Emotional disturbances, such as depression, anxiety, and phobias, are prevalent in patients with neuropsychiatric disorders, including somatization disorder (SD), major depressive disorder (MDD), general anxiety disorder (GAD), panic disorder (PD), schizophrenia, blepharospasm, and cervical dystonia –5. HowevWu F. et al. explored functional and structural connectivity abnormalities within the amygdala-prefrontal circuit in first-episode medication-naive adolescents with MDD through resting-state functional magnetic resonance imaging (rs-fMRI) and diffusion tensor imaging (DTI). Rs-fMRI and DTI imaging data were acquired from 36 patients and 37 age-and sex-matched healthy controls (HCs). The patients showed decreased connectivity between the left amygdala and ventral prefrontal cortex (PFC) and lower fractional anisotropy (FA) in the left uncinate fasciculus. These findings suggest that both functional and structural abnormalities of the amygdala-prefrontal circuit may be involved in the neuropathophysiology of adolescent MDD.Zhang et al. investigated how emotional context modulates the temporal dynamics of reward anticipation and feedback in adolescents. Electroencephalography (EEG) data from 35 patients with MDD and 37 healthy adolescents were recorded when performing a gambling task after being presented with emotional pictures. The study suggests that adolescents with MDD exhibited dissociable deficits in reward anticipation and gain or loss feedback that were distinctly modulated by emotional contexts, which might deepen our understanding of the modulation of emotional contexts on the dynamic temporal reorganization of the reward circuit in adolescents with MDD.Tao et al. investigated the subgenual anterior cingulate cortex (sACC) in patients with depression. Indeed, abnormal activity of the sACC is implicated as a potentially effective target for therapeutic modulation in treatment-resistant depression (TRD). The authors hypothesized that PFC areas with direct fiber connections to the sACC might be sites for effective treatment using transcranial magnetic stimulation (TMS). Two neuroimaging data sets were used to construct anatomic and functional connectivity maps using sACC as the seed region. One data set included magnetic resonance images from 20 HCs, and the other included MR images from 15 TRD patients and 15 additional HCs. Both left and right prefrontal cortex (PFC) is functionally connected to two regions relevant to depression, the sACC and the posterior cingulate cortex (PCC). These bilateral PFC sites may be targets for effective TMS treatment in TRD.Chen et al. examined the changes in characteristics of affective network connectivity in patients with MDD before and after repetitive TMS treatment over the left dorsolateral PFC to assess how these connectivity changes are linked to the patient's clinical characteristics. rTMS can enhance affective network connectivity in patients with MDD, which is linked to emotional improvement. This study further suggests that the insula might be a potential target region for evaluating clinical efficacy for MDD in designing rational strategies for therapeutic trials.Liu et al. examined whether the CACNA1C gene rs11832738 polymorphism and MDD had a potential interaction on untreated regional ALFF, and further determined whether the regional ALFF mediated the genetic association with MDD. With 116 patients and 66 HCs, they provided initial evidence for CACNA1C genotype-related alterations in brain function among patients with MDD, which could help better understand the neurobiological mechanisms underlying MDD. The study confirmed that the association of calcium channel dysfunction with MDD might be altered in functional brain activity.Li et al. aimed to identify the voxel-based whole-brain functional connectivity changes in patients with late-life depression (LLD). With 50 patients and 33 HCs, the study indicated that the intrinsic abnormality of network centrality exists in a wide range of brain areas in patients with LLD. Late-onset depression patients differ from early-onset depression in cortical network centrality.Krause-Sorio et al. conducted a pilot study of 22 older adults with depression who randomly received escitalopram/memantine or escitalopram/placebo treatment and tested with diffusion-weighted imaging (DWI) to investigate brain white matter integrity in the fronto-limbic-striatal tracts based on fractional anisotropy and treatment response. In bilateral anterior and posterior internal capsule tracts and bilateral inferior and right superior fronto-occipital fasciculus, higher fractional anisotropy was associated with more considerable improvements in depressive symptoms for escitalopram/memantine but not escitalopram/placebo. While the findings seemed promising, it has to be noted that the study was based on a small sample with some significant losses to follow-up. Therefore, further studies with adequate statistical power are needed in the future.Wu Y. et al. aimed to examine altruism in patients with LLD and its neurobiological mechanism. Depressive patients seemed to show less altruistic behavior, while depression and age are two primary influencing factors of altruism. Kynurenine and its metabolites can cross the brain-blood barriers, impact the central nervous system, and play a significant role in psychiatric disorders, including depression. They investigated whether metabolites in the kynurenine pathway and white matter network topological features would influence altruistic behavior in patients with LLD. With 34 patients and 36 HCs, the study showed that patients exhibited a higher level of altruism and white matter global network properties than the HC. Kynurenic acid to kynurenine ratio was associated with the Dictator Game paradigm performance and network density in the patients. Kynurenine metabolism might play an important role in altruistic behavior in LLD.Lu et al. performed a study of 27 HCs, and 36 patients with bipolar depression (BPD) treated with quetiapine. At baseline, the altered composition of gut microbiota and low B/E ratio was founded in patients with BPD, and Enterobacter spp count was significantly correlated to CD3+ T cells. Antipsychotic treatment significantly improved depressive symptoms and the balance of B/E. This finding suggests that the treatment effect on depressive symptoms might be partly through the improved gut microbiota balance.Ren et al. investigated possible age-associated alterations of white matter integrity in adolescents and young adults with BPD aged 13–30. The findings provide neuroimaging evidence supporting a back-to-front spatiotemporal directionality of the altered development of white matter integrity associated with age in patients with BPD during adolescence/young adulthood.Takahashi et al. explored abnormal melatonin secretion in patients with MDD and BPD. They employed MRI to examine pineal gland volumes and pineal cyst prevalence in 56 patients with MDD (29 currently depressed and 27 remitted patients), 26 patients with BPD, and matched HCs (33 for MDD and 24 for BD). The pineal gland was significantly smaller in the current MDD of non-melancholic depression than in the melancholic MDD. Pineal volumes negatively correlated to the severity of loss of interest in the current MDD. The medication used and the number of affective episodes were not associated with pineal volumes in the MDD or BPD. While the findings do not suggest that pineal volumes reflect abnormal melatonin secretion in affective disorders, they point to the possibility that pineal abnormalities are associated with clinical subtypes of MDD and its symptomatology.Luo et al. performed a study of dynamic functional connectivity strength (dFCS) at a different frequency with rs-fMRI data from 96 patients with schizophrenia (SZ) and 121 HCs. They found that relative to HCs, patients with SZ tend to have decreased dFCS in the salience, auditory, sensorimotor, and visual networks but increased dFCS in the cerebellum, basal ganglia, and prefrontal network consistently at low-frequency bands, which significantly interacted with disease status. However, no significant difference in dFCS was found in higher frequency. The study may provide potential implications for examining the neuropathological mechanism of SZ.Duan and Zhu aimed to examine SZ research utilizing MRI through a bibliometric analysis of literature searched in PubMed from 2004 to 2018, divided into three 5-year periods. It showed that the utilization of MRI in SZ research was relatively diverse, but the theme clusters derived from each period reflect the evolvement from (1) the brain structure and its link to functional abnormality, metabolism, and antipsychotic efficacy and pathology; (2) the physiopathology mechanism and etiology of cognitive disorders including brain structure and function, default network, and psychology; and to (3) the neurobiology between SZ and other mental disorders, including the genetic and antipsychotic effect on brain structure and function. These findings provide useful information for developing future SZ research using MRI techniques.Yang et al. examined regional Cerebral Blood Flow (rCBF) alterations and their cognitive performance in unmedicated heroin-dependent individuals (HDIs). Voxel-wise whole-brain analysis of rCBF measured arterial spin labeling (ASL) perfusion MRI showed that relative to HCs. HDIs tend to have decreased rCBF in the bilateral cortical and subcortical inferior temporal gyrus, medial frontal gyrus (MFG), orbital medial frontal cortex, precuneus, posterior cerebellar lobe/declive, and right thalamus and posterior cerebella cortex, all of which were significant between HDIs and HCs in the ROIs analysis. The rCBF at MFG was significantly associated with cognitive performance measured by Trail Making Test. These findings suggest the MFG as a critical region in HDIs and suggest ASL-derived CBF as a potential marker for use in heroin addiction studies.Sariah et al. performed an interesting study of whether chewing betel quid had acute and longer-term addictive effects in naive and dependent users. The rs-fMRI was performed in 24 male betel quid-dependent chewers and 28 male HCs before and promptly after betel quid chewing. They found that individuals who chronically used betel quid have higher functional connectivity in the frontal, parietal, and temporal brain regions relative to HCs. However, the acute effect was observed in naive Betel-quid chewers with increased functional connectivity in some visual cortical areas of the superior and right middle occipital gyrus and subcortical caudate, putamen, pallidum, and thalamus.Seok and Cheong aimed to identify gray matter deficits and functional alterations using voxel-based morphometry and fMRI analyses of 15 men with intermittent explosive disorder (IED) and 15 age sex-matched HCs. Gray matter volume and brain activation while viewing the anger-inducing films were measured using 7T MRI. Men with IED had significantly reduced gray matter volume in the insula, amygdala, and orbitofrontal area relative to HC; gray matter volume in the left insula was negatively correlated with composite aggression scores. fMRI showed that men with IED showed greater activation in the insula, putamen, anterior cingulate cortex, and amygdala during anger processing. Left