initial stem cell theory, one or a few multipotent stem/progenitor cells present in the eutopic endometrium or bone marrow translocate to ectopic sites via fallopian tubes during menstruation, vasculolymphatic routes, or through direct migration and invasion. Subsequently, they give rise to endometriotic lesions followed by differentiation into various cell components of endometriosis, including glandular and stromal cells. Recent somatic mutation analyses of deep infiltrating endometriosis, endometrioma, and eutopic normal endometrium using next-generation sequencing techniques have redefined the stem cell theory. It is now proposed that stem/progenitor cells of at least two different origins—epithelium and stroma—sequentially, differentially, but coordinately contribute to the genesis of endometriosis. The dual stem cell theory on how two (or more) stem/progenitor cells differentially and coordinately participate in the establishment of endometriotic lesions remains to be elucidated. Furthermore, the stem/progenitor cells involved in this theory also remain to be identified. Given that the origin of endometriosis is eutopic endometrium, the candidate cells for endometriotic epithelium-initiating cells are likely to be endometrial epithelial cells positive for either N-cadherin or SSEA-1 or both. The candidate cells for endometriotic stroma-initiating cells may be endometrial mesenchymal stem cells positive for SUSD2. Endometrial side population cells are also a possible candidate because they contain unipotent or multipotent cells capable of behaving as endometrial epithelial and stromal stem/progenitor cells. Many hypotheses explaining the pathogenesis of endometriosis have been proposed over the past 100 years. The retrograde menstruation theory accounts for the pathogenesis of various types of endometrioses, and it is the most widely accepted among the various hypotheses. They include the coelomic metaplastic theory, embryonic rest theory, and lymphovascular metastasis theory ,3,4,5. AThus, the retrograde menstruation theory alone cannot perfectly account for the pathogenesis of all types of endometrioses, although endometriosis in premarcheal girls is thought to result from the seeding of naive endometrial progenitor cells into the pelvic cavity at the time of neonatal uterine bleeding . The weaMost of the current theories have been based on the assumption that endometriotic cells originate from the eutopic endometrium. However, other theories are suggested by new advances in endometrial physiology. For example, over the past two decades, endometrial stem cell physiology has been extensively explored through identification, isolation, and characterization of endometrial stem/progenitor cells and clarification of their roles in endometrial structure and function ,18,19,20Since the first discovery of ways to derive embryonic stem cells from early mouse embryos , stem ceThe human endometrium undergoes cyclic repetitive changes that include proliferation, differentiation, tissue breakdown, and shedding (menstruation) more than 400 times throughout a woman’s reproductive life . The repAmong these cells, ESP cells are perhaps the most extensively studied stem/progenitor cells derived from the endometrium ,36,37,38Human endometrium contains small numbers of SP cells  with stem cell properties, including multi-lineage differentiation into a variety of endometrial cell components, including epithelial, stromal, endothelial, smooth muscle cells, and tissues when transplanted under the kidney capsule of immune-deficient mice  Figure . As illu+CD146+) are enriched in the ESP fraction ) or create (as pattern [C’]) CAMs-bearing or epigenetically changed epithelial stem/progenitor cells, they can survive and behave as active lesions similar to the red lesions of peritoneal endometriosis. Otherwise, such tentative endometriotic lesions eventually disappear, resulting in scar formation similar to the white lesion of peritoneal endometriosis.The presence of both driver and passenger mutations in epithelial and stromal cells is rare ,62,63,66This latter scenario, however, does not apply to a rare case in which three distinct and independent DIE sites have the same KRAS mutation . In thisThe dual stem cell theory seems robust, but greater elucidation of the mechanisms underlying the dual stem cell theory is needed. Furthermore, the dual stem cell theory does not exclude the possibility that endometriotic epithelial and stromal cells arise from a common hypothetical stem/progenitor cell as proposed by the initial stem cell theory ,16. It rEndometrial stem cells and their supportive niches were mostly hypothetical prior to 2000, but thereafter, through identification and characterization of those cells and systems, they have been recognized as critically important. In accordance with advances in endometrial stem cell physiology, the stem cell theory (hypothesis) for the pathogenesis of endometriosis has emerged over the past decade. The initial theory proposed that endometriosis arose from a single or a few endometrial stem/progenitor cells present in eutopic endometrium or bone marrow as a result of retrograde menstruation and/or lymphovascular dissemination. The stem cell theory may permit unification of the various theories accounting for implantation and coelomic metaplasia. More recent evidence based upon somatic mutation analyses of microdissected endometriotic and endometrial samples using next-generation sequencing has substantiated the initial stem cell theory but with some revision. At present, the most likely mechanism is that epithelial stem/progenitor cells independently acquire CAMs and that both the epithelial stem/progenitor cells with CAMs and stromal stem/progenitor cells without CAMs differentially, sequentially, but coordinately participate in the genesis of endometriotic lesions. Given that the origin of endometriosis is eutopic endometrium, the candidate cells for endometriotic epithelium-initiating cells are likely to be endometrial epithelial cells positive for either N-cadherin or SSEA-1 or both. The candidate cells for endometriotic stroma-initiating cells may be endometrial mesenchymal stem cells positive for SUSD2. ESP cells are also a possible candidate because they contain unipotent or multipotent cells capable of behaving as endometrial epithelial and stromal stem/progenitor cells. The dual stem cell origin model is particularly relevant for DIE and ovarian endometrioma, presumably via retrograde menstruation or lymphovascular dissemination. However, it remains unclear how both types of stem/progenitor cells coordinately determine their fates. Elucidation of the precise origin of endometriotic cells and possible stem cell-mediated mechanisms underlying the pathogenesis of endometriosis will contribute to the development of new modalities of diagnosis, treatment, and management of endometriosis, but such developments await further studies.


