First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Li Wang is first author on ‘ Li WangHow would you explain the main findings of your paper to non-scientific family and friends?“Our research highlights the importance of STAT3 as a central mediator of vascular permeability.”Proper vascular function is essential for maintaining good health. Our body's vascular system distributes oxygenated blood to all tissues, returns deoxygenated blood to the lungs, and helps maintain balanced function among blood cells, lipids and immune cells. During tissue injury or disease, vascular barrier integrity is impaired. A protein called vascular endothelial growth factor (VEGF) is a central mediator of vascular permeability. Increased VEGF expression promotes hyperpermeability, oedema and tissue damage, which contribute to the pathogenesis of cardiovascular disease, cerebrovascular conditions, cancer, retinal disorders and acute lung injury. However, VEGF also plays a role in tissue repair following damage by stimulating the growth of new blood vessels. Our goal is to discover molecular signalling that can be targeted to reduce the tissue-damaging effects of vascular permeability, while not affecting the tissue-repair role of VEGF. In our study, we demonstrate that VEGF signals through a mediator called signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. In mice engineered for STAT3 deletion in their vascular  cells, we observe reduced VEGF-induced vascular permeability. Similarly, in Stat3 knockout zebrafish, vascular barrier integrity is maintained despite ‘turning on’ VEGF expression. Importantly, the process of blood vessel formation stimulated by VEGF is unaffected in these animals, which suggests that the function of VEGF in tissue repair is maintained upon STAT3 inhibition. STAT3 can be effectively inhibited using a drug called pyrimethamine, which has been US Food and Drug Administration (FDA) approved to treat parasite diseases. We show that the STAT3 inhibitor pyrimethamine substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. STAT3 could be used as a target to reduce the pathological effects of vascular permeability in human diseases, including vascular conditions, cancer, eye disorders and COVID-19-associated acute lung injury.What are the potential implications of these results for your field of research?Our research highlights the importance of STAT3 as a central mediator of vascular permeability. We demonstrate that an FDA-approved STAT3 inhibitor called pyrimethamine inhibits VEGF-mediated vascular permeability, while not affecting the tissue restorative functions of VEGF. These findings suggest that targeting STAT3 may be an effective strategy to inhibit vascular permeability, oedema, and tissue damage associated with heart disease, stroke, retinal conditions, cancer and acute lung injury.What are the main advantages and drawbacks of the model system you have used as it relates to the disease you are investigating?in vivo models are quite useful for answering many important questions related to the molecular regulation of vascular permeability.In our study, we took advantage of a transgenic VEGF-inducible zebrafish model that is amenable to genetic manipulation and reproducible live imaging of vascular permeability in optically clear zebrafish embryos using fluorescently labelled tracers. This model can be used to identify novel regulators of VEGF-induced vascular permeability or to test the effects of candidate drugs on vascular barrier integrity. Additionally, the widespread recent emergence of CRISPR/Cas9 genome-editing techniques has enhanced the study of genetic regulators of endothelial barrier function. As such, we crossed CRISPR/Cas9-generated Stat3 loss-of-function mutant zebrafish to the VEGF-inducible fish to study the role of STAT3 in VEGF-mediated vascular permeability. We also used endothelium tissue-specific STAT3 knockout mice, which are healthy and fertile with no overt defects in vascular development or structure. These genetic STAT3 knockout animal models could be used in other studies related to STAT3 in other diseases. One drawback of using these model systems is the time and effort required to inject tracers into the zebrafish ventricle through the pericardium and deliver Evans Blue dye into the mice by tail vein injection. However, once an individual has mastered the injection techniques, these What has surprised you the most while conducting your research?Pyrimethamine is an FDA-approved drug used to treat microbial infections. Through a chemical-biology screen, our collaborator, Dr David Frank, found that pyrimethamine is an inhibitor of STAT3 function at concentrations known to be achieved safely in humans. Dr Frank and colleagues reported that the STAT3 inhibitor pyrimethamine displays anticancer and immune-stimulatory effects in murine models of breast cancer. As highlighted in our article, we discovered that the STAT3 inhibitor pyrimethamine reduces VEGF-induced vascular permeability in mice, zebrafish and several types of human endothelial cells. I was most surprised by the power of drug repurposing – using an ‘old’ drug to treat a different disease. I am excited that pyrimethamine is already FDA approved and can be used safely in humans, which will help translate our work into the clinic.Describe what you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years?“[…] the advent of the preprint era has benefited early-career scientists.”Discovering the molecular mechanisms underlying human disease is the first step. The next challenge is how to translate these basic science discoveries to clinical use to improve the lives of people afflicted with some of these devastating diseases. Over the next 10 years, perhaps it will become easier to synthesise drugs based on basic science findings. Pipelines to test candidate drugs in disease models will likely become more efficient and more effective. Hopefully these technical advancements will facilitate the development of new treatments and, ultimately, cures.What changes do you think could improve the professional lives of early-career scientists?At The Hormel Institute, University of Minnesota, many activities have been organised to improve the professional lives of early-career scientists, like external and internal seminars, career development opportunities, and the implementation of mentoring committees for postdoctoral fellows. My mentor, Dr Luke Hoeppner, also provided outstanding mentoring support throughout the whole project and has encouraged goal setting to facilitate my career growth. For most trainees, more time reading papers and more grant opportunities are desired. In regards to grant applications, I like that we can now cite preprints in proposals, and I think the advent of the preprint era has benefited early-career scientists.What's next for you?In our publication, we report that suppression of STAT3 activity inhibits vascular permeability mediated by VEGF in mice, zebrafish and human endothelium. Given that the STAT3 inhibitor pyrimethamine has already proven safe in humans, we hope to translate our findings to the clinic. To that end, we are currently testing pharmacological STAT3 inhibition in animal models of human diseases associated with vascular permeability. In terms of my own career progress, I am beginning to focus on postdoctoral grant-writing opportunities.


MUS81 complex, exhibiting endonuclease activity on specific DNA structures, plays an influential part in DNA repair. Research has proved that MUS81 is dispensable for embryonic development and cell viability in mammals. However, an intricate picture has emerged from studies in which discrepant gene mutations completely alter the role of MUS81 in human cancers. Here, we review the recent understanding of how MUS81 functions in tumors with distinct genetic backgrounds and discuss the potential therapeutic strategies targeting MUS81 in cancer. In recent findings, it is becoming increasingly evident that MUS81 has close relationships with cancers. Interestingly, MUS81 is helpful for tumor survival in some cases but lethal to them in other cases. For instance, endonuclease activity of MUS81 is crucial for survival of BRCA2-insufficient cancer cells , and D-loops  heterodimer of the MUS81–EME1 complex. The disordered loop of the EME1 linker becomes ordered, which subsequently unmasks the hydrophobic wedge and forms the 5′ end binding pocket of MUS81  combined with 3′ flaps demonstrates the recognition and cleavage mechanism of MUS81 complex  are difficult-to-replicate foci that preferentially form breaks in chromosomes under replication stress and frequently rearrange in tumor cells  (Kim et al., In MSI cells, TA repeats, which are highly unstable, encounter and accumulate large-scale expansions for some reason (van Wietmarschen et al., in vitro and have extremely low sequencing depth (van Wietmarschen et al., A question, which remains to be answered, will be of considerable interest. Why does MUS81 cleavage in TA repeats lead to chromosome shattering rather than triggering POLD3-dependent DNA synthesis for DNA repair? It has been found that these TA repeats cannot be amplified DDR pathways play crucial roles in genomic stability, and compromised DDR pathways are common in multiple tumors. Recent studies have unveiled the link between DDR deficiency and innate/adaptive immunity against tumor cells Barber, . Cyclic DDR-directed therapies have been introduced in clinical trials recently (Pilié et al., As this review demonstrates, human MUS81 is closely related to cancers but results in totally different fates of cancers bearing distinct mutated genes, indicating that the interactions between MUS81 and other DDR proteins master the cancer cell fate and MUS81 is a potential and intriguing target in specific cancer therapies. A further understanding of the precise mechanisms of MUS81 and the interactions among DDR pathways in discrepant cancers may pave the way toward improved cancer therapeutic strategies.SC wrote the manuscript. XG, MS, ZH, CZ, and SY provided guidance and supervised the final version of the manuscript. 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.


