Leptospira) OR (leptospirosis))”. The review identified 11 original studies that evaluated the performance of 15 kidney biomarkers related to leptospirosis. Assessment of the evidence for biomarker utility was limited because of the small number of studies and sample sizes. Although some biomarkers were associated with kidney disease, no specific biomarker appeared to be ready for clinical practice, and more research in this field is necessary.Leptospirosis is a zoonotic infection with a global distribution, though it has a greater impact on marginalized rural agricultural and urban communities in developing countries. Kidney injury, which can lead to severe and lethal infections, is the most frequent complication associated with leptospirosis. Novel biomarkers are being studied as tools for assessing kidney injury in different pathological processes to improve early detection. This review aimed to gather information on the use of novel kidney biomarkers for human leptospirosis. A search of the literature was carried out in September 2021 using the parameters “((kidney) OR  OR (chronic kidney disease) OR (acute kidney injury)) AND ((biomarker) OR (marker)) AND (( Leptospira is a genus of highly mobile spirochaete bacteria comprising both pathogenic and saprophytic species. The interaction between a pathogenic species and susceptible host can lead to leptospirosis. Human leptospirosis is a reemerging zoonosis with a global distribution, presenting an average of 1.03 million cases and 58,900 deaths each yearLeptospira-host interaction, and during acute disease, renal impairment is common, and bacterial persistence within the kidney leads to fibrosisLeptospira in the kidneyet al. identified CKD after leptospirosis-related AKI in 9% of the patients in a two-year prospective studyAcute kidney injury (AKI) is one of the most frequent complications of leptospirosis, with a reported occurrence varying between 10% and 88% of leptospirosis casesLeptospira could play a role in the etiology, as the bacteria have kidney tropism and regional hotspots are located in areas in which both diseases overlap,Chronic kidney disease of unknown etiology (CKDu) has emerged as a cause of CKD in agricultural communities in warm tropical regions, with toxins, infectious agents, and heat-related kidney stress proposed as probable causesKidney damage results in diminished function and is classified according to the time of establishment and the degree of function reductionAssessing kidney damage by quantifying renal function using traditional biomarkers such as serum creatinine (SCr), the derived estimated glomerular filtration rate (eGFR), or blood urea nitrogen (BUN) is an insensitive approach, as significant kidney injury may have already occurred prior to any change in these measures of kidney function. In contrast, the levels of kidney injury biomarkers that are produced by the pathological mechanism of injury may increase before kidney function is altered,,In the context of human leptospirosis, several kidney injury biomarkers have been studied for their potential use as tools for the early detection of kidney injury and to monitor patients after acute disease to identify those who might develop CKDLeptospira) OR (leptospirosis))”. The search did not have a year restriction for publication date. Summaries of the resulting articles were reviewed to apply the inclusion and exclusion criteria, as shown in The MEDLINE and LILACS databases were searched in September 2021 using the following search parameters: “((kidney) OR  OR (chronic kidney disease) OR (acute kidney injury)) AND ((biomarker) OR (marker)) AND  levelset al. compared biomarker levels among 46 leptospirosis cases and 31 healthy controls and found a correlation between NGAL and SCr levels. In this study, NGAL was used as an acute injury marker to study the performance of syndecam-1 and intercellular adhesion molecule 1 (ICAM-1)et al. carried out a similar retrospective study with 143 participants in which NGAL was used as a reference point to evaluate the performance of defensin α1. In this study, NGAL levels were significantly different between leptospirosis cases and control groupsNGAL was included in six studies, two of which used NGAL as a reference point for the performance of other biomarkers. NGAL, also known as lipocalin 2, is a protein belonging to the lipocalin family and is present in neutrophil granules and epithelial cells from different organs, including the kidney,,et al. published a retrospective study in which urinary NAG was used as a kidney injury biomarker in emergency department patients with leptospirosis in Thailand in 2016, and a second retrospective study was published in 2020. In both studies, NAG was strongly associated with AKI and had specificity for leptospirosis,et al., urinary NAG levels were higher in Leptospira seropositive participants than in seronegative participants; however, this was not statistically significant after further model adjustmentNAG was the second most studied kidney injury biomarker and was included in three studies. Similar to NGAL, NAG was used as a reference point to test other biomarkers,et al. evaluated its performance in a cohort study with a two-year follow-up in which leptospirosis MAT antibody