Light acts as a vital external cue that conveys surrounding information into plant growth and performance to facilitate plants to coordinate with changing environmental conditions. Upon exposure to light illumination, plants trigger a burst of molecular and physiological signaling cascades that induces not only photomorphogenic responses but also diverse adaptive behaviors. Notably, light responses and photomorphogenic traits are often associated with plant responses to other environmental cues, such as heat, cold, drought, and herbivore and pathogen attack. Growing evidence in recent years demonstrate that the red/far-red light-absorbing phytochrome (phy) photoreceptors, in particular phyB, play an essential role in plant adaptation responses to abiotic and biotic tensions by serving as a key mediator of information flow. It is also remarkable that phyB mediates the plant priming responses to numerous environmental challenges. In this minireview, we highlight recent advances on the multifaceted role of phyB during plant environmental adaptation. We also discuss the biological relevance and efficiency of the phy-mediated adaptive behaviors in potentially reducing fitness costs under unfavorable environments. Light acts as a central environmental cue that triggers a variety of adaptive behaviors in plant organs, termed photomorphogenesis . Plants While light is critical for plant growth and survival, high light intensity often accompanies temperature increases over a physiological range in nature , which pAccumulating evidence indicate that phyB also plays a role in maintaining plant fitness under abiotic and biotic stress conditions , which aThe primary task of this minireview is to summarize recent advances on the versatile roles of the phy photoreceptors, with focus on phyB, during thermomorphogenesis and adaptive responses to abiotic and biotic challenges and discuss a unified phyB-mediated signaling network that integrates multiple signaling cues into plant growth and adaptive morphogenesis. Considering that plant stress adaptation is tightly linked with managing fitness costs , we alsoArabidopsis, which mediate both distinct and overlapping photomorphogenic traits  gene encoding a fatty acid desaturase  phytohormone regulates stomatal movements and drought-responsive gene expression. Under drought conditions, ABA accumulates mainly in the leaf vascular tissues, from which it spreads to virtually all plant tissues to enhance stomatal density and closure . Recent Insect herbivores and pathogens impose a destructive threat on plant survival and propagation. Plants therefore have developed versatile defense mechanisms to cope with these biotic stressors, and a large collection of pathogen- and herbivore-inducible genes and signaling molecules have been identified in various plant species , among wIt is notable that the induction pattern and altitude of plant defense responses are widely affected by light and temperature. The coordinated linkage between plant defense and light cycles has been extensively studied . The onsphyA phyB double mutants also exhibit a significant reduction in SA sensitivity and systemic acquired resistance, indicating that phy-mediated light signaling is essential for SA-associated defense responses. In contrast, under high R/FR conditions, the light stabilization of FHY3 and FAR1, two homologous transcription factors that are important for phyA signaling  gene, which functions in JA metabolism, is elevated in a phyB/PIF-dependent manner (ST2A induction is linked with the reduction of JA accumulation, weakening JA responses. Meanwhile, low R/FR ratio and phyB mutation induce rapid degradation of DELLA proteins (Moreover, it has been demonstrated that phyB regulates JA biosynthesis and sensitivity, depending on R/FR ratio . Under lt manner . The ST2proteins , well-knproteins , to incrMolecular genetic studies and genome-scale approaches in model and crop plants indicate that they have evolved diverse mechanisms and signaling networks for efficient sensing and transducing surrounding signals into morphological, physiological, and biochemical processes, culminating in environmental adaptation and survival . The besThe intimate linkage between light and other environmental cues reflects two distinct ecological consequences. One aspect is the signaling interactions of light with other cues. In nature, light signals oscillate in accordance with other environmental cues, such as temperature and drought . TherefoWe summarized recent findings on the multifaceted role of phyB in plant environmental adaptation and discussed its potential role in maintaining plant fitness under fluctuating environments. In natural habitats, multiple environmental changes occur in a complex manner. While the patterns of temperature and drought cycles and the propagation patterns of herbivore and pathogen cooscillate in a regular manner with light cycles in many cases, others, such as temperature extremes, occur somewhat independent of light cycles. Nevertheless, light signaling is closely associated with most of abiotic and biotic signals by sharing some signaling molecular and mechanisms. It is believed that the phyB-mediated signaling networks help plants to manage fitness costs in adapting to environmental alterations. Therefore, signaling interactions and managing the growth-defense tradeoffs would have to be considered for better improvements of adaptive traits.C-MP and JYK designed the concept and organization of the manuscript. C-MP and JYK wrote the manuscript with helps of J-HL. All authors contributed to the article and approved the submitted version.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.


To date, only one single-center European randomized controlled trial3 has shown improvements in postoperative complications, pain, short-term quality of life, and functional recovery when comparing robotic with open esophagectomy. Furthermore, a recent publication from the Upper Gastrointestinal International Robotic-assisted Association (UGIRA) demonstrated promising results of this technique when undertaken in high-volume specialized centers with adequate training.4 The data concerning robotic gastrectomy is largely based on observational cohort studies5 and originates from the Far East, with a different patient population and standard of lymphadenectomy to what is commonly observed in Western centers.6Over the past decade, there has been a marked increase in the uptake of robotic surgery in patients undergoing curative esophagectomy or gastrectomy for esophagogastric cancers. Although there is an expanding evidence base for minimally invasive techniques that appears to suggest either improved or similar morbidity, without compromising oncological quality,7 included patients with nonmetastatic esophageal and gastric cancers receiving open , laparoscopic , or robotic  surgery from the US National Cancer Database (NCDB) (2010–2017). Patients receiving robotic surgery were more commonly treated within high-volume, academic centers and with advanced clinical T and N stage disease. From 2010 to 2017, textbook outcome (TO) rates increased for esophageal and gastric cancer treated by all surgical techniques. RAMIE  and RAMIG  had significantly higher TO rates compared with open surgery. For esophagectomy, TO  and RAMIE  were both associated with long-term survival. For gastrectomy, TO  and both LAMIG  and RAMIG  were all associated with long-term survival. Subset analysis in high-volume centers confirmed similar findings.The present studyMoving forwards, dissemination of robotic surgery is key to ensuring routine adoption into clinical practice to optimize patient benefits. Firstly, implementation of training programs should be safe and be adopted within high-volume centers and/or surgeons to ensure a sufficient case volume to shorten any potential proficiency gain curve. Further, embedding adjuncts such as video-based analyses of performance, telemedicine for surgical coaching, and image-based surgery with projections of preoperative imaging may allow refined surgical anatomy and dissection in cancer surgery and shorten the learning curve among surgeons. Secondly, regulators and surgical community need to have highly regulated systems in place, such as international registries. This would be useful for (i) close monitoring of performance and uptake of robotic surgeries across various specialties and (ii) generating accurate data to inform the creation of appropriate safeguards; national bodies should consider providing coverage for robotic surgery with provisions for evidence development. The UGIRA was established to facilitate the reporting of robotic procedures worldwide and analyze variation and learning curves. Use of these provisions would facilitate greater understanding of how robotic procedures are being used in real-world practice. Akin to post-market surveillance of pharmaceuticals, such provisions would also create a common data resource from which the comparative safety and effectiveness of robotic operations can be evaluated by numerous investigators and is necessary for not only RAMIE/MIG, but also other types of surgery.