Traumatic brain injury is one of the leading causes of mortality and morbidity in the world with no current pharmacological treatment. The role of BDNF in neural repair and regeneration is well established and has also been the focus of TBI research. Here, we review experimental animal models assessing BDNF expression following injury as well as clinical studies in humans including the role of BDNF polymorphism in TBI. There is a large heterogeneity in experimental setups and hence the results with different regional and temporal changes in BDNF expression. Several studies have also assessed different interventions to affect the BDNF expression following injury. Clinical studies highlight the importance of BDNF polymorphism in the outcome and indicate a protective role of BDNF polymorphism following injury. Considering the possibility of affecting the BDNF pathway with available substances, we discuss future studies using transgenic mice as well as iPSC in order to understand the underlying mechanism of BDNF polymorphism in TBI and develop a possible pharmacological treatment. Traumatic brain injury (TBI) is the leading cause of death worldwide, and in young adults under 35 years of age, the death rate is 3.5 times that of cancer and heart disease combined . TBI is Neurotrophins, in particular brain derived neurotrophic factor (BDNF), have been identified to play a prominent role in the cellular events that occur in restorative processes following TBI, such as neuronal survival, axonal sprouting, and synaptogenesis . Due to BDNF is the most abundant neurotrophin in the brain ,5, playiThe BDNF single nucleotide polymorphism (SNP) in rs6265 produces a missense mutation, Val66Met (196G/A), which causes deficits in the activity-dependent secretion of BDNF . The ValThe most studied polymorphism of BDNF in humans is rs6265 , in relation to normal cognitive function, effects on psychiatric, neurodegenerative and neuroinflammatory disease, and traumatic brain injury ,13,14,15Among healthy individuals (mean age 36 y), carriers of the met-allele had worse episodic memory measured with n-back working memory test, as well as abnormal hippocampal activity assessed by functional MRI (fMRI), compared to Met- . SimilarThe effect of BDNFval66met polymorphism on the early cognitive outcome after mild TBI (mTBI) have been assessed in two different studies and shown a worse result for met-carriers. At a subacute follow up (one month) after mTBI, both the Met+ controls and the Met+ TBI-group had a slower reaction time in both simple reaction time (SRTRT—simple reaction time reaction time) and information processing speed (measured with Gordon Continuous Processing Test—CPT) compared to Met-patients and controls . This coNevertheless, in the follow up of military veterans after mTBI in the chronic phase (mean 4.5 years), evaluating memory and executive function, the Met+ group with TBI had better results than their corresponding control group, whereas the Met- had the opposite result . This coThe risk for neurodegeneration after TBI has been discussed and evaluated in different studies . Severe In a study investigating the effect of headings in soccer players during six months on remyelination assessed by diffusion tensor imaging (DTI), it was shown that the met-carriers had significantly less remyelination compared to val-homozygot players . This maCognitive outcome after TBI is multifactorial. Age and gender affect the outcome ,19,33 anThe gender difference related to depressive and anxiety related symptoms was further addressed in a study that followed those symptoms in a group of patients one and six weeks post mTBI . This stOnly one study could be identified that investigated the effects of BDNFval66met polymorphism on early recovery after severe TBI . BagnatoThere are a few studies on long term outcome following severe, penetrating brain injury in American Vietnam veterans were correlation of BDNF polymorphism and cognitive outcome has been studied ,20,40. TThree studies have used a gene risk score based on “risk” BDNF SNPs (rs6265 and rs 7124442) ,19,33, aThree studies could be identified where BDNF levels were measured in plasma and/or CSF. Simon et al. analyzed the levels of BDNF in plasma at admission to the ICU, at mean 6.4 h post arrival to the hospital following severe TBI in 120 males  3-8). There was no correlation between BDNF plasma levels and short-term fatal outcome  or between isolated brain injury versus multitrauma . In 12 cThe studies of the effect of BDNF val66met polymorphism vary in outcome measure, timing, and type of brain injury. Generally, the studies are small and most study population are smaller than 200 individuals. As a consequence, prospective studies are scarce and the results are heterogenous. The met/met prevalence in Caucasian population is low and therefore most studies group met-heterozygote and met-homozygote together for analysis.In this section, we summarize the current knowledge of BDNF expression after traumatic brain injury (TBI) in different animal models, as well as the correlation between novel TBI treatment options and BDNF expression, and their effects on behavioral outcomes. In the section evaluating BDNF expression after TBI compared to sham, we chose to exclude studies which did not clearly state the location or time of BDNF analysis as well as those which did not report the data from sham animals.More than ten different trauma models have been used to study BDNF expression after TBI  and the In the reviewed material, the analyzed anatomical regions varied greatly. In order to compare and examine trends in the published data the anatomical regions were grouped together . StudiesBoth the mRNA and protein expression of BDNF were assessed. The most common method for mRNA analysis were in situ hybridization and quantitative real-time polymerase chain reaction (qPCR). For BDNF protein quantification Western blot and enzyme linked immunosorbent assay (ELISA) were commonly used . In mostBDNF mRNA expression in the ipsilateral hippocampus was examined at 16 different timepoints and BDNF protein expression in the ipsilateral hippocampus was examined at 23 timepoints 2 h- 56 d) a. Three  d a. ThrHippocampus contralateral to the injury was analyzed at six timepoints for BDNF mRNA expression and three for BDNF protein expression, ranging from 1 h to 56 days. Rostami et al. report that BDNF mRNA expression was increased at 1, 3, and 14 days after trauma in the contralateral hippocampus, but not at the chronic phase measured at 56 days post-injury . In the The ipsilateral cortex displays a similar pattern of expression as the ipsilateral hippocampus. During the first day post injury BDNF mRNA expression was exclusively reported as increased ,70. KoboOnce again, the contralateral side is not as well examined. Corne et al. examined the cortical mRNA-expression in the contralateral parietal lobe at 21 days post-injury and reported a decrease of all BDNF exons in the TBI group compared to sham , while YTo summarize, several studies found that BDNF mRNA expression increases in the ipsilateral hippocampus and cortex in the first 1–2 days after injury and that BDNF mRNA increases in the contralateral hippocampus in days 1–14 after injury. This increase in BDNF mRNA is not accompanied by a clear increase in BDNF protein in neither cortex nor hippocampi, however the data remains insufficient to draw definitive conclusions.Several studies examining the correlation between BDNF-expression and TBI performed behavioral tests to evaluate functional outcomes, as seen in A total of 73 studies used various interventions to examine their effect on BDNF-expression and functional outcome following TBI, as seen in HSP20 gene, a gene expressing the heat shock protein 20 which is a chaperone previously known to be expressed after tissue stress and to be protective in e.g., Alzheimer’s disease. They found that silencing of the HSP20 gene canceled the exercise induced enhancement. Three studies that examined exercise found a correlation between improved BDNF protein expression (and one study found that exercise increased BDNF mRNA expression) and improved cognitive function in the Morris Water Maze test [Exercise as a therapeutic method in traumatic brain injury was examined in 14 studies. Exactly what constituted exercise differed between the studies, but the most commonly used method was the running wheel. Some of the studies differed between voluntary and involuntary exercise, and some articles did not properly define exactly what type of exercise was performed. Out of those 14, 11 studies found that either pre- or posttraumatic exercise increased cerebral BDNF protein expression in the ipsilateral and contralateral hippocampus as well as in the ipsilateral cortex, as compared to non-exercised animals ,91,92,93aze test ,67,68,71Da Silva Fiorin et al. and Zhao et al. were the only ones that examined the effects of pre-traumatic exercise. da Silva Fiorin et al. found that while FPI alone had no significant effect on BDNF protein expression, previous physical exercise induced an increase in BDNF protein expression in the hippocampus at 1 and 14 DPI. Zhao et al. found that running wheel exercise for 4 weeks before trauma increased BDNF mRNA expression in the ipsilateral cortex of both sham and injured animals compared to non-exercised animals. They also found that pre-traumatic exercise improves motor function in the beam walking test and cognitive function in Morris water maze and novel object recognition tests.Griesbach et al, utilized a voluntary wheel exercise and examined functional outcome after lateral fluid percussion and found no significant effect on gross motor impairment. Shen et al. examined different intensity training models and examined functional outcome after severe controlled cortical impact and found that while there was no significant difference between trauma groups in the Neurologic Deficit Scores, low intensity exercised animals performed better in the MWM compared to control and high intensity exercised animals. Griesbach et al. utilized a lateral fluid percussion injury model with voluntary wheel exercise and found no significant effect of exercise on BDNF expression nor the result of beam walk test ,68,78. HA total of 15 studies examined different dietary treatment effects on traumatic brain injury in animal models. This included treatment with Astaxanthin  , blueberChandrasekar et al. examined the effect of acute ethanol intoxication in conjunction with trauma and found that trauma increased BDNF mRNA in the hippocampi bilaterally at 1 and 3 h after trauma compared to sham, and that the TBI-induced upregulation of BDNF was markedly decreased by ethanol pretreatment .Ren et al. examined Resolvin, a docosahexaenoic (DHA) essential n-3 fatty acid derivate. The study examined BDNF protein expression in the hippocampus at 7 DPI and also found that TBI induced BDNF protein expression, and that Resolvin D1 further increased BDNF expression and ameliorated the cognitive effects of TBI in fear conditioning and beam