titers >1:400 were associated with deterioration in renal function. Patients with high MAT titers also had higher urine KIM-1 and creatinine levelset al. found elevated urine KIM-1 levels in patients with leptospirosis related AKIet al. showed an association between high levels of urine KIM-1 and AKI as well as evidence that KIM-1 is an early marker of kidney injuryKIM-1, also known as cell immunoglobulin mucin domain 1 (TIM-1), is a type 1 transmembrane protein that increases rapidly during proximal tubule injury and has been linked to the pathogenesis of renal fibrosis,et al. assessed its capacity as a biomarker in a cohort study and found no link between MCP-1 levels and decreased kidney functionet al. found a link between elevated MCP-1 levels and leptospirosis-related AKIMCP-1 is a protein from the chemokine family that plays a role in the migration and filtration of monocyteset al., investigators reported higher IL-18 levels in Leptospira seropositive participants than in seronegative participants; however, this was not statistically significantInterleukin-18 (IL-18) is a member of the interleukin-1 family and has regulatory effects on innate and acquired immunityet al. with 77 participants showed that blood levels of syndecan-1 were higher in leptospirosis patients with AKI than in patients without AKISyndecan-1 is a major cell surface heparan sulfate proteoglycan in epithelial cells that acts as an adhesion target for bacteria in many infections, and is an endothelial damage biomarkerLeptospira in an in vitro model,et al. measured defensin α1 in a case-control study that evaluated AKI in 112 patients with leptospirosis, and observed higher levels of defensin α1 in AKI patients than in leptospirosis patients without AKIDefensin α1 is an antimicrobial peptide that was effective against et al. evaluated 12 patients with laboratory-confirmed leptospirosis in an emergency department and assessed oxidative stress markers and complications. In this study, GSH depletion was positively correlated with higher SCr levels; however, no significant difference was observed between leptospirosis patients with and without AKIGSH is an important antioxidant and low-molecular-weight peptideet al. included ROS in a cohort study that evaluated GSH and found that ROS levels were positively correlated with serum potassium levels. There was no significant difference between leptospirosis patients with and without AKIROS are a group of free oxygen radicals produced during different metabolic processes that are highly reactive and potentially harmful,et al. conducted a retrospective cohort study using samples from patients diagnosed with leptospirosis and found higher levels of Angpt-2, ADMA, and SDMA in patients with evidence of AKI than in patients without AKIAngpt-2, ADMA, and SDMA are markers of endothelial inflammationet al. reported higher levels of serum and urine OPN in patients with altered urinary dipstick parameters in a retrospective study of 112 febrile patients with confirmed acute leptospirosisOPN is a highly phosphorylated glycophosphoprotein that has multiple important functionset al. found an association between Gal-9 and severe leptospirosis; however, the levels were not linked to kidney damage during leptospirosisGal-9 is a lectin from the galectin family with immunomodulatory and anti-inflammatory effects that act through the suppression of TH1 and TH17 lymphocytesLeptospirosis-related CKD has been linked to renal fibrosis, secondary to persistent or recurrent kidney colonization by pathogenic species, or because of unresolved AKI,Leptospira, which causes tubulointerstitial fibrosis and, if not treated, kidney failure,With only 60 articles, the search results revealed a generally limited knowledge base on the use of these biomarkers for leptospirosis, despite no language or year restrictions. Of these, only 11 studies met the criteria for evaluating the performance of novel biomarkers associated with kidney injury during leptospirosis. Five were cross-sectional studies with no possibility of evaluating longitudinal changes in kidney function after confirmed leptospirosis infection. Studies tended to have a small sample size, which limited the ability to identify statistically significant differences or to evaluate potentially important patient and pathogen-level factors. -Leptospira, and reduced kidney functionLeptospira-related CKD,,,The limited evidence does support kidney injury biomarkers as a promising tool in the management of leptospirosis, potentially applicable in the diagnosis, prognosis, and follow-up of patients. NGAL and NAG were the two most studied biomarkers and provide supportive evidence as biomarkers of kidney injury. They have also been used in leptospirosis studies as a reference point to evaluate other biomarkers of AKI,Further evaluation of biomarkers is needed to identify their role in prognosis following leptospirosis-associated AKI, and to help prioritize patients in highly endemic regionsLeptospira are involved, as not all pathogenic species have the same virulence. For example, Leptospira interrogans is associated with aggressive episodes of leptospirosis with a higher degree of kidney impairmentLeptospira santarosai favors less aggressive leptospirosis, which tends