insula activity was positively correlated with composite aggression scores. These findings collectively suggest that structural and functional alterations in the left insula are linked to IED, thereby providing insight into IED's neural mechanisms.Chen et al. and Krause-Sorio et al. examined neuroimaging in response to antidepressant treatment, which may provide a proof of concept that neuroimaging can predict pharmacological treatment response.Emotional disturbance is mostly related to mood and anxiety disorders that are highly prevalent neuropsychiatric disorders and common in older adults of general populations . Neuroimin-vivo in physiological and pathological conditions due to both its spatial resolution compared to other methods, such as MEG and EEG, as well as the possibility of using it with poorly compliant patients due to their clinical condition. This allows us to highlight abnormalities at multiple levels across neural systems. Rs-fMRI studies have underlined that neuropsychiatric diseases show altered interactions between different RSNs, and disease progression and treatments may modify these interactions.The advances in neuroimaging techniques and biotechnology have provided an unprecedented opportunity to study precision neuropsychiatric disorders in past years. Advanced neuroimaging techniques have helped to investigate brain structural and functional changes in neuropsychiatric diseases through MRI , ; fMRI has in particular provided information about neural connectivity at rest (rs-fMRI) and during a task. Rs-fMRI represents a promising method for investigating brain Even if neuroimaging techniques  represent valid tools to investigate neuropsychiatric diseases, results are often inconsistent, showing significant variability, which may present problems for identifying specific biomarkers of pathology. The lack of confirmed biomarkers might be due to different variables. These may include the heterogenicity of the neuropsychiatric disease, the diversities of data analysis technique used, and the limits intrinsic to rs-fMRI, such as the scarce possibility of monitoring patients' real mental content during the acquisition phase. Future studies should include standardized protocols for enrollment of sample size, reducing the heterogeneity observed. Integration of temporal and spatial imaging as well as clinical and molecular data may improve the identification of brain circuits related to neuropsychiatric disorders and treatment response.Moreover, a combination of multimodal imaging tools, such as PET, structural MRI, and fMRI, can help understand brain circuits' structure and function. A PET scan can reveal metabolic and functional problems at a cellular level that MRI and fMRI cannot provide, which may be critical for some neuropsychiatric diseases. These precautions could provide more power in identifying disorder-specific neural alteration and help develop specific therapeutic interventions in future precision psychiatry.All authors listed have made substantial and intellectual contributions to the topic and approved this editorial for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mitochondrial dysfunction plays an important role in the pathogenesis and progression of diabetic kidney disease (DKD). In this review, we will discuss mitochondrial dysfunction observed in preclinical models of DKD as well as in clinical DKD with a focus on oxidative phosphorylation (OXPHOS), mitochondrial reactive oxygen species (mtROS), biogenesis, fission and fusion, mitophagy and urinary mitochondrial biomarkers. Both glucose- and non-glucose-induced mitochondrial dysfunction will be discussed. In terms of glucose-induced mitochondrial dysfunction, the energetic shift from OXPHOS to aerobic glycolysis, called the Warburg effect, occurs and the resulting toxic intermediates of glucose metabolism contribute to DKD-induced injury. In terms of non-glucose-induced mitochondrial dysfunction, we will review the roles of lipotoxicity, hypoxia and vasoactive pathways, including endothelin-1 (Edn1)/Edn1 receptor type A signaling pathways. Although the relative contribution of each of these pathways to DKD remains unclear, the goal of this review is to highlight the complexity of mitochondrial dysfunction in DKD and to discuss how markers of mitochondrial dysfunction could help us stratify patients at risk for DKD. The number of patients with diabetes mellitus is expanding, and diabetic kidney disease (DKD) is an important cause of diabetic microvascular complications that constitutes an independent risk factor of mortality and cardiovascular events .+/K+-ATPase is essential for solute reabsorption. Therefore, mitochondrial dysfunction is postulated to play a central role in the pathogenesis and progression of kidney diseases including DKD  binding to cause apoptosis in proximal tubular cells [Tubular cells in patients with DKD are likely exposed to fatty acid-bound albumin, since dyslipidemia and proteinuria often accompany diabetes mellitus. Whereas albumin itself can cause tubular cell damage on its reabsorption, FA-bound albumin was shown to induce more severe tubular damage ,60. Cytoar cells .Genetic and pharmacological enhancement of FAO could represent a new therapeutic treatment strategy for patients with DKD. Transgenic expression of PCG1α and fenofibrate treatment ameliorated tubular cell apoptosis via restoration of CPTs and/or acyl-CoA oxidases ,67.Tubulointerstitial hypoxia is known to be a final common pathway of CKD progression . Regardl+/Ca2+ exchanger in the inner mitochondrial membrane. Na+ imported into the matrix then interacts with phospholipids to reduce membrane fluidity, resulting in the inability of free ubiquinone to move between Complex II and Complex III. Thus, Complex III produces ROS.Recently, the mechanism by which acute hypoxia causes mtROS production in the ETC has been revealed . Acute hHypoxia had long been deemed a direct suppressor of OXPHOS because oxygen is indispensable as a receiver of electrons in the ETC. However, to decelerate OXPHOS acutely and directly, the oxygen concentration has to be as low as 0.3% . In mildEndothelin-1 (Edn1) was first characterized as a signaling molecule released by podocytes in TGF-β-induced FSGS . SurprisThe similar role of mtROS and EDNRA in endothelial cells and Edn1 is also described in DKD . CirculaAnother vasoactive pathway, the renin–angiotensin–aldosterone system (RAAS), is also known to participate in CKD progression, including in DKD. Aside from the detrimental effects of increasing systemic and intraglomerular blood pressure, angiotensin II treatment was found to exacerbate mtROS production and mitochondrial fragmentation in podocytes both in vivo and in vitro, which can be reversed by mitoquinone, a mitochondria-targeted antioxidant .Mitochondrial dysfunction plays a central role in the development and progression of DKD. Thus, targeting mitochondrial dysfunction in DKD could represent a novel therapeutic strategy for patients with DKD. However, as can be learned from studies investigating the beneficial effect of systemic antioxidant administration as a treatment for DKD, it seems that intervention in mitochondrial dysfunction has to be cell type- and context-specific. Another intriguing perspective is age- and sex-related difference in mitochondrial dysfunction in DKD. Although age is an important factor that affects mitochondrial function and the development and progression of DKD, not much is known about the exact mechanisms. In terms of sex, much difference is observed between males and females in the mitochondria of certain tissues, and estrogen and possibly testosterone can mediate renal mitochondrial bioenergetics . Further


Vitamin C (ascorbic acid) is a normal liver metabolite in most animals, with humans being a notable exception due to random genetic mutations that have occurred during our evolution. As such, it has become a vitamin , with requirements increasing significantly during various illnesses, particularly severe infections. Recent international clinical trials are highlighting the potential for intravenous vitamin C administration to improve clinical outcomes for patients, particularly those with severe respiratory illness and sepsis and some cancers. Furthermore, there has been an upsurge in new discoveries and new mechanistic insights, particularly around epigenetic regulation by vitamin C, that are providing rationales for future targeted clinical trials.To facilitate the translation of leading-edge research into clinical practice, we held the second “Vitamin C Symposium” in 2019 with the theme of “Vitamin C for Cancer and Infection: From Bench to Bedside” [In this Special Issue, we have collected 15 original research papers and comprehensive review articles spanning laboratory-based research, observational studies and intervention trials. These cover the following key research themes: pharmacokinetics, immune function, epidemiology, cancer research, surgical outcomes, and infection research, specifically pneumonia, sepsis, and COVID-19.Pharmacokinetics: Lykkesfeldt and colleague wrote a comprehensive review highlighting the complex pharmacokinetics of oral and intravenous vitamin C , an issuImmune function: Carr and colleagues investigated the role of vitamin C in neutrophil function, including a systematic review of randomized controlled trials carried out in different population groups and patient cohorts . The neuEpidemiology: Dr Myint and colleagues interrogated the large European Prospective Investigation into Cancer-Norfolk (EPIC-Norfolk) database to determine demographic and lifestyle risk factors for vitamin C deficiency and associations with functional health . They foCancer research: Dr Wang and colleagues have identified key molecular targets of vitamin C activity in triple-negative breast cancer metastasis in cell culture and a xenograft model , indicatSurgical outcomes: A systematic review on the effects of vitamin C administration on outcomes of Percutaneous Coronary Intervention (PCI) has indicated that intravenous infusion of vitamin C before PCI may serve as an effective method for cardioprotection against reperfusion injury . A meta-Pneumonia and sepsis: Observational research by Carr and colleagues has indicated that hospitalized patients with community-acquired pneumonia exhibit both depleted vitamin C status and elevated markers of oxidative stress . PneumonSepsis and COVID-19: Dr Fowler and colleagues wrote a comprehensive review on the emerging role of vitamin C as a treatment for sepsis , which cCollectively, this Special Issue displays the diverse areas of health and disease in which vitamin C plays important roles and the continuing efforts of researchers to unravel the underlying mechanisms of its biological functions. Although its role in scurvy has been recognized for close to a century, it appears that we still have much to learn about this small carbohydrate molecule. Well-designed observational and interventional studies that consider the baseline status and unique pharmacokinetics of vitamin C are needed moving forward to help address the current gaps in our knowledge. Underpinning mechanistic research will also further our ability to inform good clinical practice. We look forward to future discoveries of the roles of vitamin C in human health and disease.