Nature by Oren and colleagues used barcode lentiviral library labeling to prominently discern antioxidant profiles, among others, being the primary underlying cellular program that governs proliferation under tyrosine kinase inhibitor (TKI) drug pressure.1The ability of a minority of cancer cells lineages to withstand drug-induced toxicity and re-enter cell cycling in the presence of the drug has been observed for long, but the underlying mechanisms remained elusive. New work published in 1 Thus, this study design is fundamentally different from most reports investigating the eventually-arising drug-resistant cells rather than their constituting progeny .In the study, lung cancer cells were pulsed with osimertinib, a third-generation EGFR TKI. After two weeks, two main rare populations were identified: non-cycling persisters that survived drug treatment and cycling persisters proliferating under drug burden. The elegance of the experiments lies in the utilization of a lentiviral barcode library called Watermelon, allowing back-tracing and transcriptional profiling of the initial progenitor cells present before drug addition that gave rise to the surviving cells two weeks later.Cycling persisters are responsible for tumor relapse in patients. The overarching hallmark of this cell population was a redox profile shifted towards antioxidant responses, as characterized by increased glutathione metabolism and Nrf2 gene signatures and decreased ROS levels. This was seen in several cell lines in vitro, minimum residual disease lung tumors in vivo, and as collective transcriptional ROS signature in residual disease lung, melanoma, and breast tumor samples derived from patients after oncogenic kinase inhibitor treatment. In vitro, through the Watermelon system, these signatures were found to be induced through drug challenge in distinct lineages rather than being present a priori. The role of ROS and antioxidants was validated as the percentage of cycling persisters was increased by genetic suppression of the Nrf2 negative regulator Keap1, overexpression of the transcriptional Nrf2 target GPX2, and to the greatest extent by the addition of the antioxidant NAC. By contrast, a decline was observed with pharmacological inhibition of the cysteine-glutamate antiporter xCT crucial for synthesizing the antioxidant glutathione. Strikingly, non-cycling persisters did not show such (but instead other) signatures, suggesting ROS control to be the decisive element governing the fate of tumor cells not only in terms of drug resistance but also in retaining proliferate capacity.Oncogene using PC-9 cells, among others, as in the Nature study.2 They reported that EGFR-dependent proliferation in PC-9 cells could be circumvented when inducing oxidative stress generated through cigarette smoke, which inactivated Keap1 to free Nrf2, eventually unleashing its three-fold battery of cellular defense mechanism, namely antioxidant genes, phase II detoxifying enzymes, and drug efflux pumps. The results were confirmed in A549 cells harboring constitutively active Nrf2 signaling due to a mutation in Keap1, rendering the cells intrinsically insensitive to TKI-induced proliferation suppression.These results do not seem overly surprising, considering the many existing reports on Nrf2 and antioxidant defense in drug resistance. At the same time, the study is novel in the way that it shows that the inducibility of the Nrf2 pathway is critical for cell survival under drug pressure rather than its preexisting activity on a single cell level rather than bulk profiling. Moreover, cycling persisters under drug pressure were re-seeded in the absence of drugs, giving rise to progeny with similar drug sensitivity profiles as found initially, ruling out genetic mechanisms causing drug resistance in this study. However, the underlying question is where the ROS are coming from that elicit the Nrf2 pathway in the first place. The EGFR targeting of osimertinib deprives lung cancer cells of pro-survival signaling, and ROS are known to be generated during pro-apoptotic processes and autophagy. As ROS, Nrf2, and antioxidant signatures were the main hallmark of cycling persisters in this study, it can be assumed that these are decisive for drug resistance and retained proliferation. A key study supporting these findings was made by Yamadori and colleagues published in 2012 in Nature study to spur NRf2 activity? Second, why is such a mechanism only active in the small subset of cycling persisters but not all cells? The cellular dynamics of the second question remain to be unraveled, as the Nature study showed that drug-resistant cyclers’ transcriptional profile did not significantly differ from other cells before drug addition and that they regained drug-sensitivity during prolonged culture in the absence of TKI. Regarding the first question, several ROS sources are known to contribute to altering intracellular redox states, such as NADPH oxidases, dual oxidases, mitochondrial superoxide leaking from the respiratory chain activity, and reduced antioxidant activity. Moreover, excess ROS can also stem from extracellular sources, such as dyeing cells, releasing hydrogen peroxide into the microenvironment that can enter live cells through aquaporins. The ROS levels, however, must be low enough to serve as signaling molecules (oxidative eustress)3 to activate the Nrf2 pathway that then serves as an EGFR-independent driver of proliferation. Apart from where the initial ROS are coming from, it is unclear how the ROS levels are sustained to promote continuous Nrf2 activation. As TKI-resistance was retained only presence of osimertinib, it is conceivable that the substance may have off-target effects on the activity of ROS-generating enzymes or other proteins, such as endogenous uncouplers of the mitochondrial respiratory chain to promote ROS leakage.This proliferation seen under TK inhibition with concomitant Nrf2 activation prompts two key questions. First, where is the ROS coming from in the Over the last years, the plot thickened on the critical roles of spatiotemporal ROS level changes and modulation in their removal capacity in cancer drug resistance. At the same time, the omnipresence of ROS and technical hurdles in their identification and quantification leads to an often underestimated role of ROS in setting pathways in motion. Thus, it is time to call for a dedicated field of Redox Oncology focusing on the roles of ROS and their regulation in preclinical and clinical cancer biology and medicine.4 Another innovative local ROS-generating treatment modality is gas plasma technology, with first cancer patients benefiting during early clinical trials.5 However, consensus-based broader clinical application and therapy approval of such approaches is still awaited.Finally, the question arises of how ROS can be actively engaged and locally generated to promote tumor cell demise in concert with standard therapies. Besides classical photodynamic therapy, new approaches continuously arise from the field of nanomedicine and functionalized nanomaterials. For instance, surface-oxidized nanosheets were recently fabricated and provided both ROS-dependent antitumor efficacy and fluorescence-guided imaging in preclinical cancer models as a novel theranostic platform.