to persist in the kidneyThe kidney disease model may also vary about which species of et al. on renal fibrosis biomarkers not related to Leptospira identified that urine transforming growth factor-beta (TGF-β), blood and urine matrix metalloproteinase-2 (MMP-2), and MCP-1 were associated with adverse renal outcomes based on a longitudinal follow-up of patientsTGF-β and MMP-2 in leptospirosis were identified in this review. Considering their potential association with renal fibrosis in CKD, future research should consider their use in leptospirosis-associated CKD. Urine MCP-1 has been evaluated in leptospiral-related kidney damage by Yang et al. with mixed resultsA 2017 review by Mansour Kidney damage biomarkers could be a potential tool for the early detection of kidney impairment, with evidence from other disease models showing promise as a tool for identifying patients who may develop AKI


In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed. Seventy-one new biological drugs have been approved and launched into the market from 2014 to 2018, of which 62 are recombinant therapeutic proteins (RTPs) . RecombiTo meet the increasing demand in biopharmaceutical market, it is necessary that innovating the production process with a higher production capacity, high quality product, and reducing production costs. Vector frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors can infect a target cell naturally and effectively, with no additional reagent or equipment required, but it may elicit unwanted cellular consequences and the maximum vector size of viral vectors is restricted, hampering gene delivery of larger genes. In contrast, nonviral DNA plasmid-based vectors are largely nonimmunogenic and can carry large amounts of DNA, but the low transfection and poor integration rates are their major problems . AlthougThe main recombinant protein expression systems contain prokaryotic, yeast, insect, and mammalian cell expression systems. For the expression of the complex recombinant proteins with high molecule weight, proper folding and post-translational modifications are required to display their biological activities due to their complex structure. Therefore, mammalian cells mainly including CHO cells and human embryonic kidney (HEK293) cells, have become the main expression hosts for RTPs production due to their PTM system.Compared with prokaryotic, yeast and insect cells, CHO cells have the following advantages : 1) CHO Besides, the transformed cell line HEK293 derived from human embryos have unparalleled advantages in comparison to other engineered cells in recombinant protein expression, 1) high efficiency in transfection ; 2) no p8 cells/mL, titers of secreted protein levels of 8 g/L in fed-batch or 25 g/L in perfusion cultures  (PiggyBac (PB)  , and theBac (PB) . AlthougBac (PB) . Tol2 anBac (PB) . In contBac (PB) .in vitro with purified PB transposase and DNA elements  to the N-terminal end of PB transposase. This manipulation capacitated the transposon system in targeted integration by combining the “cut-and-paste” mechanism of the transposon system with the target recognition property of ZFP, and successfully realized the targeted integration of ZFP-PiggyBac compared with the natural PiggyBac . After ZMore attentions have been paid to how to further optimize the transposable subsystem, not the problem of the transposon system itself. Given the endogenous transposases are also present in the target cells and there is no lack of transposase recognition sequences in the genome, we believe that either endogenous or exogenous transposases might have potential adverse influence on the cellular genome and GOI. However, it has been demonstrated that the introduction of exogenous transposase did not result in genome breakage and reorganization in the host cell and that the cell’s own transposase also did not destroy the successfully integrated GOI . HoweverCell type, co-transfection ratio of transposon and transposase, transfection efficiency, and expression regulatory elements in the vector are all factors influencing the transposition efficiency of different transposon systems . Only whTypically, GOI can only be inserted into the genome of mammalian cells by classical random integration manner, requiring a complex antibiotic-resistance screening step to obtain monoclonal cell lines. Moreover, the expression of GOI is often also influenced by the genomic DNA elements near the random insertion site, leading to significant variation in transcription level . In addiTo address the drawbacks of classical integration methods, researchers have developed dihydrofolate reductase/methotrexate based gene amplification method , as wellTransposon vector systems usually require only the successful screening of a pool of stably expressing cell lines  which arIn addition, cell sorting is also a way to quickly obtain high expression cells. Matabaro et al. expressed glycosylphosphatidylinositol (GPI) anchoring protein in HEK293 cell line, while PGAP2 gene determines whether GPI can be secreted or not. First, endogenous PGAP2 was knocked down in HEK293 using CRISPR/Cas9 technology to construct the PGAP2-KO cell line, and then the exogenous PGAP2 gene was integrated into PGAP2-KO by establishing the PB transposon