Wound healing is a complex tissue regeneration process involving many changes in multiple physiological parameters. The pH and temperature of a wound site have long been recognized as important biomarkers for assessing wound healing status. For effective wound management, wound dressings integrated with wearable sensors and systems used for continuous monitoring of pH and temperature have received much attention in recent years. Herein, recent advances in the development of wearable pH and temperature sensors and systems based on different sensing mechanisms for wound status monitoring and treatment are comprehensively summarized. Challenges in the areas of sensing performance, infection identification threshold, large-area 3-dimensional detection, and long-term reliable monitoring in current wearable sensors/systems and emerging solutions are emphasized, providing critical insights into the development of wearable sensors and systems for wound healing monitoring and management. Our skin is important in protecting internal organs from external environmental hazards . HoweverThe wound healing process is complex and multi-stage, and different stages are often accompanied by different changes in the physiological environment of the wound, such as the increase of the local temperature, pH alkalization, volatile organic compounds (VOCs) and the abnormal release amount of metabolite, etc. ,11,12,13Wearable sensors and systems refer to the devices that can deal with trace biological or chemical analytes and convert the chemical reactions or physical changes into usable signals  according to certain rules ,20,21. WThe pH within wound milieu is a significant biochemical marker, which has been proved to be an important contributing factor throughout the wound healing process ,27. In gOptical pH sensors/systems contain indicator dyes, which show color changes depending on the pH, can be used to monitor pH variation . These sIn addition to microfluidic spinning, electrospinning technique was also developed to produce pH-responsive fibers with improved sensitivity for wound monitoring ,54. ReceFor more precise detection, electrochemical approaches that have innate selectivity and sensitivity towards target analytes were applied to transduce wound biomarkers concentration into potential, current or/and impedance. In terms of the flexible electrochemical sensors used for healthcare applications, organic and polymer materials have been widely employed as sensing element for pH detection ,56. PolyPooria et al., have developed a conductive thread-based pH sensor with a microfluidic splitter which was used to deliver the sample to the sensing chambers a 58]. I. I58]. IComparison of sensitivity/accuracy of wearable pH sensors based on different sensing mechanisms are summarized in Temperature variation has been recognized as an important parameter associated with wound inflammation and infection, since a series of chemical and enzymatic actions involved in the wound healing process are affected by abnormal temperature changes ,65,66,67Nowadays, infrared sensors-based portable devices that provide objective thermal images for clinical tissue structural monitoring are becoming increasingly important in wound assessment ,76,77. FIn order to make up for the above defects in traditional infrared sensors, wearable sensors with the ability of temperature visualization offers a promising approach with in-situ monitoring of temperature change and real-time diagnosis of the health status by seamless contact with human skin . ThermocWith the development of wearable electronics, multifunctional wearable devices are widely used in healthcare and intelligent monitoring . To cont2+ (Gr/SF/Ca2+) that could be transferred onto the skin [S/V) ratio of PEDOT:PSS. The fastest response (<3.5 s) was achieved by using nanowires with a maximum S/V ratio. By doping PEDOT: PSS nanowires with graphene oxide (GO), its thermosensitivity can be maximized at a specific doping ratio. For the monitoring system based on wearable flexible temperature sensors to monitor temperature and other vital signals, secure attachment and strong biocompatibility are essential in obtaining accurate physiological and pathological information. Yamamoto et al., proposed a gel-free adhesive conductive electrode for electrocardiogram (ECG) recording and studied the influence of film thickness of the substrate on the temperature detection of the skin [Wang et al., have described a temperature-responsive self-healable electronic tattoos (E-tattoos) based on a graphene/silk fibroin/Gathe skin . The fabthe skin ,92,93,94the skin . They fothe skin . The resthe skin . This tyBy adding additional flexible wearable sensors or/and actuators to measure other biochemical health markers in/out the body and deliver the drug with precise control, the potential applications of the wearable sensors can now be widely developed and realized for human healthcare. In addition to monitoring human skin temperature, research shows new functions of wearable smart wound dressing. Kim et al., described a system containing two ultrathin and bendable temperature sensors and microscale heating elements used for medical sutures . One sutBesides the resistive temperature sensors, the wearable temperature sensors based on other mechanisms  are also being widely studied. Recently, Lu et al., developed an LC (inductance-capacitance) oscillator that can measure regional body temperature through changes in capacitance . The capThe development and implementation of wearable sensors and systems used for wound healing-related pH and temperature detection have been growing rapidly, and have begun to revolutionize the approach to wound diagnosis and treatment. Smart wound dressings integrated with wearable sensors to monitor the pH and temperature at the wound site in a noninvasive and real-time manner are promising in facilitating efficiencies in clinical resource use and management of distant patients. In addition, progresses in material science and manufacturing capacity have led to an endless emergence of new wound medical devices. Along with these advances, new types of wound dressing based on flexible, self-healable, absorbable materials and integrated with on-demand triggerable drug delivery systems will provide better and more efficient wound management and treatment.Despite significant advances in recent years, the field of smart wound dressing integrated with wearable sensors and systems, which is really helpful for wound biomarkers  monitoring and patient management, still needs to solve many bottlenecks to meet the requirements of modern healthcare. Due to subtle changes in wound pH and temperature when early infection occurs, the detection sensitivity of wearable sensors and the accuracy of the infection identification threshold require further improvement. Therefore, the next generation of medical wound dressings should focus on the improvement of sensitivity by applying novel materials or/and sensor structures, and the improvement of accuracy of wound infection judgment by integrating more biosensors to monitor multiple wound-related biomarkers apart from pH and temperature, providing mechanistic insights about the wound healing process and reliable basis for on-demand drug delivery. Moreover, the wound is a 3D structure, so 3D wound information is critical in the precision diagnosis of early infections. Thus, it is necessary to pay more attention to the development of large scale of pH or/and temperature sensors, which are fabricated through novel techniques and new protocols. Finally, wound rehabilitation is a long-term process in chronic wounds, which can always lead to the possibility of infection in the wound site. However, the wearable sensors mentioned above were tested in vitro or on human skin for only very short periods of time. Therefore, the long-term stability and reliability of the wearable sensors for wound monitoring are other important factors in ensuring long-term accurate detection. To overcome this obstacle, wearable sensors and systems with self-healing and self-adjustment properties need to be developed.Overall, wearable sensors and systems have shown great potential in monitoring wound healing status through wound-related pH and/or temperature detection, which can effectively diagnose early infections and give timely on-demand therapeutics. Emerging solutions for each of the above limitations are also creating new opportunities for the advancement of wound dressings, and with the joint efforts made by researchers and doctors worldwide, the next generation of wound dressings based on wearable sensors and systems will provide more accuracy and reliable wound status monitoring and treatment.