Metabolic insufficiency and neuronal dysfunction occur in normal aging but is exaggerated in dementia and Alzheimer’s disease (AD). Metabolic insufficiency includes factors important for both substrate supply and utilization in the brain. Metabolic insufficiency occurs through a number of serial mechanisms, particularly changes in cerebrovascular supply through blood vessel abnormalities , alterations in neurovascular coupling providing dynamic blood flow supply in relation to neuronal demand, abnormalities in blood brain barrier including decreased glucose and amino acid transport, altered glymphatic flow in terms of substrate supply across the extracellular space to cells and drainage into CSF of metabolites, impaired transport into cells, and abnormal intracellular metabolism with more reliance on glycolysis and less on mitochondrial function. Recent studies have confirmed abnormal neurovascular coupling in a mouse model of AD in response to metabolic challenges, but the supply chain from the vascular system into neurons is disrupted much earlier in dementia than in equivalently aged individuals, contributing to the progressive neuronal degeneration and cognitive dysfunction associated with dementia. We discuss several metabolic treatment approaches, but these depend on characterizing patients as to who would benefit the most. Surrogate biomarkers of metabolism are being developed to include dynamic estimates of neuronal demand, sufficiency of neurovascular coupling, and glymphatic flow to supplement traditional static measurements. These surrogate biomarkers could be used to gauge efficacy of metabolic treatments in slowing down or modifying dementia time course. These physiological experiments are highly novel, assessing the dynamic nature of substrate supply and neuronal demand on a second by second basis with progressively more severe conditions to elucidate metabolic insufficiency and whether neurovascular coupling or glucose uptake  are limiting features of the substrate cellular supply . These dImportantly, the onset and progression of reduced neurovascular coupling and efficacy of metabolic supply in the CVN-AD model and AD may precede or aggravate histological amyloid changes expected at each age. However, as in humans, the Alzheimer’s model progresses on a background of “normal” aging, hence the critical comparison to the WT and genotype control animals with age, which also demonstrate progressive alterations in metabolism, neurovascular coupling and substrate supply in older mice. Many of these approaches are translatable to humans with imaging techniques to estimate metabolism on a real-time basis , 32, 97.These novel results show a significant reduction in neurovascular coupling in response to robust metabolic demand in middle-aged and aged CVN-AD mice compared to age-matched controls, confirming that a significant aspect of metabolic deficiency in AD may arise from insufficient, dynamic substrate delivery to the brain . These rWith aging we have found reduced oxidative capability and enhanced dependence on glycolysis using a brain slice approach , 39. TheThe understanding of metabolic insufficiency in AD will first require defining substrate availability and limitations to cells from the extracellular space, as detailed in There is a critical need to differentiate the stages between normal aging and dementia syndrome, particularly the early onset of mild cognitive impairment (MCI), so that early treatment could applied most effectively to the appropriate candidates. For this reason, a number of biomarkers have been tested, including imaging studies, cerebrospinal fluid samples (CSF), and more recently blood tests that may be easier to perform. Imaging studies, for example, include glucose metabolism (measured by PET 18fluoroglucose uptake into the brain) , 104, stCSF biomarkers have focused on lactate, amyloid, and tau, which filter down through the extracellular space and drain into CSF as a filtering system , 119. TaDirect, dynamic recordings of brain glucose and oxygen under a variety of metabolic activation paradigms may reveal the underlying mechanisms of metabolic deficiency in comparison to the degeneration noted as a function of animal age , 78, 124As these biomarkers are tested the concept of early, pre-clinical AD may be enhanced and confirmed, allowing earlier, pre-symptomatic treatment in which treatment schemes may show improved efficacy, as compared to more severe Alzheimer’s states , 6, 126.Critical deficiencies in metabolism are likely to include reduced neurovascular coupling, which can lead to insufficient metabolite being delivered at the capillary level to the brain, as well as decreased substrate supply . There aEven though reduced glucose metabolism has been consistently noted in clinical studies, treatments to address this issue remain empirical, though some are already in initial (but mixed outcome) clinical trials , 132. OnKey residual questions include the mechanisms underlying the defective neurovascular coupling, and whether even earlier changes occur in hippocampus, which shows a more progressive degeneration process than the cortical surface of the brain. If glucose transport is a critical limitation, this may be upregulated through glucagon-like-peptide (GLP-1) agonists to possibly prevent degeneration if caught earlier . For exaMore detailed understanding of time-related progression of the dementia in relation to neurovascular coupling and substrate supply are critical diagnostic aspects. Using surrogate markers to estimate neurovascular coupling (in addition to baseline metabolism at rest) may help understand another dimension of time related progression from normal aging to mild cognitive impairment . These sThere is a cascade of neurovascular, glucose transport, and cellular metabolic changes early in AD progression, limiting substrate availability and utilization. But, specific mechanisms are needed to correlate the dynamic metabolic cascade with neuropathological worsening, particularly identifying rate-limiting steps. Understanding physiological metabolic demand, extracellular space, and pathological limitations leading to degeneration are optimally explored through physiological substrate demand/supply experiments at early stages of the Alzheimer’s-like progression. The dynamic nature of neurovascular coupling, substrate flow  may also influence whether there is sufficient immediate metabolism for neuronal function. Progression, aging, and gender factors are critical to understand mechanisms of substrate availability and metabolic sufficiency.In summary, the significant changes in neurovascular coupling that we report in the CVN-AD model of dementia show both early age onset compared to control animals and involvement of the neocortex, with regional susceptibility of the hippocampus to be more completely evaluated in later studies. These results may be translated using dynamic measurements of neurovascular coupling to provide additional time and region- based biomarkers in predicting early onset of mild cognitive impairment and degeneration in humans. There are several potential avenues of translational, metabolic therapy which could be applied early, such as enhanced glucose transport and ketone availability. Further, depending on the security of early diagnosis, more aggressive, invasive treatments may be tested, in parallel to another degenerative condition, Parkinson’s disease.