walking tests .Agrawal et al. found that FPI reduced the BDNF protein expression in the frontal cortex at 7 DPI specifically in animals exposed to n−3 fatty acid deficiency, but that n−3 fatty acid pretreatment prevented this. They also showed that n-3 fatty acid treated groups spent more time in the open arms of the elevated plus maze, indicated decreased anxiety . Ji et a6 and 8 × 106) significantly increased BDNF-protein levels compared to low dose (2 × 106) and vehicle. They also found that the high and intermediate dose (4 × 106 and 8 × 106) improved NSS compared to low and vehicle treated groups. Finally, they found a dose-dependent increase in perilesional GFAP expression [In the reviewed material, 9 studies examined stem cell treatments and the effect on BDNF expression after TBI. Mahmood et al. examined intravenous treatment with marrow stromal cells labeled with bromodeoxyuridine (BrdU). They found increased BrdU-positive cells in the perilesional regions, indicating the migration of marrow stromal cells (MSCs). Furthermore, they found that MSC treatment significantly increased BDNF at 8 DPI but not 2 or 5 DPI compared to vehicle. Finally, they found that the MSC-treated group had improved scores in mNSS and rotarod compared to control groups . Mahmoodpression . Feng etpression . Deng etpression . Kim et pression . Qi et apression . Wang etpression . Cheng epression . Xiong epression .In summary, all the reviewed studies examining stem cell treatment found that several types of stem cells increased BDNF expression. Five out of nine studies did not include a sham group separate from vehicle or trauma. Regarding functional outcomes, the two studies examining umbilical cord stem cell transplantation together with the study examining Wharton’s Jelly transplantation found improved neurological severity scores in treatment groups compared to vehicle ,104,105.The number of studies examining direct intervention of the BDNF pathway in TBI are few, and this review included four studies. Sen et al. found that TBI decreased BDNF protein expression in the ipsilateral cortex 21 days following injury. Furthermore, they examined the Protein kinase-like endoplasmic reticulum kinase (PERK), a kinase in the endoplasmic reticulum activated by stress such as TBI, which mediates the downstream inhibition of translation. Previous studies have found that phosphorylation of PERK leads to an increased activation of CREB and thus the downregulation of BDNF. They found that a PERK antagonist GSK2656157 increased BDNF expression and improved cognitive performance in the Morris Water Maze test. This indicates that the inhibition of this pathway increased BDNF protein expression which could contribute to the improved performance in the MWM . Alders BDNF have a short half-life and low blood brain barrier permeability, and one group used nanoparticles coated by surfactant, poloxamer 188 (PX), to increase BDNF concentration in target areas. They found that TBI increased BDNF protein expression in ipsilateral and contralateral hemispheres 4 h after injury. Furthermore, BDNF expression was increased ipsilaterally in animals treated with BDNF together with nanoparticles with and without PX compared to vehicle and BDNF without nanoparticle treatment groups. Contralaterally BDNF expression was only increased in the group treated with BDNF together with the combination of nanoparticles and PX. In the functional evaluations, they found a spontaneous improvement of NSS in day 1 to 6, with no difference between groups. However, at day 7 there was a significant improvement of NSS in the group treated with BDNF together with both nanoparticles and PX group compared to the other treatment groups. In the Passive avoidance test, the sham group and group treated with BDNF together with both nanoparticle and PX outperformed the other groups which performed no better than the non-treated animals .Recently, the synthetic flavonoid 7,8-dihydroxyflavone  was discovered following a screening for small molecules that could selectively activate BDNF receptor TrkB. This means that 7,8-DHF may cause similar effects, as BDNF in the brain, and be more therapeutically useful due to its better absorption and ability to cross the blood–brain barrier. The 7,8-DHF has shown an ability to promote the growth of these dendrites into synapses to help restore communication between neurons in animal models of cognitive decline.In an experimental TBI model administration of 7,8-DHF prior to injury reduced cell death of neurons in the hippocampus. Reduced cell necrosis and apoptosis could also be seen upon administration of  after simulated TBI in adult mice . RecentlAdditionally, a selective antagonist of p75NTR, EVT901, was recently identified . EVT901 These two newly discovered drugs showed no harmful effect in animal models and, offer a promising opportunity for complementary pharmacological treatment of TBI.BDNF expression in transgenic animals after TBI is a novel field, and we have included a total of three studies. The results of these studies naturally vary from non-transgenic animals and because of this the results and methods of these studies have not been included in the previous graphs.Giarratana et al. examined Val66Met-transgenic mice (Met+) and utilized a repeated mild TBI model using a lateral fluid percussion injury model. Giarratana et al. found that the total BDNF protein was decreased in Met+ injured animals in the ipsilateral cortex at 21 DPI, but that pro/mature-BDNF protein was increased in the ipsilateral hippocampus at 1 DPI compared to Met-. Furthermore, they found that Met+-animals had larger volume of inflammation compared to Val66Val at 21 DPI and that Met+animals had an increased activation of microglia in both hippocampal and cortical tissues at both 1 and 21 DPI. Met+ also have increased activation of Caspase-3+ cells (a marker for apoptosis) compared to Met- at 1 DPI, and have increased levels of FluorojadeC+ cells (a marker for neurodegeneration) compared to Met- at 1 and 21 DPI. Finally, they also found that Val66Met-injured animals had an increased number of phosphorylated tau+ cells (a marker for neurodegenerative pathology) compared to Met- at 1 and 21 DPI, and an increased number of GFAP+ cells in the ipsilateral cortex in Met+ compared to Met- at 21 DPI, but not 1 DPI, indicating increased astrocyte activation and risk of glial scarring .Gao et al. utilized a cre/flox conditional knockout (KO) of BDNF which enables a site-specific knockout of BDNF in the granular neurons of the dentate gyrus of the hippocampus. In the flox/flox control animals, they found that TBI increased BDNF protein expression in the hippocampus. In the conditional KO animals, they found significantly decreased BDNF levels in the dentate gyrus in sham treated animals, and that TBI increased the levels of BDNF protein in the dentate gyrus of KO mice to a lesser extent that the increase of flox/flox control animals. Furthermore, they found significantly increased number of FJB+-cells in KO animals compared to injured flox/flox control animals, which indicates that the conditional knockout of BDNF leads to increased cell death in the dentate gyrus after trauma. Moreover, they showed that TBI injury significantly induces newborn neuron death 24 h following moderate TBI injury, and that the BDNF conditional KO further increases newborn neuron death in the dentate gyrus .TSP-1 KO BDNF increased only in the ipsilateral cortex and not in the contralateral cortex. This might hint at a TSP-1 gene depletion-associated resistance of BDNF. Moreover, they found that measurement of synaptophysin (a marker for synapse quantification) showed no difference between KO and WT groups before TBI, but that TBI similarly significantly decreased synaptophysin in the contralateral cortex compared to sham and WT. There was no significant difference in synaptophysin expression in the ipsilateral cortex between groups. Furthermore, TBI increased extravasation in the ipsilateral hemisphere, which was significantly exasperated in TSP-1 KO mice compared to WT. In the functional tests, TSP-1 KO significantly worsened performance in NSS compared to WT post-TBI, indicating worse motor-sensor response. Wire grip and corner test was not significantly different in KO and WT groups and returned to normal at 10 DPI. In the MWM, TSP-1 KO mice had increased latency to find the platform compared to WT, but no significant difference in entry times or target quadrant. TSP-1 KO might worsen spatial memory recovery after TBI [Cheng et al. studied thrombospondin-1 (TSP-1) KO animals after controlled cortical injury. TSP-1 is an extracellular matrix protein secreted by astrocytes in the brain and has been linked to several cerebral pathologies. Chang et al. found that in wild type (WT) animals, TSP-1 increased in the ipsilateral cortex at 6 h to 3 days, then returned to normal levels. Examining the relationship with BDNF expression, they found that TBI increased BDNF protein expression in both the contra- and ipsilateral cortex in WT at 21 days after trauma. However, in fter TBI .The reviewed material is very heterogenous regarding the examined brain regions, temporal analysis of BDNF-expression after injury, type of trauma model, and functional tests, as well as whether a sham group was presented or reported. There is an urgent need for the standardization of experimental design in order to provide more reproducible results and solid conclusions. Nevertheless, there is an overall pattern of transiently increased BDNF-mRNA expression in the first day after trauma in the ipsilateral hippocampus followed by an ipsilateral decrease and a contralateral increase. Similarly, in the ipsilateral cortex, BDNF-mRNA increased in the first day after trauma, followed by a tendency of decreased expression.Regarding the human studies, there is a similar need for standardization and larger cohorts. Generally, the studies are small, most have a study population <200 individuals and a control group has not always been used. Outcome measures differ among the studies, especially when evaluating cognitive function. Additionally, the time point for follow up varies between the studies and the access of prospective studies are scarce. The met/met prevalence in Caucasian population is low and therefore most studies group met-heterozygote and homozygote together for analysis which begs the question if the functional effect is the same and whether the met+ result in a lower baseline of cognitive function but offer a protective quality of cognition post TBI.As previously described, TBI is a heterogenous and complex condition involving multiple CNS cell types. Cellular interactions and subcellular processes follow temporal and spatial patterns that vary between affected individuals, and between different injuries. Accordingly, experimental TBI is usually studied using in vivo models, typically rodents, recapitulating many of the aforementioned features. However, certain aspects of TBI, such as the contribution of cell autonomous versus non-cell autonomous