system. So, then the target protein could be expressed in the cell membrane to sort the highly expressed cells. Finally, the target protein can be secreted into the culture for subsequent isolation and purification after removing the PGAP2 gene by adding the transposase again . The fleMatasci et al. first used PB vector to express a tumor necrosis factor receptor: Fc fusion protein (TNFR-Fc). In CHO cells, transposase and transposon were co-transformed at a ratio of 1:9, and then a pool of stably expressed cells were obtained through screening with different concentrations of puromycin. The results showed that the protein yield of the cell pool obtained by screening with high concentration of puromycin was higher. The increased productivity correlated with the GOI copy number per cell by RT-qPCR analysis, suggesting that cell lines showing high efficiency in protein production can be established using PB vectors and stringent screening process .Balasubramanian et al. expressed TNFR-Fc in PB using CHO DG44 cells as hosts. The yield of TNFR-Fc reached 600 mg/L after 14 days of batch culture in a 1 L culture system. And the volumetric productivity of the cell pool can be maintained for up to 3 months in the absence of selecting pressure. This indicates that the production of recombinant proteins could be successfully improved using PB transposons . To furtBarnard et al. used the PiggyBac transposon vector to express four antibodies in a CHO (CHO K1SV GS Knockout) cell line . The titIn recent years, mammalian cells have played an irreplaceable role in RTPs expression, and transposon vector systems which could improve yields without affecting the quality of RTPs deserve to be studied in greater depth. Compared with conventional expression vector systems, transposon system enables rapid construction of protein-expressing cell lines and can increase antibody yield without compromising quality. It also can shorten the production period for monoclonal antibodies against the SARS-CoV2 and accelerate the development of clinical drugs.Besides, insertion of transposon vectors into matrix attachment regions could enhance the gene expression and insertion of cHS4 DNA could prevent the gene silencing phenomenon from affecting the target gene. To provide the transposon system with the ability to target integration, coupling the specific nucleolar localization signal NoLS with the transposon and transposase allows GOI to be localized to the rDNA-containing nucleoli, or fusing of the ZFP with the transposase enables the transposon system to have the ability to integrate in specific genomic regions. Other than that, DNA binding domains such as Gal4 DBD, Rep protein of adeno-associated virus, and TALE have been associated with the piggyBac transposase with the goal of targeting various chromosomal loci, with varying success. Unlike ZFNs and TALENs, CRISPR/Cas9 system does not rely on the recognition between the protein and the target DNA. The ribonucleotide complex is formed between the guide RNA and the target DNA. The GOI is recognized by ribonucleotide, then cleaved by Cas enzyme, and the DNA chain is repaired by homologous repair, combining transposon system with CRISPR/Cas9 technology, can establish a cell sorting method that allows proteins to be localized in the membrane to select highly expressed cells, also can target integrations based on Nols-FokI-dCas9 endonuclease coupled to NolS, although has lower integration efficiency compared to PB system coupled to NolS, CRISPR/Cas9 technology is still worth being investigated for transferring some specific GOI or specific loci. The combination of CRISPR/Cas9 and transposon systems can overcome their respective limitations, which will have high specificity and efficient integrative capacity double effects. It remains to be explored whether combining transposon vectors with CRISPR/Cas9 technology will bring more significant advantages in protein expression using mammalian cells.The advantages of using transposon systems in RTPs production are obvious. It overcomes the limitations of conventional stable transfection and has practical value for the development of novel biological drugs and their industrial production in the future. In addition, it has been shown that transposon system can be used for gene therapy and cancer gene screening. Nevertheless, the understanding of transposon vectors is still at the research stage, there are still some problems to be solved. Although neither exogenous nor endogenous transposases affect the host cell genome and the successfully integrated GOI, the transposon vector backbone might still integrate with the genome of host cell and affect the normal expression of endogenous genes. The disadvantage using transposon vector system for recombinant protein production is the random integration of transgene, if inserted into the structural gene, it may lead to transcriptional read-through, insertional mutagenesis, structural changes and other negative effects. To further enhance RTPs expression and meet industrial production requirements, even to expand the application of transposon in gene therapy, transposon vectors are required to be optimized by investigating their interaction with multiple genetic elements and other technologies.