Biomedicines aims to outline nucleic-acid-based strategies that have emerged as tools to regulate specific gene expression and, more recently, as a new class of medicines.This Special Issue of The possibility to down-regulate gene expression  with short complementary (or antisense) chemically synthesized oligonucleotides was first established in a series of publications by the group of P.Zamecnik at the Worcester Foundation in the late 1970s . The eleAntisense oligonucleotides (ASO) down-regulate their mRNA targets either stoichiometrically by steric hindrance or through the activation of the cellular RNAse H, thus leading to a more efficient enzymatic-like mechanism of mRNA degradation. Rapid progress in nucleic acid chemistry gave rise to many ASO analogs with improved pharmacological properties, including increased resistance to nucleases degradation, enhanced binding to the RNA target, improved bioavailability, or reduced toxicity. Mechanisms of action and chemical modifications of ASOs have been comprehensively reviewed by Gagliardi and Ashisawa  in this New applications have emerged that include the regulation of pre-mRNA splicing  or the down-regulation of micro RNAs.ASOs can also be designed to interfere with the expression or activity of proteins specifically. As an example, the expression of the STAT3 transcription factor can be inhibited by short double-stranded DNA fragments named decoy ODNs, as described by Kim et al.  in this However, translating these promising data gathered from cell cultures or animal (mostly murine) models of human diseases remained disappointing for a long time, even leading to skepticism about the clinical interest of nucleic acids-based strategies. The main limitations have included poor delivery to ASO intracellular (cytoplasmic or nuclear) targets across cellular membranes or initially unexpected in vivo toxicity and immunogenicity. Most nucleic acids indeed elicit innate immune responses by binding to Pattern Recognition Receptors (Toll-like receptors or TLR in particular). Detailed studies have shown that both sequence motifs and chemical structures are responsible for these interactions. They can be avoided, as sought in most applications, to decrease off-target effects. Alternatively, ASOs can be designed as TLR agonists for applications in immunotherapy, as described by Agrawal .The first FDA approval of an ASO-based drug (Formivirsen) was granted in 1998 to ISIS Pharmaceuticals  for the treatment of ocular cytomegalovirus retinitis in severely immunocompromised patients (AIDS-infected patients in particular). Of broader application was Mipomersen, approved in 2013 for the treatment of homozygous familial hypercholesterolemia. ASO-mediated regulation of the complex pre-mRNA splicing machinery has also led to several interesting clinical applications with the 2016 FDA approval of ASO-based drugs to treat Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).Further studies aiming at a better understanding of the underlying splice switching mechanisms allowed optimization of the ASO design, which could ultimately be translated clinically—as illustrated here by Flynn et al. —for the Capitalizing on RNA interference pathways with small interfering RNAs (siRNAs) has also led to potent strategies to down-regulate specific gene expressions. Several such drugs have been approved to treat liver-associated human diseases, such as TTR-mediated amyloidosis or acute hepatic porphyrias, both developed by the Boston-based Alnylam company.The registration of several nucleic-acid-based drugs in the US and Europe  is charStrategies to overcome the key issue of endosome entrapment after endocytosis has been described extensively by Juliano  with oriDelivery to specific organs or cell types remains a major hurdle, with major successes achieved in the liver using LNP association or asialoglycoprotein conjugation. Many studies concentrate on extra-hepatic  or topical  delivery. Specific problems dealing with kidney delivery and potential clinical applications were illustrated here by Carton-Garcia et al. . Along tRecent developments have included manipulating the human genome using the CRISPR strategy, which allows the targeted inclusion of DNA sequences, thus revolutionizing gene therapy. This would deserve a complete issue and has therefore not been covered here.Finally, mRNA has become a tool allowing the generation of proteins for protein replacement therapies or new vaccination strategies, as evidenced by the recent successes in SARS-COVID vaccination with approved mRNA drugs from BioNTech/Pfizer  and ModAlthough approved drugs concern eucaryotic cells and viruses, research aiming at targeting bacterial pathogens are in development given the increased number of antibiotic-resistant strains, as extensively reviewed by Jani et al.  or illusThis fast-growing list of approved drugs has demonstrated the great potential of nucleic acids-based medicines that will complement low molecular weight drugs or protein-based drugs in treating various human diseases, including previously undruggable ones. Key issues encountered in their chemical synthesis, design, and delivery have found solutions, and initial successes will probably lead to rapid developments. As pointed out already, however, targeting extra-hepatic tissues and improving endosomal escape remain major roadblocks that are now extensively studied.Lipid nanoparticles (LNPs) have been developed by Alnylam in partnership with Inex Pharmaceuticals, leading to Patisiran, the first FDA-approved siRNA drug. In addition, a slightly different lipid formulation has been engineered by BioNTech  for mRNAAn alternative strategy has involved the chemical conjugation of ligands, as already largely documented for delivering siRNAs and antisense oligonucleotides. For example, GalNac conjugation targets the asialoglycoproteins receptors at the surface of hepatocytes, a strategy that has led to the approval of several drugs treating liver-associated human diseases. The conjugation of low molecular mass ligands, aptamers, or antibodies is being investigated to improve cellular uptake, ease endosomal release, or target specific tissues . It will