As an evolutionarily conserved cellular process, autophagy plays an essential role in the cellular metabolism of eukaryotes as well as in viral infection and pathogenesis. Under physiological conditions, autophagy is able to meet cellular energy needs and maintain cellular homeostasis through degrading long-lived cellular proteins and recycling damaged organelles. Upon viral infection, host autophagy could degrade invading viruses and initial innate immune response and facilitate viral antigen presentation, all of which contribute to preventing viral infection and pathogenesis. However, viruses have evolved a variety of strategies during a long evolutionary process, by which they can hijack and subvert host autophagy for their own benefits. In this review, we highlight the function of host autophagy in the key regulatory steps during viral infections and pathogenesis and discuss how the viruses hijack the host autophagy for their life cycle and pathogenesis. Further understanding the function of host autophagy in viral infection and pathogenesis contributes to the development of more specific therapeutic strategies to fight various infectious diseases, such as the coronavirus disease 2019 epidemic. Autophagy, or cellular self-digestion, is an evolutionarily conserved cellular process through which long-lived proteins, damaged organelles, or invading pathogens could be degraded by the lysosome . AccordiAutophagy begins with the sequestration of cargoes into a cup-shaped double membrane known as the isolation membrane or phagophore, which stems from several cellular compartments . The seqAutophagy is involved in a variety of mammalian physiological processes such as the maintenance of energy homeostasis, cell differentiation and development, and innate immunity against invading pathogens . Given tAs a multi-step and tightly regulated cellular process for maintaining eukaryotic cellular homeostasis, autophagy is the only pathway that is able to degrade whole cellular organelles  and various invading pathogens (including viruses) in either a selective or a non-selective manner . Upon viBy delivering the invading pathogens to the lysosomes for degradation so as to discard them, autophagy is believed to be an important part of the host defense system. The process of degrading foreign microbial invaders by autophagy is known as xenophagy . The funin vitro study found that p62 could interact with Sindbis capsid protein and targets the viral capsid to the autophagosome  in neurons could inhibit the Sindbis virus spread, reduce cellular apoptosis, and protect against fatal Sindbis virus encephalitis , whereashagosome . Orvedahhagosome . FANCC phagosome . Besideshagosome .Picornaviruses are archetypical non-enveloped viruses, which are a major cause of human and veterinary infections that lead to various diseases, such as polio and the common cold . Host gaAs a small, enveloped RNA virus that mainly targets human hepatocytes, hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide . The preAs a sensor of cytosolic DNA that activates the type I interferon pathway, cyclic GMP-AMP synthase (cGAS) could bind to microbial or self-DNA in the cytoplasm and therefore supervises infections or tissue damage . SpecifiHow is autophagy induced in mammalian cells during viral infection? By using genome-wide short interfering RNA screens, the endosomal protein sorting nexin 5 (SNX5) is found to be required merely for virus-induced autophagy, but not for basal level autophagy or stress-induced autophagy . DeletioCollectively, host cells detect the invading viruses and activate the autophagy, which targets viruses to the lysosomes for degradation through the interaction of host protein with the viral protein. Future studies should focus on exploring additional viruses that can be selectively degraded by autophagy and clarifying the specific mechanisms involved in this process.As the first line of defense against virus infection, the host innate immune system plays essential roles in recognizing invading viruses and inducing anti-viral responses to prevent viral invasion and pathogenesis before the generation of more specific protection by the adaptive immune system . The hosThere are several classes of PRRs with specific functions; virus-derived nucleic acids with distinct features are recognized by these specific host transmembrane or cytosol PRRs . As an iAs an essential DNA virus sensor, cGAS could prompt IFNs production by generating cGAMP, which binds to and activates an endoplasmic reticulum-associated adaptor protein STING . Chen etThese studies suggest that host autophagy plays an essential role in activating the innate immune response to eliminate the invading viruses by promoting the infected cells and other immune cells to produce IFNs. However, there are also some studies reporting that autophagy or its components contributed to the negative regulation of host innate immune response . Therefo+ or CD4+ T lymphocytes  molecules at the cell surface of antigen-presenting cells (APCs) such as dendritic cells and macrophages, which then can be recognized by CD8phocytes . In genephocytes . Howeverphocytes . In addiphocytes .+ T cells aided by proteasome function and the secretory pathway.Macrophages infected with the Herpes simplex type 1 virus could activate the process of autophagy, which plays an essential role in the targeting of viral proteins to lysosomes and then loaded on MHC class I molecules for presentation . During An endogenous human cytomegalovirus (HCMV) latency-associated protein, pUL138, could be presented by MHC-I through both the conventional TAP-dependent and the non-conventional TAP-independent pathways . The TAP+ T cells  infection resulted in the impairment of CD4nfection . Moreovenfection . These r+ T-cell responses , could inhibit host autophagy activation through binding with the host autophagy protein Beclin 1 . HSV-1 vMatrix protein 2 of influenza A virus could inhibit autophagy function through blocking the autophagosome–lysosome fusion, causing the enhanced virus-induced cell death of infected cells and elevated viral antigen release . Human pSevere Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), an enveloped, single-stranded and positive-sense RNA β-coronavirus, is the cause of the COVID-19 pandemic . ORF3a, As a DMV formed during autophagy, autophagosome provides a perfect place for the RNA viral replication through concentration of essential intermediates for viral package and protection of viral RNAs away from the detection by innate immune supervision and degradation. The host autophagy has been required by several viruses such as Coxsackieviruses (CVB) 3, CVB 4, foot and mouth disease virus, HCV, and poliovirus for their own replication, as genetic or pharmacological inhibition of autophagy could decrease viral yields . MoreoveIn summary, host autophagy is a double-edged sword during viral infection and pathogenesis. On the one hand, hosts could utilize their own autophagy to prevent viral infection and pathogenesis; on the other hand, viruses have evolved various strategies by which they hijack and subvert host autophagy to aid their infection and pathogenesis.As an evolutionarily conserved cellular process, autophagy is essential for both hosts and invading viruses. On the one hand, it could prevent viral infections and pathogenesis mainly by degrading viruses, initiating innate immune response, and facilitating antigen presentation. On the other hand, a mass of viruses have evolved strategies to hijack host autophagy for their own benefits. Although the function of host autophagy in viral infection and pathogenesis has been widely studied in the past two decades, current knowledge of autophagy in viral infections is still in its infancy and many important questions remain. For example, why some viruses have evolved strategies to evade host autophagy, whereas others have not? What is the driving force for this evolution? What roles do a variety of selective autophagy (such as mitophagy and lysophagy) play in viral infection and pathogenesis? What roles do microautophagy and chaperone-mediated autophagy play in viral infection and pathogenesis? Moreover, the specific function of autophagic proteins and the mechanisms controlling autophagy during viral infection and pathogenesis are still unclear. Further research is needed to elucidate the specific functions of different types of autophagy and the specific function in viral infections and pathogenesis, to develop more specific targeted drugs to combat epidemic viral infections, such as COVID-19.J-XT and H-FL formulated and conceived the study. Y-SW, SL, D-YL, J-XT, and H-FL wrote the manuscript. All authors helped to interpret results and approved the final version of the manuscript.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.