factors may also be studied in vitro, allowing for more mechanistic studies of isolated processes. In addition, a disadvantage with the current in vivo models is possible differences between human and animal cells regarding gene and protein expression or response to pharmacological interventions. These differences may underly some of the difficulties in translating results from basic research to clinical applications.In line with the literature, we propose that in vitro models using neuronal cell types differentiated from induced pluripotent stem cells (iPSCs) from human subjects could be used as a complementary model, e.g., for studying cell to cell interactions, diffuse axonal injury (DAI), neuroinflammation, and the screening of neuroprotective drugs ,114,115.More advanced models include the use of brain organoids derived from iPSCs, which better resemble the three-dimensional environment in the brain and allow for more complex analyses ,117, butBDNF val66met polymorphisms could give clues about how this genetic variation influences, e.g., secretion and signaling of BDNF, synaptic plasticity and neuronal and glial response to injury. Moreover, such a model would be well-suited for pharmacodynamic and pharmacokinetic studies of the effects of the two potential neuroprotective compounds 7,8-DHF and EVT901.We propose that studies of iPSC-derived neurons and glia from TBI patients with However, since the val66met polymorphism has been linked to various neurodevelopmental and neurodegenerative disorders ,119, we It should be considered that the monumental leap from patient to cells in a dish may result in subtle, artefactual or clinically irrelevant phenotypes . TherefoMoreover, it should be considered that single nucleotide polymorphisms (SNP) often result in subtle and multifactorial phenotypes, which may involve “multiple hits” in patients. Certain phenotypes associated with SNPs may therefore not manifest in vitro.Since iPS cells derived from humans are genetically heterogenous, phenotypic differences between a patient line and a control line may be due to other factors than the ones intended to study. One approach to overcome this problem could be to use multiple control lines, but as proof-of-concept, one would modify the genetic variation in the patient line of interest using targeted gene correction in order to create an isogenic control line.BDNF gene affects neuronal and glial function, and to evaluate new drug candidates, but should be used wisely in order to generate clinically relevant result.In summary, iPSC based TBI models could be useful in the studies of how genetic variations in the Regarding the treatment of traumatic brain injury, several of the studies showed promising results and there is evidence of a positive correlation between increased BDNF expression and improved functional outcome, at least in animal studies. This is made especially clear in the cases where the positive effects of the treatment were cancelled by a BDNF antagonist, but unfortunately this was not often utilized in the reviewed material.Traumatic brain injury is a global health issue with potentially devastating life-long consequences for the individual patient. Both injury and rehabilitation are very complex, and more research is necessary in order to understand the pathological mechanisms, and to provide novel treatment options of the primary injury. The treatment of the BDNF pathway could provide a novel treatment option in improving functional outcomes. Although treatment potential with the BDNF-molecule itself is limited because of low permeability of the blood brain barrier and a short half-life, an option could be TrkB-agonist treatment, such as 7, 8-dihydroxyflavone.


Methyl-CpG-binding protein 2 (MeCP2) is a transcriptional regulator that is highly abundant in the brain. It binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. MeCP2 has mainly been studied for its role in neurodevelopmental disorders, but alterations in MeCP2 are also present in stress-related disorders such as major depression. Impairments in both stress regulation and synaptic plasticity are associated with depression, but the specific mechanisms underlying these changes have not been identified. Here, we review the interplay between stress, synaptic plasticity, and MeCP2. We focus our attention on the transcriptional regulation of important neuronal plasticity genes such as BDNF and reelin (RELN). Moreover, we provide evidence from recent studies showing a link between chronic stress-induced depressive symptoms and dysregulation of MeCP2 expression, underscoring the role of this protein in stress-related pathology. We conclude that MeCP2 is a promising target for the development of novel, more efficacious therapeutics for the treatment of stress-related disorders such as depression. In the eukaryote cell, genomic DNA is organized as a nucleoprotein complex in which approximately 200 bp of DNA wraps in two left-handed superhelical turns about a heterotypic histone octamer core comprising two H2A, H2B, H3, and H4 core histones. These repetitive discrete nucleoprotein subunits connect to each other by linker DNA domains (ranging from 20–60 bp), to which linker histones histone H1) are bound. These complexes are referred to as nucleosomes  are boun also binThe notion that postsynthetic chemical modifications of histones and DNA could alter gene expression arose as early as 1964 with the seminal observation made by Vincent Allfrey about the potential involvement of histone acetylation and methylation post-translational modifications (PTMs) in the regulation of RNA synthesis . The speIn 1983, Feinberg and Vogelstein reported that DNA (gene) hypomethylation could distinguish several human cancers from their normal counterparts , indicatIn 2000, Strahl and Allis put forward the histone code hypothesis, according to which, PTMs  occur at discrete, well-defined residues, and their combinations generate a code that could be read by functional protein effectors (readers). This provides a plethora of markers (more than 16 histone PTMs have been described to date) and combinations of markers with enormous potential for gene regulation that surpasses by orders of magnitude the 5 mC DNA potential. Moreover, although histone PTMs have a highly pervasive distribution in eukaryotes, the extent and function of DNA methylation vary extensively among different organisms within this group ,22.In 2005, Esteller’s group clearly showed that lifetime differences between monozygotic human twins are the result of epigenetic changes in DNA methylation and histone acetylation that affect the landscape of their gene expression. It was subsequently shown that such epigenetic molecular drivers could have long-term functional implications. For example, it is now clear that the quality of maternal postnatal care has specific epigenetic effects  on gene expression within the brain hippocampal region in rats, leading to lasting functional changes in learning, memory, anxiety, and stress regulation .DNA methylation and histone PTMs constitute the main epigenetic markers that are established by a dynamic set of highly specific enzymes (writers) with downstream effects elicited by equally specific individual proteins and/or protein complexes (readers), and they can be removed by enzymatic complexes (erasers) ,25.The brain has a set of “exceptional and unique epigenetic” players . At the A rather unique noncanonical DNA methylation component of the brain is the occurrence of mCH  particularly mCpA, which is present after birth . This DNMeCP2 has a 60% unstructured organization , and henThe levels of MeCP2 in the brain change during the circadian cycle , and alterations affecting this cycle can have a subtle influence on the regulation of gene expression. Considering this and the high abundance of MeCP2 in neurons, the important role of this protein in both normal and altered functions of the brain is not surprising .There is an epigenetic uniqueness that transcends DNA methylation and its readers. Quite recently, evidence has been presented for a new class of histone modifications, serotonylation of glutamine, at position 5 (Q5ser) on histone H3 tri-methylated at lysine 4 (H3K4me3), [H3K4me3Q5ser] . FurtherNoncoding RNAs, which can be classified as small  or long , have also been shown to contribute to depression ,50, and Stress is described as a state of threatened homeostasis to the body, created by exposure to a variety of different types of stressors. These can range from environmental factors  to homeostatic challenges . The ability to cope with stressors relies upon the activation of the hypothalamic–pituitary–adrenal (HPA) axis. The hypothalamus comprises many heterogeneous nuclei that control distinct physiological functions, among which is the paraventricular nucleus (PVN), a main HPA axis regulator . The PVNSex is an important determinant of human health, as evinced by the observation of sex-specific prevalence rates of various mental and physical disorders. There are also sex-specific patterns of HPA axis activation following perceived, or psychological, threats or stressors . FemalesStress activation in the PVN is also dependent on the nature of the stressor, and it involves a complex system of inhibitory and excitatory projections originating in multiple brain regions, thereby making it a highly plastic nucleus ,64. In nSynaptic plasticity is influenced by stress and impairments in synaptic plasticity mechanisms could contribute to stress-related pathology ,65.MeCP2 has been widely studied for its involvement in Rett syndrome (RTT), a neuronal disorder characterized by a loss of function resulting from mutations in the MeCP2 gene that result in neurodevelopmental and cognitive alterations . The assAs the research that can be performed on human postmortem brains is limited, animal models of RTT have been developed and used to further elucidate the role of MeCP2 in brain synaptic plasticity ,72. HippMeCP2 gain of function is associated with MeCP2 duplication syndrome (MDS), which causes severe mental retardation, stunted motor development, early-onset hypotonia, epileptic seizures, and progressive spasticity , and int+-Cl− cotransporter 2 expression, a target of MeCP2, and a critical transporter for regulating the polarity and efficacy of GABAergic neurons [Similar effects in synaptic plasticity have also been observed in vitro . For ex neurons .All these studies show that MeCP2 affects synaptic strength and plasticity, but the specific underlying mechanisms are not completely understood. Phosphorylation of MeCP2 at Serine 421(pS421) occurs upon neuronal depolarization and can lead to an activity-dependent gene transcription important for neuronal connectivity and synaptic plasticity in the nervous system. Indeed, results obtained in vitro and in vivo, using a highly specific antibody recognizing this phosphorylation site (pS421) and using transfections with plasmids expressing an S421A mutation, found that such mutation prevents MeCP2 phosphorylation, which, in turn, causes an impaired