The application of modern PCR approaches for the diagnosis of bacterial gastrointestinal pathogens is on the rise due to their rapidly available results combined with high sensitivity. While multiple studies describe the ongoing implementation of this technique for routine diagnostic purposes in laboratories in Western industrialized countries, reports on successful and also sustainable respective approaches in resource-poor tropical settings are still scarce. In order to shed light on potential reasons for this marked discrepancy, this narrative review summarizes identified challenges for the application of diagnostic PCR targeting bacterial gastrointestinal pathogens from stool samples in the tropics. The identified and discussed issues comprise the lack of generally accepted definitions for (1) minimum standards regarding sample acquisition, storage and transport time for diagnostic PCR analyses in the tropics, (2) nucleic acid extraction standards allowing an optimum detection of all types of pathogens which may be responsible for gastroenteritis in the tropics, (3) validation standards to ensure comparable quality of applied diagnostic assays, and (4) cut-offs for a reliable discrimination of infection and mere colonization in areas where semi-immunity due to repeated exposition associated with poor hygiene conditions has to be expected. Further implementation research is needed to solve those issues. Among those articles, only about 2% are focused on the specific situation in the tropics. This obvious neglect reflects the situation of scarce availability of this technology in the laboratories of resource-poor tropical countries; however, it does not necessarily indicate a lacking local need.As of April 2021, academic interest in PCR-based diagnosis of gastroenteritis has been documented by the more than 7000 articles on the NCBI  PubMed database  recommend against the diagnostic use of traditional gel-based or SybrGreen-based PCRs with melting curve analysis, because cross-reaction cannot be reliably excluded based on just two DNA-binding oligonucleotides . AccordiHowever, such technical quality control is still insufficient for diagnostic purposes. In silico validation of the applied oligonucleotides and in vitro validation of the whole assay are also mandatory prior to diagnostic use ,96. UnfoThe test validation scheme as suggested by Rabenau and colleagues for viral pathogens  is interIt might be argued that diagnostic stool culture could be applied as a reference standard. However, diagnostic yield of stool culture is known to be low ,100,101 Without a reliable reference standard, however, indirect methods for diagnostic accuracy estimation have to be considered as summarized elsewhere . Such inWithin the European Union, steps are being taken regarding the standardization of diagnostic test validation by the imminent enforcement of the Regulation (EU) 2017/746 ,107. In Even in case of well-validated test assays with good sensitivity and specificity values of 99% or more, the predictive values depend on pretest probability according to Bayes’ theorem . AccordiFurther, when interpreting diagnostic PCR results, one has to keep in mind that the target structure of each PCR assay is just a defined nucleic acid sequence, not the pathogen as a whole. The pathogen is the meta-structure, for which this sequence acts as a sensitive surrogate parameter . TherebyIf reliable sequence databases can be considered as granted, sequencing of the amplicon is a good option in order to confirm the specificity of a positive PCR result . Due to In case of surveillance or study settings, imperfect test accuracy of PCR assays can be compensated by accuracy-adjusted prevalence estimators ,110 or LApart from the abovementioned issues of uncertain test specificity under circumstances different from the validation setting and low positive predictive value in case of low pretest probability , there iS. enterica, Campylobacter spp., diarrheagenic E. coli [Shigella spp. [When PCR from stool of patients with diarrhea is performed in resource-rich Western industrialized countries like Germany  or Switz E. coli ,13,14,30lla spp.  or enterlla spp. ,30,113 clla spp. . Even inlla spp. ,11,13.Next to asymptomatic colonization ,115,116,Shigella spp., there have been repeated reports indicating higher likeliness of etiological relevance in case of recorded low Ct values [In particular, the discrimination of etiologically relevant infection from harmless colonization is a considerable challenge for PCR applied with stool samples . Some aut values ,120. Howt values ,30,116 at values  but alsoDiscrimination of colonization from infection in case of positive PCR results will most likely remain a challenge in settings where semi-immunity due to repeated pathogen exposition  associatThe review was performed to focus on some yet to be resolved challenges for the reliable application of diagnostic PCR for bacterial agents causing gastroenteritis with focus on the situation in the tropics, where infectious diarrhoea is a particular issue of concern as repeatedly shown for both the local population ,13 and tIf well-equipped laboratory infrastructure is scarce, automated cartridge-based PCR approaches provide an easy-to-implement solution for PCR in resource-poor tropical settings, as successfully proven by the rollout of GenXpert-based tuberculosis screening in sub-Saharan Africa ,131,132.The choice of the most appropriate PCR assays for the tropics remains a challenge, because validation studies specifically performed in the tropics are still scarce. Although even broad confidence intervals regarding the expected test accuracy due to a paucity of validation data can be included in the calculation of test accuracy-adjusted prevalence on a population-based level, such considerations ,110 are At least specificity-associated problems with the interpretation of PCR results can in part be compensated by sequencing if a sufficient amount of amplicon DNA is available. Some authors even argue that unbiased sequencing approaches directly from primary sample materials may provide useful hints in the future ; howeverSchistosoma spp. in peripheral blood [PCR-based diagnosis of causative agents of bacterial gastroenteritis in other compartments such as, e.g., in blood in case of bacteremic shedding, remains challenging due to low pathogen concentration in the bloodstream. As detailed above, assessment by PCR with or without subsequent sequencing is possible after blood culture incubation as well ,139, butal blood , are usuThe broad variety of potential causative agents of infectious gastroenteritis remains another challenge. This includes emerging new pathogens, as recently demonstrated by the causal association of SARS-CoV-2 detection in a stool sample of a child with diarrhoea in Iran .In a more abstract epistemiological sense, medical diagnosis is always a variant of the art of coming close to an unknown truth . As descIn summary, PCR for the diagnosis of gastrointestinal pathogens is a widely applied approach, whose potential for a diagnostic routine application under tropical conditions requires additional evaluation. Further investigations are particularly needed to standardize the pre-analytic, analytic and post-analytic steps with special emphasis on the climatic, infrastructural, epidemiological and socioeconomic situation in the tropics. With adequate academic support by the disciplines of implementation science, however, it will most likely be possible to define optimum conditions for beneficial use of this technique under tropical conditions as well.