Genome instability may play a role in severe cases of male infertility, with disrupted spermatogenesis being just one manifestation of decreased general health and increased morbidity. Here, we review the data on the association of male infertility with genetic, epigenetic, and environmental alterations, the causes and consequences, and the methods for assessment of genome instability. Male infertility research has provided evidence that spermatogenic defects are often not limited to testicular dysfunction. An increased incidence of urogenital disorders and several types of cancer, as well as overall reduced health (manifested by decreased life expectancy and increased morbidity) have been reported in infertile men. The pathophysiological link between decreased life expectancy and male infertility supports the notion of male infertility being a systemic rather than an isolated condition. It is driven by the accumulation of DNA strand breaks and premature cellular senescence. We have presented extensive data supporting the notion that genome instability can lead to severe male infertility termed “idiopathic oligo-astheno-teratozoospermia.” We have detailed that genome instability in men with oligo-astheno-teratozoospermia (OAT) might depend on several genetic and epigenetic factors such as chromosomal heterogeneity, aneuploidy, micronucleation, dynamic mutations, RT, PIWI/piRNA regulatory pathway, pathogenic allelic variants in repair system genes, DNA methylation, environmental aspects, and lifestyle factors. Infertility (defined as the inability to conceive within a year of unprotected coitus) is a global health and social issue affecting close to 15% of couples. In half of the couples seeking medical treatment for infertility, male factor infertility is identified together with abnormal semen characteristics [Increasing infertility is considered to be mostly dependent on the lower fecundity of women planning their first pregnancy at a more advanced age, coupled with decreasing semen quality in men . In 1992The focus of current clinical practice is laid on the presence of sufficient sperm count in the ejaculated specimen with adequate motility and morphology of spermatozoa capable of giving fertilization a chance . However, semen analysis fails to predict the fertilizing potential of the male gamete precisely. Indeed, a substantial overlap of semen parameters between fertile and infertile males has been reported . MoreoveAn abnormal sperm chromatin structure (defective histone-to-protamine replacement) has been found to be associated with the incomplete maturation of sperm during spermiogenesis . HoweverThe central biological event of fertilization is the transition of the paternal and maternal genomes to the offspring. Genome instability—understood as reduced fidelity with which genetic information is passed on to daughter cells—can impair both mitosis and meiosis. Changes taking place in the course of DNA replication, DNA repair, and cell division  provide a natural source of genome instability. Depending on the nature of processes involved, genome instability can lead to (a) single nucleotide variants and microsatellite expansions caused by errors in DNA synthesis and defective DNA repair mechanisms; (b) variations in the number of chromosomes (aneuploidy) due to mistakes of the chromosome segregation apparatus; (c) other types of genetic modifications, such as gross chromosomal rearrangements, copy number variants (CNVs), hyper-recombination and loss of heterozygosity, triggered in the majority of cases by single-strand DNA gaps or double-strand breaks (DSBs) occurring as a consequence of replication stress . Apart fIt was recently suggested that genome instability could lead to decreased general health and increased morbidity . It is pAn array of genetic and epigenetic factors of male infertility, including single nucleotide variants, CNVs, protamine content, methylation characteristics, protein content, and small RNAs, have been analyzed in various studies. Currently, the above assays are only used within the research setting being not routinely carried out by fertility clinics . In the The content of the review is schematically summarized in A literature search in Pubmed and Google Scholar databases for MeSH terms referred to genome instability, male infertility, genetic variations, epigenetic regulation, repair system, senescence, and environmental agents was performed until October 2020. The most important results were further described in this review article.In this section, well-established genome changes associated with reduced male fertility are reviewed, beginning with genome abnormalities visible by microscope (cytogenetic variants), followed by microdeletions (azoospermia factor (AZF) region variants) and microsatellite instability (MSI), then finally, single-gene allelic variants. Aneuploidy  is the most prevalent cause and consequence of genome instability. The most common chromosomal aneuploidy in azoospermic patients is Klinefelter syndrome , accounting for approximately 11% of azoospermia cases . Up to 4microdeletions in the AZF locus of the Y chromosome [Another cause of genome instability is CNVs—a phenomenon characterized by the presence of repeated genome sections with the number of repeats in the genome varying between individuals. A classic and simple example of CNVs related to male infertility is romosome ,33,34. Tromosome ,36. AZF romosome ,38.The frequency of Yq microdeletions in different parts of the world varies slightly. This could be explained by differences in sample size, methodology used, and the population screened. The lowest prevalence of Yq microdeletions is estimated in Europe (3%) and Australia (5%), while the rest of the world has an average of 8–9% .Genome instability can be caused by MSI, when the number of repeated DNA bases in a microsatellite differs from what it used to be when the microsatellite was inherited. MSI represents a state of genetic hypermutability (predisposition to mutation) resulting from impaired DNA mismatch repair (MMR) . The dynAR) gene [AR gene contains a region with variable numbers of copies of the trinucleotides CAG and GGN. Increased copy numbers of CAG and GGN are associated with impaired spermatogenesis [AR gene have been found to be correlated with a higher risk of prostate cancer, especially in Caucasian men [A variable MSI-related region is found in the androgen receptor (AR) gene ,43. The AR) gene . The AR ogenesis ,43. Howesian men .CFTR gene, displaying a strong association with azoospermia. The protein this gene encodes operates as a channel across the membrane of cells with exocrine function, thereby besides pulmonary and digestive system dysfunction affecting the reproductive function as well. In recent years, several other allelic variants have been reported to have a strong association with non-obstructive azoospermia  and sever genes ,48).Mobile genetic elements in humans have been categorized as DNA transposons and retrotransposons (RT). DNA transposons move driven by a cut-and-paste mechanism, whereas the process of retrotransposition involves RT mobilization by a copy-and-paste mechanism via an RNA intermediate . DNA traRT are abundant in all types of organisms, comprising 45–50% of the human genome. RT are the most widespread class of transposons in mammals and represent a potential source of genome instability, mutations, and polymorphisms ,53. RT dThe silencing of RT in the germline is a unifying mechanism of PIWI-interacting RNAs (piRNAs) observed in all animal species. Moreover, piRNAs have recently been proven essential for successful spermatogenesis controlling transcription .−8. Each additional year of the father’s age at conception has been found to increase the germline mutational load of the offspring by one to three de novo mutations. Due to the fact that said process involves recurrent rounds of DNA replication and cell division, random copy-error mutational events are expected to occur mainly in the male germline [The average mutation rate of single base substitutions in humans per generation has been calculated to reach ~1–1.5 × 10germline .MSH genes can be responsible for the MMR failure in sperm DNA and male infertility [MSH3 is associated with idiopathic male infertility, which makes it a possible risk factor for idiopathic male infertility [MSH3 gene located at 5q11-13 encodes an MSH3 protein playing an essential role in the MMR system. MSH3 also recruits other MMR proteins to the base mismatch loci to fix the DNA damage [MLH1 gene showed an association with severe oligozoospermia [MLH1 promoter is significantly more methylated in oligozoospermic patients. Furthermore, a statistically significant positive association was observed between elevated reactive oxygen species (ROS) levels and MLH1 methylation, with no such association found in the case of MSH2 methylation [Repair of damaged DNA occurs only in early spermatogenesis. The oocyte is capable of repairing sperm DNA to maintain sperm/embryo genomic integrity and stability ,59. One ertility ,61,62. TA damage . In addiThe most deleterious type of DNA damage is breaks in DNA double-strand (DSBs). They can occur endogenously by virtue of DNA transcription and replication through the impaired activity of topoisomerases I and II ,65 or exOxidative stress directly damages guanine residues in DNA. Even though this process is initially reversible, it is capable of impairing DNA double-strand architecture and ultimately inducing single-strand breaks (SSBs). Male germ cells are not equipped with molecular SSB repair mechanisms, but nevertheless, at the spermatid stage, sperm can rely on the base excision repair capability for the removal of oxidative stress-induced transversion mutations. In this mechanism, altered DNA bases are identified, and an abasic site is generated, the next step being cleavage by an endonuclease. After zygote formation, paternal SSBs and DSBs can be effectively repaired by the oocyte when the DNA damage in sperm is below 8% . Male paDuring embryonic development, the mammalian genome is exposed to two waves of global demethylation and remethylation. The first wave occurring after fertilization includes the erasure of most methylation marks inherited from the gametes, followed by the establishment of the embryonic methylation pattern with the onset of differentiation determination . DevelopSpermatogenesis is a set of processes setting into motion in the postembryonic period of ontogenesis with the appearance of primordial germ cells (PGCs) that are destined to migrate to and populate the genital ridge. PGCs undergo various epigenetic modifications that could be further subdivided into two events—global erasure and paternal reestablishment of the imprinted regions. Male PGCs undergo several mitotic division rounds in the embryonic testes until they enter mitotic arrest  .The role of histones in spermatogenesis has been widely considered in the literature. There are two basic histone subtypes, so-called canonical histones, that are involved in genome packaging and gene regulation, and non-canonical histones (histone variants) regulating the initiation of transcription DNA repair. In mammals, 11 histone H1 subtypes are known, with H1t, H1T2, HILS1 being expressed specifically in the testis. The testis-specific H1T2 variant expression is observed in haploid male germ cells until histone-to-protamine transition . Loss ofThe PIWI/piRNA pathway is recognized as a major mechanism protecting the germ cell genome from insertional mutagenesis by RT . The gerFailure to suppress RT during spermatogenesis often results in DSBs in sperm DNA. Therefore, it is important to identify pathogenic allelic variants in repair system genes. Allelic variants in the two DNA recombinases RAD51 and DMC1—indispensable in meiosis mediating DSB repair system genes and DNA MMR genes in the case of MSI—could be important as they play decisive roles in key pathways of DNA repair. Furthermore, given these genes are crucially involved in mitotic DNA repair and meiotic recombination, also being highly expressed in the testis, any decrease of the activity of their encoded protein may disrupt the DNA repair system and induce the accumulation of mutations and breaks in sperm DNA. Accordingly, variants in these MMR genes may affect sperm quality and reduce fertility .Aging is an ontogenesis phase characterized by the deterioration over a period of time of an organism’s physiological functions that are necessary for survival . In the Male aging manifests itself in a higher occurrence of meiotic errors, chromatin fragmentation, telomere shortening, epigenetic changes, and impaired control of mobile elements. The major biological markers of genome stability are telomeres with their sheltering proteins protecting chromosomes against DNA damage and ensuring correct chromosome alignment during DNA replication. Drastic telomere shortening is associated with decreased sperm parameters. DNA methylation and telomere integrity are also important for normal fertilization and embryo development .Werner syndrome (WS) illustrates the link between cellular aging and male infertility. Besides features associated with premature aging , the phenotypic hallmark of Werner syndrome is male infertility due to hypogonadism leading to complete azoospermia . The cauAt the end of the long arm of the human X chromosome encompassing the region from Xq27.2 to the q telomere, including the chromosomal band Xq28, there exists a fragile X-chromosome region of the DNA breakpoints, possibly due to the enrichment with LTR retrotransposons. This region is of particular interest since it contains the highest density of genes related to genetic diseases . SubteloThere is accumulating evidence that harmful environmental  and lifestyle factors  impact DNA methylation in sperm and could consequently lead to male infertility . It is uMaintenance of genome stability relies on the DNA damage response executed through a functional network consisting of signal transduction, cell cycle regulation, and DNA repair.Various approaches can be used to investigate different types of genome instability in different tissues. These methods, such as sperm DNA fragmentation tests, micronucleation assays, or MSI investigation are successfully applied in clinical diagnostics and are further described in this article.Genome instability in sperm cells can be assessed by (1) sperm chromatin structural probes, (2) sperm nuclear matrix assays, (3) direct assessment of DNA strand breaks , (4) ALU/LINE transcription. The processes inducing DNA damage in ejaculated spermatozoa are interrelated. Abnormal spermatid protamination and disulphide bridge formation as a result of inadequate thiols oxidation during epididymal transit, leads to diminished sperm chromatin packaging and makes sperm cells more susceptible to ROS-induced DNA fragmentation .Detailed investigation can determine transposon content and activity after stressful events . A simplVarious molecular genetics approaches can be employed to anticipate genome instability. Allelic variants and microdeletions in genes controlling MMR, RT, and other important mechanisms can be detected by Sanger sequencing, next-generation sequencing, and third-generation sequencing . HistoriA micronucleus is an erratic (third) nucleus that develops during the last step of cell division. As micronuclei contain fragments of chromosomes, their formation proceeds in a daughter cell with an aberrant chromosomal set. An increased number of micronuclei is a sign of chromosomal damage caused by genotoxic agents . MicronuThe COMET assay measures DNA strand breaks at the individual cell level, and it can be applied both in sperm (as described above) and peripheral blood cells. The fluorescence is calculated to measure the level of DNA damage . The COMIn summary, we have presented extensive data supporting the notion that genome instability may lead to severe male infertility (OAT), whereas genetic tests recommended for routine clinical investigation  provide a diagnosis in only 15–25% of cases. We have detailed that genome instability in men with OAT depends on several other genetic and epigenetic factors such as chromosomal heterogeneity, aneuploidy, micronucleation, dynamic mutations, RT, PIWI/piRNA regulatory pathway, pathogenic allelic variants in repair system genes, DNA methylation, and environmental aspects .