dendritic growth and spine maturation . It has Evidence regarding the role of MeCP2 in regulating proteins involved in neurotransmitter release also exists. For instance, MeCP2-mutated neurons derived from hiPSCs , neuronsIn summary, there is a wide body of evidence for the involvement of MeCP2 in brain synaptic plasticity, suggesting a bidirectional relationship between short-term plasticity and MeCP2 levels. MeCP2 levels could, either directly affect the calcium concentration in the presynaptic terminal or perhaps may alter proteins involved in neurotransmitter release. Moreover, it can influence postsynaptic plasticity by modulating GABAergic and glutamatergic transmission, leading to alterations in neuronal activity and connectivity. Such alterations could underlie the loss of motor and cognitive abilities and impaired social interactions seen in individuals with RTT, as well as the anxiety and depressive symptoms present in individuals with depression.Stress-related disorders such as depression share similar synaptic plasticity alterations to those observed in MeCP2 loss- or gain-of-function models . In thisEarly life stress (ELS) typically results in sustained HPA-axis deregulation and is a major risk factor for stress-related diseases, in particular depression. Interestingly, MeCP2 has been shown to contribute to ELS-dependent epigenetic programming of genes that enhance HPA-axis activity . For insSimilar alterations have been observed in other stress paradigms. For example, chronic unpredictable stress (CUS) in rodents causes depression-like behaviors that are accompanied by a decrease in MeCP2 and BDNF levels in the hippocampus, as well as an increase in miR-132, which is negatively correlated with MeCP2 and BDNF levels in blood samples from patients with depression . The samFurther evidence of MeCP2 involvement in stress-related pathologies such as depression is provided by the observation that the therapeutic effects of most antidepressant drugs rely on changes in MeCP2 levels. For example, MeCP2- phosphorylation at Ser421 was recently reported to be essential for the sustained, but not the rapid, antidepressant effects of ketamine and scopolamine in mice . MoreoveAlthough these observations in animal models are exciting, it is important to acknowledge that they may not translate to the human condition. There is almost no literature looking at alterations in MeCP2 levels in depressed patients. We have only been able to find two studies that assessed this: An early study examined blood samples of individuals suffering from depression and found a negative correlation between MeCP2 and miR-132 expression levels, which is a microRNA associated with depressive-like behavior . A more Altogether, these data indicate that MeCP2 may play a key role in early and adult life stress-induced epigenetic modifications that lead to the development of depression-like behaviors via MeCP2-dependent transcriptional regulation. This might provide more insight into the mechanisms of antidepressant drug action and even provide a new target for novel antidepressants. More clinical studies that focus on examining changes in MeCP2 and other markers of epigenetic change in patients with depression are badly needed.Reelin is a pleiotropic extracellular matrix protein that regulates neural migration and maturation, and dendritic sprouting during brain development, and is involved in neural plasticity in the adult brain ,128,129.The human RELN promoter can bind several transcription factors such as Sp1 and CREB and provides several susceptible methylation sites (CpGs) ,135,136.Human and animal studies of bipolar, autism spectrum disorder (ASD), and schizophrenia have revealed the presence of hypermethylated RELN promoters, which correlate with a decrease in reelin protein levels ,141,142.Finally, although we demonstrated that stress alters MeCP2 levels and that RELN expression is epigenetically regulated by MeCP2, most of the literature only focuses on the effects of MeCP2 in modulating BDNF. Even though BDNF can also modulate reelin receptor levels  Figure  and thusAltogether, in this study, we described the known functions of MeCP2 as an epigenetic regulator, and we provided evidence for its role in modulating synaptic plasticity via the transcriptional regulation of BDNF or other proteins involved in synaptogenesis and synaptic strength like reelin. We also revealed that environmental factors such as chronic stress cause the development of a depressive phenotype that can be reversed with common antidepressants and parallel with alterations in MeCP2 levels. Chronic stress is a potent risk factor for depression, and patients with depression show abnormal synaptic plasticity and reelin levels, which, according to the evidence gathered here, seem to be mediated, in part, by the epigenetic actions of MeCP2. Given the clear need for better treatment options for depression, as up to 50% of patients do not respond to current antidepressants, we believe additional studies of the role of MeCP2 in depression are necessary and may open new opportunities for the development of novel antidepressants.


Cancer has posed a major threat to human life and health with a rapidly increasing number of patients. The complexity and refractory of tumors have brought great challenges to tumor treatment. In recent years, nanomaterials and nanotechnology have attracted more attention and greatly improved the efficiency of tumor therapies and significantly prolonged the survival period, whether for traditional tumor treatment methods such as radiotherapy, or emerging methods, such as phototherapy and immunotherapy, sonodynamic therapy, chemodynamic therapy and RNA interference therapeutics. Various monotherapies have obtained positive results, while combination therapies are further proposed to prevent incomplete eradication and recurrence of tumors, strengthen tumor killing efficacy with minimal side effects. In view of the complementary promotion effects between different therapies, it is vital to utilize nanomaterials as the link between monotherapies to achieve synergistic performance. Further development of nanomaterials with efficient tumor-killing effect and better biosafety is more in line with the needs of clinical treatment. In a word, the development of nanomaterials provides a promising way for tumor treatment, and here we will review the emerging nanomaterials towards radiotherapy, phototherapy and immunotherapy, and summarized the developed nanocarriers applied for the tumor combination therapies in the past 5 years, besides, the advances of some other novel therapies such as sonodynamic therapy, chemodynamic therapy, and RNA interference therapeutics have also been mentioned. Cancer has severely threatened human life worldwide, and malignant tumors are still a serious problem that needs to be solved urgently . TraditiThe application of nanomaterials has further optimized traditional tumor therapies and provided more options for emerging tumor therapies such as phototherapy, immunotherapy, sonodynamic therapy, chemodynamic therapy and RNA interference (RNAi) therapeutics . The uniThe review is aim to outline the recent advances in nanomaterial-mediated radiotherapy, phototherapy, and immunotherapy, and some other therapies . Besides, we will present innovate strategies for combined tumor treatment based on nanomaterials in the past 5 years.Radiotherapy utilizes high-dose radiation to induce DNA destruction and generate free radicals to kill tumor cells, including internal and external radiotherapy, usually in conjunction with chemotherapy or surgery . However2W18O62)6– (W18) polyoxometalates through electrostatic adsorption to further enhance the sensitivity of radiotherapy (•OH), singlet oxygen (1O2) and superoxide (O2−), which is an excellent radio-enhancer for the treatment of MC38 and CT26 tumor bearing mice  have received extensive attention due to the good biocompatibility and surface modifiability. Studies have further enhanced its superiority in radiotherapy sensitization by changing the size, structure or surface properties . Dong et2 to decompose the over-expressed H2O2 into oxygen, thereby alleviating the tumor hypoxia and enhancing the sensitivity of radiotherapy  is stably expressed under hypoxic conditions, which has been shown to participate in the proliferation and metastasis of tumor cells by regulating its downstream signaling molecules . Based ootherapy . Meng etl tumors .Photothermal therapy and photodynamic therapy are currently the most common phototherapy for tumor treatment. Photothermal therapy is to gather materials with high light-to-heat conversion efficiency in the tumor tissue and heat it to ablate cancer cells under the irradiation of external light source . And phoin vitro, and showed excellent anti-tumor effect in vivo with no recurrence in 20 days -bismuth (Bi) Z-type heterostructure with a good electron-hole separation ability, simultaneously generated ROS and O2 under NIR irradiation, improved photodynamic therapy efficiency with excellent biocompatibility and biodegradability  to emit visible light under NIR excitation and stimulate photosensitizers to effectively produce ROS, overcome the limitation of weak tissue penetration of visible light, and improve the efficacy of photodynamic therapy . Liu et adiation . Liang eadiation .2@RP nanorods with red phosphorus (RP) as the shell and TiO2 as the core. After being irradiated with 808 nm NIR light for 5 min, the nanorods can reach 39.3–44.1°C and induce the production of 1O2, effectively killing cancer cells in deep tissues of renal cell carcinoma  or major histocompatibility complex class II (MHCII) molecules extracellularly . For tumentation . Suitablentation . The mosentation , 1,2-dioentation  and dimeentation , inducinentation . Recent entation . MOFs haentation . In anotentation .Although tumor vaccines have shown excellent anti-tumor effects, the immunosuppressive microenvironment presents a major challenge for tumor immunotherapy, and immune checkpoint inhibitors have been considered as a promising strategy in recent years . BlockinNanomaterials have exhibited great potential in tumor treatment and obtained remarkable results in both traditional and emerging therapies. However, there are still some limitations in monotherapy such as the inability to completely eliminate the tumor or the potential risk of tumor recurrence. The application of nanomaterials in combination therapy is emphasized to achieve synergistic effects for tumor treatment .18O49 nanosphere which showed excellent radiation sensitization and photothermal performance, promoted the production of 1O2 and ⋅OH, increased radiation-induced DNA damage and significantly inhibited tumor cell proliferation and metastasis , which can penetrate muscle tissue to achieve effective afterglow imaging in mice. Under X-ray irradiation, the nanoparticles emitted NIR long afterglow thus activating the photosensitizer to kill cancer cells and effectively inhibiting the growth of liver tumors in situ  nanoparticles, and modified