Salinity stress is a major environmental impediment affecting the growth and production of crops. Finger millet is an important cereal grown in many arid and semi-arid areas of the world characterized by erratic rainfall and scarcity of good-quality water. Finger millet salinity stress is caused by the accumulation of soluble salts due to irrigation without a proper drainage system, coupled with the underlying rocks having a high salt content, which leads to the salinization of arable land. This problem is projected to be exacerbated by climate change. The use of new and efficient strategies that provide stable salinity tolerance across a wide range of environments can guarantee sustainable production of finger millet in the future. In this review, we analyze the strategies that have been used for salinity stress management in finger millet production and discuss potential future directions toward the development of salt-tolerant finger millet varieties. This review also describes how advanced biotechnological tools are being used to develop salt-tolerant plants. The biotechnological techniques discussed in this review are simple to implement, have design flexibility, low cost, and highly efficient. This information provides insights into enhancing finger millet salinity tolerance and improving production. Eleusine coracana (L.) Gaertn.] is a valuable cereal crop that is a staple food crop by a large segment of population living in marginal areas of sub-Saharan Africa and Asia  plays an adaptive role in protecting sub-cellular structures and mediating osmotic adjustment during salt stress conditions . Plants LEA2 and LEA3 (late embryogenesis abundant proteins) as well as better recovery development. Increased levels of LEA2 and LEA3 genes were discovered during the study of stress-sensitive genes. In another study, EcNAC67 transcription factor increased rice salinity tolerance. Recently, a novel finger millet endoplasmic reticulum-specific bZIP TF gene (EcbZIP17) was isolated and overexpressed in tobacco  encoding gene. PcSrp expression was discovered in the salt-stressed roots and rhizome of P. coarctata. To determine its function, the PcSrp gene was cloned downstream of the rice actin-1 promoter and introduced into finger millet using the particle-inflow-gun method. Under 250-mM NaCl stress, transgenic plants expressing PcSrp were able to mature and set seed. The untransformed control plants, contrast, did not withstand similar salt stress. Transgenic plants’ stressed roots invariably accumulated higher Na+ and K+ ion contents than untransformed plants “roots, while transgenic plants” shoots accumulated lower amounts of both ions. mtlD (mannitol-1-phosphate dehydrogenase) gene in finger millet in another study. Salinity tolerance testing revealed that transgenic plants expressing the mtlD gene grew faster than wild-type plants under salinity stress. Similarly, SbVPPase gene isolated from Sorghum bicolor and studied the biochemical and physiological parameters of control and transgenic plants. In control plants, the relative water content, plant height, leaf expansion, plant length, width, and grain weight were all severely reduced, and flowering was delayed by 20%. By contrast, transgenic plants had higher proline, chlorophyll content, enzyme activity, and lower malondialdehyde levels (MDA). These three examples demonstrate how genetic engineering can substantially aid salinity stress management in finger millet. As effective regeneration protocols in finger millet have been developed , osmotic stress, and oxidative damage, which in turn impedes the growth and development of the plants  isolated from saline regions on salinity sensitive finger millet seeds. In their study, finger millet seeds were treated with isolates and exposed to various concentrations (0–350 mM NaCl) of salt stress. Isolates were screened for growth-promoting characteristic qualities and growth parameters under greenhouse conditions. Under increased salt conditions, strain SPF-33 showed increased enzymatic antioxidant activity and increased proline content, lower lipid peroxidation and hydrogen peroxide, and increased plant height and spikelet number. Under 350-mM NaCl, treatment with SPF-37 increased germination, vigor index, plant height and the number of spikelets, total chlorophyll, phenolics, flavonoids, proteins, and relative water content of the leaf, considerably more than the control. These results demonstrated that fluorescent Pseudomonas strains SPF-33 and SPF-37 are potential PGPR for improving finger millet salinity tolerance. Currently, there is limited information on an array of rhizobacteria that can be used to mitigate salinity stress in finger millet as well as information on complex plant–microbial interactions in various agro-ecosystems. This knowledge is needed in order to implement this management strategy in finger millet. More work needs to be done to verify the current outcomes of PGPR usage and to improve our knowledge and understanding about the value of microbial agents. Furthermore, PGPR should be used in conjunction with other techniques to protect finger millet from salinity stress over time.For decades, soil microorganisms have been used in crop production . They arMoringa oleifera leaf extract , compounds of biological origin that are applied to embellish nutrient uptake, stimulate growth and enhance stress tolerance or quality traits of crops , through extract , and Ascextracts  have beeBrassica spp. and discovered a more efficient salt overly sensitive pathway made up of SOS1, SOS2, SOS3, and the vacuolar Na+/H+ antiporter. The conserved structure of these genes, as well as their intra and intergenic relatedness, was revealed by sequence analyses of partial cDNAs. Based on their findings, they concluded that the existence of an efficient SOS pathway, which results in a higher K/Na ratio, could be a major factor, among others, in deciding the salinity stress tolerance of Brassica juncea genotypes CS 52 and CS 54 as compared to Varuna and T 9. In another study, OsHsp17.0 and OsHsp23.7 to test stress tolerance. When exposed to mannitol and NaCl, both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines showed higher germination potential than wild-type plants. Transgenic rice lines showed higher resistance to drought and salt stress than wild-type plants, according to a phenotypic study. Furthermore, under drought and salt stress conditions, transgenic rice plants had lower MDA and higher free-proline levels than wild-type rice lines. These findings showed that OsHsp17.0 and OsHsp23.7 are important in rice salt adaptation to salinity and dehydration stresses, and that they can be used to engineer rice that is drought and salt-tolerant.The advancement of plant genomics