In cancers and other complex diseases, the fusion of two genes can lead to the production of chimeric RNAs, which are associated with disease development. Several recurrent chimeric RNAs are expressed in different cancers and are thus used for clinical cancer diagnosis. Rheumatoid arthritis (RA) is an immune-mediated joint disorder resulting in synovial inflammation and joint destruction. Despite advances in therapy, many patients do not respond to treatment and present persistent inflammation. Understanding the landscape of chimeric RNA expression in RA patients could provide a better insight into RA pathogenesis, which might provide better treatment strategies and tailored therapies. Accordingly, we analyzed the publicly available RNA-seq data of synovium tissue from 151 RA patients and 28 healthy controls and were able to identify 37 recurrent chimeric RNAs found to be expressed in at least 3 RA samples. Furthermore, the parental genes of these 37 recurrent chimeric RNAs were found to be differentially expressed and enriched in immune-related processes, such as adaptive immune response and the positive regulation of B-cell activation. Interestingly, the appearance of 5 coding and 23 non-coding chimeric RNAs might be associated with regulating their parental gene expression, leading to the generation of dysfunctional immune responses, such as inflammation and bone destruction. Therefore, in this paper, we present the first study to demonstrate the novel chimeric RNAs that are highly expressed and functional in RA. Chimeric RNAs are produced by the fusion of exons/introns from two different genes . SeveralRheumatoid arthritis (RA) is an auto-immune disorder exemplified by the chronic and persistent inflammation of the joint synovial tissue associated with the destruction of the affected joints . The patTo understand the potential impact of chimeric RNAs on RA development and heterogeneity, in this paper, we analyze chimeric RNA expression in publicly available RNA-seq data on synovial fluids from 151 RA patients and 28 healthy controls. We identified 37 recurrent and RA sample-specific chimeric RNAs whose parental genes are predominately involved in immune-related processes, such as “adaptive immune response” and the “positive regulation of B-cell activation”. Furthermore, we found that parental genes of 20 of 37 such chimeric RNAs are significantly differentially expressed in RA samples. This points to these chimeric RNAs being associated with the dysfunctional immune response generation that leads to inflammation and bone destruction. Significantly, our study provides the signature of chimeric RNA expression in RA and highlights the potential clinical importance of this signature in personalized treatments.A total of 217 publicly available total RNA sequencing samples isolated from joint synovial biopsies were downloaded from the GEO database (GEO accession: GSE89408). These contained a total of 151 samples from RA patients, a total of 28 samples from healthy subjects (HT), and a total of 38 samples from other arthritis patients . We alsoAll 217 samples were further used to identify chimeric RNAs using our in-house reference-based method ChiTaH , previouRNA sequencing reads of all 217 samples were subjected to analysis by STAR aligner ; a humanGene ontology, pathway, and gene enrichment analysis of the parental genes of recurrent chimeric RNAs were performed using the online Metascape server . In thisThe functional classification of recurrent chimeric RNAs was performed based on their protein-coding abilities. Three different tools .Assessing differential gene expression among samples of joint synovial biopsies from 28 healthy individuals and 151 RA patients was performed using DESeq2 . Such anTo understand further the structure and function of recurrent chimeric RNAs in RA, these were classified as “coding” or “non-coding”. For this analysis, we considered common predictions made using three tools, namely, CPAT , CNIT 4, and LncRecent studies identified several chimeric RNAs in different cancers that are associated with oncogenesis, cancer heterogeneity, and the evolution of cancer drug resistance ,43. The In the present study, we analyzed publicly available RNA-seq data from disease-relevant and healthy synovial tissues. Our systematic integrative analysis identified 37 recurrent RA-specific chimeric RNAs and several RA sample-specific chimeric RNAs. Previous studies demonstrated recurrent cancer-specific chimeric RNAs to be involved in cancer development. Moreover, chimeric RNAs expressed in specific cancer samples are important for cancer heterogeneity. Therefore, our finding suggests that the expression of recurrent chimeric RNAs in RA patients could be associated with RA development, whereas the expression of RA sample-specific chimeric RNAs could be important for generating disease heterogeneity. Furthermore, to understand whether recurrent chimeric RNAs are associated with RA development, we analyzed the expression of their parental genes and the pathways to which these genes contribute. Interestingly, we observed the parental genes of the 37 recurrent chimeric RNAs to be mainly involved in the immune-related processes and adaptive immune responses, namely, “adaptive immune response” and the “positive regulation of B-cell activation”. We also found that at least one parental gene is differentially expressed in most cases. Therefore, this could indicate that the appearance of chimeric RNAs is associated with the generation of dysfunctional immune responses in RA. Further, we performed the disease enrichment analysis to check the most enriched diseases where these parental genes of RA-specific recurrent chimeric RNAs are involved. We found the most enriched diseases are gout, rheumatism, and reactive arthritis. Therefore, the dysregulation of these genes could induce the common clinical signature of gout and RA, such as pain and swelling and stiffness of joints. Next, the functional characterization of RA-specific recurrent chimeric RNAs predicted the majority of them could act as lncRNA and involved in regulatory functions. In summary, we hypothesize that these recurrent chimeric RNAs could regulate the expression of their parental gene, leading to the generation of dysregulated immune responses in affected tissues, the development of RA, and subsequent inflammation in osteoclast formation and bone destruction of affected tissues .In summary, our study provides an expression landscape of chimeric RNAs in the affected synovium of RA patients. Our results also highlight the potential functional association of the novel chimeric RNAs in RA development and heterogeneity. The limitations of our study are that it lacks experimental evidence, due to the unavailability of synovium tissue samples from RA patients. Nonetheless, this study opens a new paradigm to explore further chimeric RNA-mediated transcriptional dysregulation leading to RA development and heterogeneity that could help in the design of new treatment strategies.