the photosensitizer Ce6 to maximize the synergistic effect of phototherapy under 980 nm laser irradiation. Higher concentrations of cytokines such as IL-6 and TNF-α and lower level of IL-10 were induced, which proved the activation of a stronger immune response and the reduction of M2 suppressive macrophages, respectively. When combined with anti-PD-1 treatment, the survival rate of mice reached up to 77.8%, significantly higher than the group without PD-1 blockade antibody. Besides, the activation of CTLs and T memory cells can inhibit tumor metastasis and recurrence effectively . Jin et 2 is a traditional sonosensitizer and its modification has always been the focus of research  is an important gene expression regulation method, which is a gene silencing process induced by endogenous or artificially transfected small interfering double-stranded RNA . A majorr growth .Here we reviewed the progress of nanomaterials used in radiotherapy, phototherapy, immunotherapy and some other therapies such as sonodynamic therapy, chemodynamic therapy and RNAi therapeutics in the past 5 years, listed some creative designs of nanocarriers and summarized the major development direction of tumor combination therapies. The application of nanomaterials has been a promising strategy in both traditional and emerging therapies, and a variety of suitable nanomaterial-based carriers with specific properties have been gradually developed to effectively accumulate at the tumor site and overcome the adverse tumor microenvironment, which have shown great potential for improving the efficiency of tumor treatment. Recent studies have been focused on the development of the combination of multiple therapies in order to maximize the therapeutic effect. The combination of radiotherapy and phototherapy can effectively combat deep-seated tumors with minimal side effects, and the oxygen perfusion lead by photothermal therapy is conducive to oxygen-dependent therapies such as radiotherapy and photodynamic therapy. In addition, various therapies have been proved to activate the immune response to a certain extent in the process of tumor treatment. Therefore, combining immunotherapy can synergistically strengthen the therapeutic outcome and dramatically prevent tumor metastasis and recurrence.Despite satisfactory progress in the development and application of nanomaterials, some challenges remain to be addressed. The clinical translation of nanomaterials is the most critical issue for tumor treatment, so the materials should possess excellent biocompatibility and biodegradability, and FDA-approved materials ought to be prioritized for consideration. Similarly, the scale-up process from the laboratory to the clinic usually requires optimization or changes of the preparation method, so the efforts should be made to establish nanocarriers that facilitate the scale-up production while ensuring the effectiveness of tumor treatment. Besides, the toxicity and safety of nanomaterials are still pressing issues, and research should give more consideration to the long-term and potential toxicity of nanomaterials. In addition, it is of great importance to develop nanomaterials with specific physicochemical properties to meet the needs of different tumor therapies. Currently combination therapy is favored due to its synergistic anti-tumor efficacy, accordingly, nanomaterials with multiple functional properties are gradually developed to simplify the design of the carriers and achieve better therapeutic effect. It is vital to explore multifunctional nanomaterials to trigger multiple therapeutic effects in further research. The complexity of the tumor microenvironment presents a great challenge for the design of nanocarriers. With the continuous deepening of current research, the relationship between tumor therapies and its further mechanism has gradually become clear. According to the actual tumor conditions, suitable carriers can be designed to reverse unfavorable microenvironment to enhance the therapeutic outcome through monotherapies or combination therapies. The development of nanomaterials with suitable properties and biosafety to achieve remarkable therapeutic effect is the vital topic for tumor treatment in the future.


Metagenomics studies have shown that type 2 diabetes (T2D) is associated with an altered gut microbiota. Whereas different microbiota patterns have been observed in independent human cohorts, reduction of butyrate-producing bacteria has consistently been found in individuals with T2D, as well as in those with prediabetes. Butyrate is produced in the large intestine by microbial fermentations, particularly of dietary fiber, and serves as primary fuel for colonocytes. It also acts as histone deacetylase inhibitor and ligand to G-protein coupled receptors, affecting cellular signaling in target cells, such as enteroendocrine cells. Therefore, butyrate has become an attractive drug target for T2D, and treatment strategies have been devised to increase its intestinal levels, for example by supplementation of butyrate-producing bacteria and dietary fiber, or through fecal microbiota transplant (FMT). In this review, we provide an overview of current literature indicating that these strategies have yielded encouraging results and short-term benefits in humans, but long-term improvements of glycemic control have not been reported so far. Further studies are required to find effective approaches to restore butyrate-producing bacteria and butyrate levels in the human gut, and to investigate their impact on glucose regulation in T2D. Type 2 diabetes (T2D) is a global concern and is projected to affect 700 million people by 2045 . Althoug‘Gut microbiota’ is a term used to describe the trillions of microbes that live in the gastrointestinal tract. The gut microbiota has been identified as a virtual organ interacting locally with the gut and systemically with other organs in the host to facilitate multiple physiological processes . The intButyrate is one of the short chain fatty acids (SCFAs) produced as end-products of intestinal microbial fermentations , 27. ButRoseburia and Faecalibacterium prausnitzii; these findings were later extended to individuals with T2D in Indian  are also decreased in T2D and might play additional roles for the modulation of gut barrier function, inflammation and metabolism, as reviewed elsewhere , impaired glucose tolerance (IGT) or combined glucose intolerance (CGI) . Microbibut) is the primary enzyme, whereas the butyrate kinase (buk) is present in a few strains (4-hbt) and butyryl CoA:acetoacetate CoA transferase (ato)] ] , but the13C labelled acetate, propionate and butyrate in healthy subjects revealed that only 36% of the acetate, 9% of the propionate and 2% of the butyrate could be recovered in blood samples collected over different time points during the day  than in peripheral blood (79µmol/l) . However the day . Interes samples . These r samples , and thu samples .Salmonella and Escherichia species  inhibitor to regulate the expression of genes by epigenetic mechanisms. Supernatants from cultures of butyrate-producing bacteria from the human gut microbiota express HDAC inhibitory activity to class I and II HDACs . T2D is ro study . Consistro study  and lowero study . Finallyro study . These ain vitro studies mentioned here report different effects after stimulation with a mix of SCFAs or with individual SCFAs, indicating that the effects on EECs are not exclusively mediated by butyrate. Nevertheless, in human cell lines, butyrate selectively stimulates PYY secretion through mechanisms largely driven by HDAC inhibition (Butyrate (as well as the other SCFAs) also acts as a signaling molecule and is identified as a ligand for G-protein coupled receptors (GPCRs), such as FFAR3, FFAR2 and GPR109A. Human orthologs of FFAR2 have similar affinity for acetate and propionate followed by butyrate, while FFAR3 has higher affinity for propionate and butyrate compared to acetate . Niacin hibition .In addition to signaling in the gut, activation of SCFAs-binding receptors might be important also in the adipose tissue. Selective chemical agonism of GPR109A in individuals with T2D decreased fasting glucose, but not Hb1Ac, through inhibition of lipolysis in adipocytes as demonstrated by the decreased circulating levels of non-esterified fatty acids in the patients , 78. ConIn vitro experiments have showed that addition of butyrate in culture media reduced streptozotocin-induced islet cell death  may play important roles in metabolic control, particularly  tissues . Additio tissues Figure 2Restoration of the intestinal levels of butyrate might be a novel strategy for the treatment of T2D, that could also be added to conventional therapy with lifestyle management and glucose-lowering drugs. In recent years, a number of studies have attempted to replenish butyrate levels and butyrate-producers in the gut using different approaches as discussed below: direct supplementation of butyrate or butyrate-producing bacteria, dietary supplementation of fibers to feed microbial butyrate production, and fecal microbiota transplantation.Butyrate can be supplemented as sodium conjugate or as tributyrin (a triglyceride in which glycerol is esterified with three butyrate molecules). In mice with diet-induced obesity, oral supplementation of 400mg/kg of butyrate improved glucose tolerance and increased the expression of phosphorylated adenosine monophosphate kinase (AMPK) as well as glucose transporter-4 in the adipose tissue, and reversed some of the gut microbiota alterations caused by the high-fat diet (HFD) . In anotWith convincing results in mouse studies, butyrate supplementation was also tested in individuals with and without metabolic syndrome who were given 4g sodium butyrate in capsules for a period of 4 weeks . In thisLactobacillus probiotics have demonstrated some efficacy for hyperglycemia and insulin sensitivity in human cohorts  to individuals with insulin resistance improved insulin sensitivity only in individuals with a specific gut microbiota at baseline , with other gut bacteria (A. muciniphila and Bifidobacterium infantis) and inulin as fermentable fiber modestly increased butyrate levels and improved oral glucose tolerance and glycated hemoglobin levels in individuals with T2D  in feces and in the small intestine was observed in both studies, but increase in butyrate levels were observed only in one  and/or mediated by multiple undefined mechanisms besides butyrate production. FMT seems promising for the restoration of the gut microbiota and to improve insulin sensitivity, but it is impractical to perform such a highly invasive procedure in humans for short-term benefits. Future studies are required to gain a better understanding of the intestinal conditions that might influence butyrate production in individuals with T2D, in relation both to the diet and the individualized gut microbiota; for example, ingestible electronic capsules able to monitor microbial fermentations directly in the gut .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.