has played a key role in the release of vital genomic data for trait improvement in crops. Using a genomics approach, various genes that are involved in salinity stress have been identified and characterized in plant systems. For example, Agrobacterium-mediated transformation has been achieved, it becomes a challenge with an increase in the number of transgenes and the size of the transfer-DNA (T-DNA), and has the potential of gene silencing if the same promoter is repetitively used. A biolistic transformation system can simultaneously introduce many transgenes encoding for multiple strains. However, this system comes with the limitations of a complex genome integration of transgenes and an unstable linkage inheritance between transgenic generations -CRISPR-associated protein (Cas) system has emerged as an effective robust tool for site-specific genome editing and precise induction of mutagenesis in many organisms including plants. Given its precision, efficiency, simplicity, and high cost-effectiveness, the CRISPR/Cas system has been extensively applied to the edit genomes of several crops including those closely related to finger millet such as foxtail millet , sorghumThe fundamental principle of CRISPR/Cas and other genome editing tools, such as meganucleases , zinc-fiin vitro regeneration and transformation protocols that are cultivar-independent. Although attempts have been made for tissue culture regeneration and transformation of finger millet using different explants and different delivery methods, their efficiency is cultivar-dependent with low regeneration and transformation efficiency (in vitro regeneration through another culture, protoplast culture, and protoplast fusion in finger millet.Theoretically, the CRISPR/Cas system can be used to manipulate all genomes with high accuracy. However, the gRNA can find complementary positions within the genome and cause off-targets, although they are rare in plants . To avoificiency . CurrentAgriculture and applications of bioengineering techniques must be used to help feed the burgeoning global population in a sustainable manner under climate change perturbations . The mosin vitro propagation, genetic manipulation, and screening of crops. While there has been a boost to plant biotechnology following the development of novel and revolutionary techniques such as CRISPR/Cas-mediated genome editing and speed breeding, the whole sequencing and annotation of orphaned crops like finger millet and the application and growth of synthetic biology as a field remain a challenge. Nevertheless, the adoption of synthetic biology tools will be vital in remodeling future progress in agricultural biotechnology especially when dealing with multigenic stresses such as salinity.Over the years, plants have evolved elaborate and complex mechanisms for avoiding or tolerating the effects of salinity stress. Avoidance mechanisms prevent exposure and include the capability to reduce sodium ions accumulation in the cell through water-loss minimization and optimization of water uptake. Salinity tolerance allows plants to endure elevated cellular sodium ion concentrations by preserving cell turgor and raising protoplasmic resistance . With anThe technology advancement in the last three decades has exposed contemporary plant breeders to a plethora of innovative tools, such as genomic selection , high-th– ions move the Na+ ions from soil colloids which leached by rains or irrigation (+ ions inhibit the membranes’ sodium channel and decrease cytosolic salt accumulation rate, thereby allaying plant salinity stress. Moreover, if the calcium used is in the form of nitrates, the Cl– ions compete with Na+ ions for accumulation in the cytosol vacuoles. The application of nitrates to sodic soils may reduce salt uptake, although this aspect requires validation.Agronomic methods to reduce salinity stress during cultivation can be directed on either soil or crops. The simplest approach to lower soil sanity is to increase the soil water content, which can be achieved through frequent irrigation especially the modern irrigation systems with high efficiency. Increasing of the soil water content reduces the salt concentration to level which crops can withstand. However, this approach is not feasible for smallholder finger millet farmers. Further, finger millet is grown in arid and semi-arid regions of the world. Dry periods of finger millet cultivation are the most critical period for salinity stress. Survival of crops during such periods can be increased by the application of calcium nitrate or chloride because Clrigation . SimilarMany studies have emphasized the necessity for crop production to double by 2050 to meet the anticipated demands of a burgeoning global population. At present, soil salinity is the most serious threats among abiotic stresses caused by climate change and agronomic practices that impede crop production. Although meaningful gains have been made in breeding salt-tolerant crops, more work is needed. Salinity tolerance is a polygenic trait, and this is further complicated in several types of abiotic stress, such as drought, high temperatures, and nutrient deficiencies and toxicities, that may simultaneously impact the crop, making breeding for salt-tolerant cultivars a challenge. From this review, it can be summarized that finger millet production is adversely affected by salt stress, which alters its physiological, biochemical, and molecular mechanisms. The genes, metabolites, and pathways responsible for the diverse mechanisms of salinity tolerance in finger millet must be profiled, taking advantage of the discovery of the whole genome sequence of finger millet. Based on the advancement of biotechnological tools at present, multidisciplinary approaches are encouraged for the development of salt-tolerant finger millet cultivars. Several new techniques such as genomic selection, high-throughput phenotyping, genome editing using the CRISPR/Cas, speed breeding, and synthetic biology have recently attracted attention among scientists and plant breeders globally. All these strategies promise to revolutionize comprehensive trait prediction and integration of various salinity management options, including agronomic approaches, conventional breeding, and modern biotechnological advances, for the sustainable improvement of finger millet yield and nutritional quality under salt stress.WM conceptualized and critically edited the manuscript for publication. WM and AM undertook the literature review and analysis and wrote the manuscript. Both authors contributed to the article and approved the submitted version.Views expressed herein do not necessarily reflect the official opinion of the funding organization.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.