Liquid biopsy (LB) is a rapidly evolving diagnostic tool for precision oncology that has recently found its way into routine practice as an adjunct to tissue biopsy (TB). The concept of LB refers to any tumor-derived material, such as circulating tumor DNA (ctDNA) or circulating tumor cells that are detectable in blood. An LB is not limited to the blood and may include other fluids such as cerebrospinal fluid, pleural effusion, and urine, among others.The objective of this paper, devised by international experts from various disciplines, is to review current challenges as well as state-of-the-art applications of ctDNA mutation testing in metastatic non-small-cell lung cancer (NSCLC). We consider pragmatic scenarios for the use of ctDNA from blood plasma to identify actionable targets for therapy selection in NSCLCs.Clinical scenarios where ctDNA mutation testing may be implemented in clinical practice include complementary tissue and LB testing to provide the full picture of patients’ actual predictive profiles to identify resistance mechanism (i.e. secondary mutations), and ctDNA mutation testing to assist when a patient has a discordant clinical history and is suspected of showing intertumor or intratumor heterogeneity. ctDNA mutation testing may provide interesting insights into possible targets that may have been missed on the TB. Complementary ctDNA LB testing also provides an option if the tumor location is hard to biopsy or if an insufficient sample was taken. These clinical use cases highlight practical scenarios where ctDNA LB may be considered as a complementary tool to TB analysis.Proper implementation of ctDNA LB testing in routine clinical practice is envisioned in the near future. As the clinical evidence of utility expands, the use of LB alongside tissue sample analysis may occur in the patient cases detailed here. •LB is a rapidly evolving diagnostic tool that may be an adjunct or an alternative to TB in clinic.•Pragmatic scenarios for ctDNA mutation testing to identify actionable targets in NSCLC are explored.•ctDNA mutation testing may identify resistance mechanisms, discordant clinical history, and intertumor/intratumor heterogeneity.•ctDNA mutation testing may be useful if the tumor location is hard to biopsy or if an insufficient tumor sample was taken.•Proper implementation of ctDNA mutation testing in routine clinical practice is envisioned in the near future. Liquid biopsy (LB) is a rapidly evolving diagnostic tool for precision oncology that has recently found its way into routine practice as an adjunct or an alternative to tissue biopsy (TB). The concept of LB refers to the analysis of any tumor-derived material detectable in the blood and other bodily fluids, such as circulating tumor DNA (ctDNA) or circulating tumor cells.Although TB is the gold-standard diagnostic method used to obtain or to confirm the diagnosis of cancer, to define the histological subtype, or to identify actionable targets, molecular profiling from ctDNA in patients with non-small-cell lung cancer (NSCLC) has several advantages, including its minimally invasive nature, a continuous monitoring of genetic alterations, and a better reflection of intratumor and intertumor heterogeneity.5EGFR), repeat biopsy and/or plasma testing should be performed.,,,10The National Comprehensive Cancer Network (NCCN) 2021 and European Medicines Agency guidelines recommend that when there is insufficient tissue to allow for testing for clinically relevant predictive mutations and rearrangements  concluded that ‘LB approaches have significant potential to improve patient care, and immediate implementation in the clinic is justified in a number of therapeutic settings relevant to NSCLC’.Despite a continuously growing body of knowledge of ctDNA biology, alongside many advances in the technology used for testing, uptake into clinical daily practice still represents only a small proportion of cases. There are currently few approved tests for the use of ctDNA LB in routine clinical practice in NSCLC .Table 1EThe objective of this review is to consider pragmatic scenarios for the use of ctDNA from blood plasma to identify actionable targets for therapy selection in metastatic NSCLC.The high concordance rates of 91%-95% for relevant predictive markers between TB and LB allow exploration into the clinical applications of LB across the patient journey.20https://doi.org/10.1016/j.esmoop.2022.100399). At present, consensus is needed on which methodologies may provide the best answers at various stages of the patient journey -based methods and next-generation sequencing (NGS) technologies interrogating single genes, hotspot mutations, or large gene panels  and other targeted therapies can target a broad range of molecular alterations resulting in specific gene fusions or altered expression levels and are associated with multiple mechanisms of resistance that can be better captured by larger panels.At the time of diagnosis, testing for clinically actionable and well-characterized alterations might already reveal predictive information . The NCCIdeally, combining liquid and tissue testing captures heterogeneity and can be highly sensitive without compromising specificity; however, this may be biased by the location of the tumor.Regardless of the platform, well-established and validated ctDNA LB workflows are essential for routine use.,The preanalytical steps of ctDNA mutation testing can be impacted by many factors,,,,,Recommendations for the analytical validation of NGS-based ctDNA assays were recently published by Godsey and colleagues.,,,,,,,Recent data from various intra- and inter-laboratory comparisons indicate a substantial variability of NGS assays with respect to analytical sensitivity and specificity, highlighting the importance of extensive validation of the test performance before offering these tests in clinical practice.All these inter- and intra-assay comparisons stress the importance of external quality assessment (EQA) participation, a critical aspect of laboratory quality management, because EQA participation is mandatory to be accredited.,A pilot project demonstrated the feasibility of establishing a ctDNA mutation testing EQA scheme and highlighted the need for such actions in light of high error rates in detecting clinically relevant low-frequency variants.,,The field is building on tests that target single genes to whole-genome sequencing-based panels and, more recently, combined mutational, methylation, and proteomic profiling approaches.TP53, KRAS, GNAS, NRAS, and PIK3CA.,,,Clonal hematopoiesis (CH) involves the accumulation of somatic mutations in hematopoietic stem cells. This leads to clonal expansion of mutations in blood cells and may account for non-tumor-derived mutations detected in the plasma. CH is associated with the normal process of aging in both healthy individuals and those with cancer.,,Whether or not PBMCs should be sequenced to correct for CH-related variants is still a matter of debate, mainly due to the extra costs involved in paired sequencing. For focused panels interrogating well-defined, clinically relevant mutations, it might not be necessary, because hotspot oncogenic events that are relevant for solid malignancies are usually not causative for CH.,In addition to a paired plasma and PBMC analysis, variant filters based on association of a mutation with CHIP as well as functional annotation of a somatic variant as an oncogene activating event can be used to filter for CH-related variants.An important step in the delivery of precision oncology to patients with lung cancer is the interpretation and reporting of variants in the clinical context.,KRAS G12.,,,With NSCLC being a low ctDNA tumor entity,ATM (50%), BRCA2, or TP53.EGFR mutations (e.g. T790M) were reported that may be difficult to discriminate from their targetable somatic counterpart in ctDNA mutation testing when no PBMC analysis is included in parallel.Although lung cancer is not commonly associated with hereditary cancer susceptibility and is environmentally provoked, a subset of lung adenocarcinoma patients (2.5%-4.5%) may be linked to germline variants in well-known predisposing cancer genes such as Another key challenge includes which specific alterations should be classified as ‘actionable’. To support classification, the Association for Molecular Pathology (AMP), American Society of Clinical Oncology (ASCO), and College of American Pathologists (CAP) jointly published a four-tiered system classification system for the interpretation and reporting of sequence variants in cancer.Moreover, for clinical decision making, a variety of tools and platforms have recently been developed. Ever-growing knowledge bases such as the Cancer Genome Interpreter Cancer Biomarkers Database (CGI), Clinical Interpretation of Variants in Cancer (CIViC), Jackson Laboratory Clinical Knowledgebase (JAX-CKB), MolecularMatch (MMatch), OncoKB, and the Precision Medicine Knowledgebase (PMKB) facilitate interpretation. To overcome the dramatic differences in the components of variant interpretations, efforts are being made to develop a framework for structuring and harmonizing clinical interpretations across these knowledge bases.,The final call for choice of therapy is still made by a molecular tumor board, and decision support software tools can support the tumor board’s discussion and ultimately clinician’s decision on treatment strategy.Certain minimum requirements are needed for the reporting of molecular profiling results for all CAP-accredited laboratories.,,,,,,Approximately one-third of patients with advanced NSCLC may die within the first 2 months after initial diagnosis.,In cases with discordant clinical history (e.g. nonsmoker but squamous cell carcinoma) of suspected inter- and intra-tumor heterogeneity, cfDNA testing may be a good option. The usefulness of LB in oncogene-addicted first-line TKI refractoriness is linked to availability  of a specific drug to overcome the resistance.Moreover, ctDNA analysis may have additional benefit in patients who have inappropriate locations for samples to be taken, such as lesions within the bone or adrenal glands , case 2 On occasion, ctDNA mutation testing can reveal mutations that are not present in the initial TB—one case reveals discordant genotypes between tumor biopsy and blood-based analyses that may result from technological differences, as well as sampling different tumor cell populations , case 5.Testing treatment-naive patients for possible targets is also of interest, in particular if time matters or if TB is refused. A patient questionnaire revealed that up to 33% of participants rejected a rebiopsy, mainly due to initial biopsy being painful. Furthermore, up to 40% of the cohort would consider a noninvasive option if it were made available to them.,Taken together, there is strong evidence to support the inclusion of LB in the management of NSCLC during the early treatment phase.https://doi.org/10.1016/j.esmoop.2022.100399).,,,ctDNA can have a role to play for treatment decision making, but also other potential applications are emerging ; however, reimbursement for broad coverage is limited.,In some countries, reimbursement is in place for ctDNA mutation testing  to enhance early detection by LB screening.,93,,https://doi.org/10.1016/j.esmoop.2022.100399.ctDNA mutant allele fractions can be extremely low, so mutation-based detection of ctDNA has clear limitations for MRD and monitoring within the plasma.It should be mentioned that other LB analytes, such as circulating tumor cells, circulating microRNAs, tumor-educated platelets, or tumor-associated proteins, might provide complementary information for patients with cancer.The future of ctDNA LB testing opens up many potential avenues to assist in both research- and clinical-based settings. Currently the field utilizes ctDNA to identify single genes and is evolving to integrate omics data from proteomes or genome-wide sequencing where needed—combined application tests are not currently needed to address clinically relevant applications.Proper implementation of ctDNA mutation testing in routine care can be envisioned in the near future. As the clinical evidence of utility expands, the use of LB alongside tissue sample analysis may occur in the patient cases detailed above.,ctDNA LB mutation testing offers both challenges and benefits.As the clinical evidence of utility expands, routine molecular profiling using ctDNA LB alongside tissue sample analysis improves clinical sensitivity in clinical practice for improved treatment decision making for people with NSCLC.