Excitatory amino acid transporters (EAATs) optimize the temporal resolution and energy demand of mammalian excitatory synapses by quickly removing glutamate from the synaptic cleft into surrounding neuronal and glial cells and ensuring low resting glutamate concentrations. In addition to secondary active glutamate transport, EAATs also function as anion channels. The channel function of these transporters is conserved in all homologs ranging from archaebacteria to mammals; however, its physiological roles are insufficiently understood. There are five human EAATs, which differ in their glutamate transport rates. Until recently the high-capacity transporters EAAT1, EAAT2, and EAAT3 were believed to conduct only negligible anion currents, with no obvious function in cell physiology. In contrast, the low-capacity glutamate transporters EAAT4 and EAAT5 are thought to regulate neuronal signaling as glutamate-gated channels. In recent years, new experimental approaches and novel animal models, together with the discovery of a human genetic disease caused by gain-of-function mutations in EAAT anion channels have enabled identification of the first physiological and pathophysiological roles of EAAT anion channels. Glial and neuronal excitatory amino acid transporters (EAATs) ensure low resting neurotransmitter concentrations in the synaptic cleft and prevent glutamate excitotoxicity by transporting glutamate from the synaptic cleft into neuronal and glial cells Danbolt, . EAATs aXenopus oocytes and electrophysiological analysis revealed the existence of a current component carried by anions that is not coupled to electrogenic glutamate transport  accessibility for GltPh mutants with a single tryptophan substitution  was experimentally verified using tryptophan-scanning mutagenesis and by combined Ph, captured via crosslinking of an inserted cysteine pair was obtained by cryo-electron microscopy  with a case of a human genetic disease  in glial cells are larger than expected from passive distribution  with the Cl−-sensitive dye MQAE. The experiments revealed a mean resting concentration of 35 mM in juvenile animals  carrying the disease-causing P290R mutation  causes glial apoptosis  led to complete loss of transport and channel function, accompanied by decreased protein expression. Only one of the tested mutations, threonine to alanine substitution at position 318 in hEAAT1 (T318A: RefSeq NM_004172.4: c.952A>G), increased anion conductance and L-glutamate uptake through increased membrane insertion by factors of 2.4 and 1.6, respectively ; this mutation prevents glutamate transport by impairing K+ binding  was also found postsynaptic to photoreceptors on dendrites of certain ON-bipolar cell types that receive a mixed input from rods and cones , serves important cellular functions.PK, ME, FM, and CF wrote the manuscript. PK and FM generated the figures. All authors approved the submitted version.This work was supported by the Deutsche Forschungsgemeinschaft  to CF (FA 301/13-1) as part of the Research Unit FOR 2795 (Synapses under stress) and by the German Ministry of Education and Research .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.


Pathological mutations in homology-directed repair (HDR) genes impact both future cancer risk and therapeutic options for patients. HDR is a high-fidelity DNA repair pathway for resolving DNA double-strand breaks throughout the genome. BRCA2 is an essential protein that mediates the loading of RAD51 onto resected DNA breaks, a key step in HDR. Germline mutations in BRCA2 are associated with an increased risk for breast, ovarian, prostate, and pancreatic cancer. Clinical findings of germline or somatic BRCA2 mutations in tumors suggest treatment with platinum agents or PARP inhibitors. However, when genetic analysis reveals a variant of uncertain significance (VUS) in the BRCA2 gene, precision medicine-based decisions become complex. VUS are genetic changes with unknown pathological impact. Current statistics indicate that between 10–20% of BRCA sequencing results are VUS, and of these, more than 50% are missense mutations. Functional assays to determine the pathological outcome of VUS are urgently needed to provide clinical guidance regarding cancer risk and treatment options. In this review, we provide a brief overview of BRCA2 functions in HDR, describe how BRCA2 VUS are currently assessed in the clinic, and how genetic and biochemical functional assays could be integrated into the clinical decision process. We suggest a multi-step workflow composed of robust and accurate functional assays to correctly evaluate the potential pathogenic or benign nature of BRCA2 VUS. Success in this precision medicine endeavor will offer actionable information to patients and their physicians. Breast Cancer Susceptibility Gene 2 (BRCA2) was identified in 1994 at the Institute of Cancer Research by the Stratton Laboratory . GermlinRapid advances in DNA sequencing technology coupled with public awareness and quick adoption of genetic testing for HBOC genes in genetic counseling clinics have resulted in vast database repositories of BRCA mutations and variants. While the majority of BRCA mutations are associated with breast, ovarian, pancreatic, and prostate cancers, mutations have been documented in other tumor sites such as lung and malignant melanoma.https://www.wsj.com/articles/geneticists-call-on-myriad-to-share-proprietary-data-to-aid-gene-tests-11578851248, accessed on 5 May 2021).Soon after the BRCA gene sequences were determined, Myriad Genetics initiated a vigorous intellectual property protection strategy providing them with the exclusive rights to provide all diagnostic testing. A supreme court decision in 2013 overturned Myriads’ patents and opened up the genetic testing landscape to several commercial and academic laboratories. As a result, thousands of BRCA variants have been deposited into various databases. While the majority of BRCA sequencing information has been released, Myriad still maintains a large database of BRCA variants not accessible by the public research community (Genetic testing of patient germline DNA and primary tumor DNA (or circulating tumor DNA (ctDNA)) has resulted in more than 30,000 genetic changes in the BRCA genes that lack functional interpretation. This knowledge gap has led to uncertainty in the ability of genetic counselors to predict cancer risk and impedes the successful clinical management of patients who may benefit from targeted therapies. International collaborations and consortiums have joined forces to design integrative approaches to combine familial and clinical data, in silico predictions, and functional analysis of BRCA2 variants of uncertain significance (VUS). However, the challenge to evaluate extremely rare VUS looms large and functional assays may be the only avenue for some patients to rely on for fundamental life-changing preventative clinical options.In this review, we briefly cover the role of BRCA2 in HDR repair of DNA double-strand breaks (DSBs), pathogenic BRCA2 mutations and their impact on cancer risk and treatment options, database and classification methods for VUS, current clinical management strategies for BRCA2 patients, and the challenges of adopting functional analyses into clinical practice.2 phase of the cell cycle. If a cell encounters a DNA DSB and commits to the HDR pathway, several nucleases will resect the DNA end resulting in single-stranded DNA (ssDNA) 3’ terminated tails. The ssDNA is immediately coated with Replication Protein A (RPA) to remove the secondary structure and prevent self-annealing. BRCA2 then delivers RAD51 to the ssDNA displacing the RPA protein while simultaneously preventing RAD51 from binding to dsDNA and downregulating the ATPase activity of RAD51 .,77.76,77Wall Street Journal described a family where multiple women underwent prophylactic surgeries based on a “likely” pathogenic BRCA VUS finding that was later re-classified as a benign variant . This failure of precision medicine underscores the need to cautiously interpret VUS findings and to bolster VUS evaluations with additional evidence for pathogenicity. We suggest that rigorous functional assays performed by laboratories with expertise in BRCA2 biology could provide meaningful criteria for evaluating rare VUS when familial, co-segregation, co-occurrence, and other classical genetic linkage data are lacking.Sequencing-based genetic tests to screen for BRCA mutations are offered to individuals with a family history of breast or ovarian cancer. As a risk-reduction strategy, pathogenic BRCA mutation carriers, who are cancer-free, will be informed of surgical interventions such as a double mastectomy and/or unilateral salpingo-oophorectomy. In many cases, female BRCA carriers during their early reproductive years may opt for careful surveillance in order to delay prophylactic surgeries that would prevent them from having children. In 2013, the so-called “Angelina effect” sparked an acute public awareness of genetic testing for hereditary cancer risk genes, which may have resulted in many people undergoing unnecessary testing without significant associated risk factors such as a family history of unusually high cancer incidence ,79. In aFor patients who learn they are BRCA2 VUS carriers, the situation is complex and frustrating. Not only does the patient now know their BRCA2 gene is not normal (deviates from the wild-type sequence), the clinical significance of the VUS findings are rarely known, leaving the patient with unknown future cancer risk. Universally accepted standards for how to deal with BRCA VUS have yet to be established, revealing a shortcoming in our ability to make informed clinical decisions ,79,80. TPatients diagnosed with cancer and for whom a germline or somatic pathogenic BRCA2 mutation is found often respond well to chemotherapy agents such as PARPi and platinum drugs. LOH of the wild-type BRCA2 allele is usually associated with a robust clinical response to these drugs but not in every case . A tumorSeveral international groups and consortiums have joined efforts to create databases reporting BRCA2 genetic variants generated from sequencing analysis . While pThere are several commonly used online database resources that provide some interpretation of BRCA2 sequence variants . For exaIn the past decade, enormous effort has been dedicated to classifying BRCA2 VUS using a variety of clinical genetic methods and probability models. A standardized method of classifying BRCA2 variants was created by the International Agency for Research on Cancer (IARC) and the American College of Medical Genetics and Genomic (ACMG) based on a 5-tier system with a multifactorial likelihood model ,86,87 F. MultifaComputational methods have been developed to help predict amino acid changes in protein function ,97,98. PModels hold the most utility in BRCA2 variants where the frequency and/or linkage analysis of the variant is high. However, rare variants  are impossible to classify due to a lack of statistical power. Another layer of complexity is that the guidelines governing BRCA2 variant classification change very rapidly necessitating reinterpretation and leading to debate. Finally, if the clinical, genetic, and in silico data for BRCA2 VUS are not deposited in a freely accessible database, it becomes an almost impossible barrier for researchers and clinicians to overcome. In summary, functional studies accompanied by clinical annotation, and the aforementioned tools, may improve classification of BRCA2 variants.BRCA2 is a large and complex protein, and the literature to date has ascribed multiple functions to the protein. Loss of one particular activity does not necessarily directly link to cancer risk, and thus, evaluation of germline variants by a single assay should be interpreted carefully and in combination with clinical and familial data. In contrast, somatically mutated BRCA2 variants identified by sequencing of tumor DNA may correlate well with functional assays designed to measure chemotherapeutic response. For example, confirmation of tumor HDR deficiency is clinically actionable by stratifying patients for treatment with PARPi or platinum agents. Multiple studies have validated that in vitro cell culture models of BRCA2 deficiency respond in a similar manner to patients who receive PARPi therapy ,103,104.The low frequency of rare VUS coupled with limited or no family history of cancer can make the correct evaluation of BRCA2 pathogenic classification very challenging. We and others have developed numerous biological and biochemical assays to assess the consequences of BRCA2 variants on protein function. These assays require high specificity and sensitivity to complement genetic linkage and epidemiological classification methods mentioned above successfully.Functional complementation assays in BRCA2 deficient hamster, mouse, and human cells have been used extensively by several groups ,116,117.In total, while functional assays have been instrumental in the classification of BRCA2 VUS where familial and genetic information is scarce, clinical geneticists have been slow to incorporate lab-based results into counseling decisions for patients. Lack of standardization and uniformity, assays that can only measure specific regions of the protein, low throughput, uncertainty as to which functions of BRCA2 are necessary and sufficient for tumor suppression, and discordance between functional assay results with known variants of clinical significance have all likely contributed to the slow adoption. Furthermore, as with any clinical diagnostic assay, the risk of false negatives or false positives must be carefully addressed. However, in situations where rare VUS lack any clear genetic linkage data, we suggest that functional assays should be considered if used judiciously and with full disclosure.−/− cell line for cell-based and genetic evaluation [In our laboratory, we have designed a simple multi-step workflow to cover many BRCA2 functions described to date. We have taken advantage of biochemical, genetic, cellular, and molecular complementation analysis to investigate the functionality and cellular localization of the BRCA2 protein . To do saluation . Constru−/−) that tolerates a genetically BRCA2 null background is that we can evaluate pathogenic BRCA2 variants directly. Cellular complementation systems that rely on viability are limited to benign, or in some cases, hypomorphic BRCA2 alleles for further analysis as pathogenic variants do not give rise to clones. While we acknowledge that our CMV-driven cDNA complementation system forgoes expression by the endogenous promoter, evaluation of several hundred stable cell clones has revealed little difference in complementation ability due to variability in expression levels. Using our approach, we have identified multiple missense mutations predicted to be pathogenic that are mislocalized to the cytoplasm (manuscript in preparation). This unexpected observation was only possible through the expression of full-length BRCA2 proteins in the DLD1 BRCA2−/− cell line. The phenotypic consequences of pathogenic BRCA2 allele cellular localization significantly impact our workflow decision process of whether further biochemical workup is warranted. A further advantage of our ability to interrogate deleterious BRCA2 alleles is that considerable uncertainty still exists as to which functions of BRCA2, HDR versus fork protection, are critical for chemotherapeutic response in patients. In summary, the potential for biological insight derived from analyzing separation-of-function alleles, and the capability to directly measure drug sensitivity for somatically mutated BRCA2 alleles in patient tumors, highlight the importance of our approach.The advantage of using a human cell model  or GTPases  including: foci assays, anchorage-independent growth in soft agar, genetically engineered mouse models (GEMM), or tumor xenograft studies. To date, no equivalent experimental approach exists for either BRCA gene. Hopefully, in the future, we will be able to parse out the exact functions of BRCA2 that are required to spark tumorigenesis, and this knowledge can be adapted into an assay that will accurately quantify the tumorigenic capacity of BRCA2 variants.In the future, the volume of BRCA2 VUS findings will undoubtedly increase as sequencing technology becomes more economical, higher throughput, and adopted into clinical practice as a routine diagnostic. Patients, clinicians, and researchers will hopefully demand more open access to this information, with appropriate privacy controls in place, thus that VUS have a better chance of being correctly classified. The end goal of functional analysis is to derive a highly sensitive, specific, and predictive score that can be integrated with prior methods to correctly classify the large number of BRCA2 VUS and unmask both cancer risk and response to chemotherapeutics.The current diversity and non-standard resources and databases for deciphering BRCA2 VUS information is a constant challenge for physicians and genetic counselors. Without proper curation and standardized guidelines, clinical decisions surrounding VUS are complex at best and irresolvable at worst. To improve classification efforts, a universal workflow could be adopted with agreed-upon expert guidelines to evaluate BRCA2 variants in a systematic fashion. These universal guidelines will need to escalate quickly as VUS are rapidly clogging the variant pipeline in step with clinical genetic testing panels and sequencing technology. Commercial companies such as Myriad, Ambry, deCODE, and Labcorp are adding to this VUS pipeline, and public awareness has further increased findings of VUS as more and more people seek out their own genetic information.The collective sequences of BRCA variants are being centralized in databases such as BRCA Exchange and are growing exponentially. Just a few days before the submission of this review, BRCA Exchange announced on social media that the number of BRCA variants in their database would increase from 40,000 to over 60,000. The pace of VUS findings concurrent with inconclusive assessments of cancer risk or potential response to targeted therapy will undoubtedly accelerate in the future. Hence, the need for functional clarification intensifies.We conclude that rationale, rigorous, and statistically sound functional analyses of BRCA2 VUS will unleash the current lack of clinical significance for thousands of rare VUS. Although laborious, biochemical and cellular functional assays have the potential to correctly assign a pathogenic or benign classification to a BRCA2 VUS. Functional information combined with familial, co-segregation, co-occurrence, and other classical genetic epidemiological data will reveal the full catalog of BRCA2 pathogenic mutations. Additionally, we will continue to leverage patient-derived BRCA2 pathogenic variants to enhance our understanding of the molecular events that lead to tumorigenesis. Our long-term goal is to remove the frustration associated with clinical BRCA2 VUS findings such that patients feel empowered with clinically actionable information fulfilling the modern promise of delivering precision medicine.


Despite significant progress in past decades, crop production still depends largely on weather , SNORKEL1 (SK1), SNORKEL2 (SK2), TREHALOSE 6-PHOSPHATE PHOSPHATASE 7 (TPP7), deepwater rice–specific variant of SEMIDWARF1 (SD1-DW) etc. have been revealed in the past two decades   and New York University, published in the Plant Cell  when they usually flower  is necessary to develop climate-smart rice varieties for rained-fed agro-ecosystems.After the recent confirmation of our hypothesis, we believe it is high time to identify the genomic speciality of drought tolerance in deepwater rice and mine the key genes/QTLs from Bhadoia 303 and other deepwater rice varietiesIn our opinion, more systems biology studies integrating morphological, molecular and physiological responses to water deficits are necessary for better understanding of underlying mechanisms of drought tolerance developed among rice sub-populations. This approach may close the translation gap between laboratory findings and field performance  is necessary in screening for drought tolerance. The severity and timing of droughts can be easily controlled in rain-out shelters. However, most universities do not possess sufficient resources to handle large field studies, which require special facilities like rain-out shelters, a large number of trained-technicians for simultaneous uniform sampling, etc. Oppositely, rice research institutes usually possess such facilities, expertise, etc., but are typically less interested in basic research. Therefore, we think more research collaboration between rice research institutes and universities may speed up the development of climate-smart rice varieties. The identification of the genetic basis of submergence tolerance  is predominantly available in deepwater rice varieties of Bangladesh whereas deepwater rice in other countries only harbor SK1/2. Moreover, we have earlier showed that abiotic stress tolerance accessions are predominately originated from Bangladesh  and Nepal   to JZ.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.