Glaucoma, its early diagnosis, and monitoring of interventions remain an ongoing challenge. We here review developments in functional assessment and its relation to morphology, evaluating recent insights in electrophysiology in glaucoma and highlighting how glaucoma research and diagnostics benefit from combined approaches of OCT and electrophysiological investigations. After concise overviews of OCT and non-invasive electrophysiology in glaucoma, we evaluate commonalities and complementarities of OCT and electrophysiology for our understanding of glaucoma. As a specific topic, the dynamic range (floor effects) of the various techniques is discussed. Glaucoma is a globally leading cause of irreversible blindness  and is cThe loss of RGCs and their axons are the hallmarks of neurodegenerative processes in glaucoma . OCT hasDiagnostic performance of pRNFL and macular GCIPL and GCC was ascertained in glaucoma, where most of the studies reported comparable area under the curve (AUC) of pRNFL vs GCIPL/GCC measures, i.e., the discriminative performance between glaucoma and controls. In a recent meta-analysis of 150 studies , the pooBoth peripapillary and inner macular thickness might also have a critical role in the timely identification of glaucoma progression to implement therapy and avert permanent visual loss. However, many studies demonstrated the beneficial role of either GCC or GCIPL over pRNFL in monitoring glaucoma—see reviews , 9. At pOptical coherence tomography angiography (OCT-A) is a nascent imaging modality assessing non-invasively the microvasculature of the macula, the peripapillary area, and the optic nerve head that might improve glaucoma management, as reviewed in –14. It iAt the peripapillary area, most of the studies, see reviews , 20, fouWe will review methods offered in clinical visual electrophysiology and their relation to glaucoma management, drawing heavily on previous reviews , 22, 23 Clinical visual electrophysiology comprises a large set of methods with an associated alphabet soup of acronyms. Table In accordance with Table The PhNR , 27 is rMildly invasive, electrodes at the cornea or near the eye.With current protocol, pupil dilation is required.Patient preparation ½ h, recording time ≈5 minNo high-quality optical imaging required .Direct indicator of ganglion cell function.Along glaucoma progression, PhNR amplitude may floor later than pattern electroretinogram (PERG) (see section “Floor effects and dynamic range” below).Recent progress with rapid stimulation suggests improved sensitivity.The PERG –37 is reIt is well established that the PERG  originates from the RGCs , 42. TheMildly invasive, electrodes at the cornea or near the eye.Adequate optical imaging necessary .Patient preparation ½ h, recording time ≈5 min.Sensitive early, then saturated; thus not useful as a marker in later glaucoma stages.For stimulation, no LCD displays can be used.A direct indicator of ganglion cell function.Affected very early (probably before marked field changes are present).Large literature body available including longitudinal studies that find success in early diagnosis.More sensitive with rapid pattern changes (>10 times per second), allowing Fourier-based automatic. analysis without the need for individual peak assessment.The VEP, being a long-standing well-developed tool with little invasiveness, has been assessed many times for its use in glaucoma. It is a given that glaucoma will affect the VEP: Glaucoma leads to ganglion cell loss, and the VEP, representing visually-driven cortical activity, will necessarily be affected. The crucial aspect is its sensitivity and specificity. The VEP signal can be delayed and/or reduced in amplitude. For instance, a very positive report  reportedThe blue-sensitive pathway is thought to be especially vulnerable in glaucoma, probably due to its relatively low dynamic range . Targeti“ic-VEPs”  sweep ct-tests and further 16 correlation tests without correction for multiple testing, these findings await rigorous re-testing.The “SD-tVEP” : a typiHigh interindividual amplitude variability.Very few longitudinal studies are available.LCD displays should not be used for stimulation (but are).Little invasiveness .Useful for monitoring (no saturation like PERG).In principle, the multifocal VEP (mfVEP) could serve as perfect objective perimetry. Small obstacles are the necessity of good retinal imaging with respect to refraction and accommodation and steady correct fixation. A major problem false-positive scotomata. Due to the complicated folding of V1, for some field locations, the corresponding activity in V1 does not sufficiently project to the scalp electrodes, even if multiple channels  are usedFalse-positive scotomata due to the convoluted cortical folding and the ensuing loss of dipole projection on surface electrodes.Objective perimetry.Electroretinographic measures of RGC function might help to disentangle the interplay of the RGC function and risk factors for glaucoma, specifically the intraocular pressure (IOP), not only for elucidating damage mechanisms but also for defining the onset of glaucoma. An attractive maneuver to decipher the IOP-RGC relationship is to manipulate IOP by employing body-position-induced physiological changes and to manifest its impact on PERG, i.e., as an indicator of RGC function.Ventura et al.  probed tAnother recent study  utilizedAs evident from the above, both OCT and electrophysiology bear great potential for improved management of glaucoma. Assessing overlap and complementarities of both approaches will help to identify rewarding targets of combined applications of both techniques with the aim to establish reliable endpoint biomarkers along the disease continuum, particularly for glaucoma conversion and advanced glaucoma. This might offer a better understanding and quantification of glaucomatous damage with respect to the functional and structural relationship. This will in turn optimize glaucoma detection by tailoring tests according to the stage of the disease. In order to identify these targets, we firstly evaluate relevant application fields for commonalities and subsequently review recent studies of combined OCT and electrophysiological research in glaucoma.At the earliest stages of glaucoma, functional damage might not be detectable on SAP, albeit being the most commonly used to monitor glaucoma progression, due to its subjective nature and variability of results . TherefoThe PERG is a well-established and sensitive biomarker of RGC function , 64 emplOCT has been widely utilized to diagnose glaucoma , 72 and We conclude that functional and structural assessment through ERG measures and OCT technologies provide complementary information that might, in combination, optimize the early detection of glaucoma.Following progression in advanced glaucoma is quite challenging due to the variability of VF defects. To reduce test-retest VF variability in advanced damage, some , 78 receThere is a growing body of evidence that pRNFL thinning is not observable anymore in advanced glaucomatous cases, again a floor effect , 83. It In a recent review of longitudinal studies in glaucoma progression , it was Electrophysiological approaches in glaucoma research and diagnostics benefit from the combination with other techniques. Given the considerable variability of structural and functional tests , multimoTraditionally, glaucoma was thought to affect the peripheral VF; however, Bach et al. , 90 inveYears later, a substantial body of literature established early involvement of and even initial defects in the macula in glaucoma , 93. HooAs a consequence of the prominent developments in OCT technology, OCT might be of considerable help for the characterization not only of structural retinal changes in glaucoma  but alsoRGC damage in glaucoma might be attributed to elevated IOP, exerting mechanical stress on the optic nerve head, i.e., “mechanical theory” , 97. HowTo elucidate these pathomechanisms, ERG measures of vision function might play a pivotal role. In a study combining PhNR of ERG with OCT-A in NTG , macularElectrophysiology functional measures of vision provide an integral pivotal role in glaucoma management along with state-of-the-art ophthalmology technologies, i.e., optical coherence tomography/angiography as evidenced by the detection of early glaucoma and elucidations of damage mechanisms. Future directions employing longitudinally designed studies might corroborate the utility of multimodal approaches in the current standards of glaucomatous care for the sake of early implementation of therapy and prevention of blindness. The translation of multimodal approaches into practice might potentially add a major diagnostic dimension to the currently established tests in glaucoma as well as provide insights to several unanswered inquiries about glaucoma pathogenesis.