The “omics revolution” has totally changed the scientific research approach and is contributing to the development of personalized therapies. In motor neuron diseases (MNDs), a set of complex, multifactorial, late-onset and chronic neurodegenerative diseases, the use of multi-omics approaches in clinical trials is providing new opportunities to stratify patients and develop target therapies. To show how omics science is gaining momentum in MNDs, in this work, we review the interventional clinical trials for MNDs based on the application of omics sciences. We analyze a total of 62 clinical trials listed in the ClinicalTrials database where different omics approaches have been applied in an initial phase, for diagnosis or patient selection, or in subsequent stages to cluster subjects, identify molecular signatures or evaluate drugs security or efficacy. The rise of omics sciences in clinical experimentation of MNDs is leading to an upheaval in their diagnosis and therapy that will require significant investments and means to ensure the correct and rapid evolution of personalized medicine. The Human Genome Project (HGP), devised in the 1980s, began in 1990 and completed in 2003, made it possible to map and sequence the entire human genome. The rapid advancement of knowledge, together with high-throughput sequencing technologies, have laid the foundation of the omics sciences. Several research areas can be classified as omics sciences: genomics examines the genetic composition, transcriptomics investigates gene expression, proteomics focuses on the final protein product, metabolomics analyses metabolites and epigenomics evaluates epigenetic modifications.In the last years, the application of omics technologies to clinical research has provided a more comprehensive understanding of the pathogenesis of complex diseases and the identification of biomarkers for patient stratification is simplifying the development of target therapies . The fieMNDs represent a group of neurological progressive and degenerative disorders, which selectively affect motor neurons in brain and spinal cord . They inTo show how omics science is gaining momentum in MNDs, in this manuscript we highlight the rise of the “omics” approach in clinical trials. Following a brief introduction to the MNDs of interest, we review all omics-based clinical trials performed to date to investigate the diagnostic and therapeutic complexity of these rare diseases.The term “clinical trial” defines a human clinical pharmacological, biomedical or health study that follows predefined protocols. Clinical studies can be observational or interventional: the first tends to demonstrate the possible effects of various risk or protective factors on a group of people by observing events that occur without any intervention by the investigator. Interventional studies are research studies that aim to evaluate a new treatment , with the aim of evaluating if this represents an improvement compared to what is normally practiced.https://www.clinicaltrials.gov/, 22 April 2022). To date, 62 omics-based clinical trials for MNDs can be found in this database: 41 (66.1%) are for ALS  studies are currently in Phase I-II, in which the drug’s safety and toxicity as well as the effect produced by the treatment on the pathology under study are assessed; 36 (58.1%) studies are in Phase II-III, in which the drug is tested in a wider number of patients; whereas only one (1.61%) study is in Phase IV, called post-marketing surveillance trial. A genomic approach is used in 23 (37.10%) clinical trials for patient stratification; transcriptomics is used in 16 (25.81%) clinical studies and aims to monitoring therapy, while the remaining 23 (37.10%) trials integrate a multi-omics approach to evaluate the effect of treatments. Finally, grouping all trials according to the purpose for which the omics approach was used, we find that 52 (83.87%) studies aim at drug testing using a high-throughput technology, of which 17 (27.42%) trials aim at patient stratification and 35 (56.45%) at the evaluation of pre- or post-treatment clinical outcomes. The remaining 10 (16.13%) studies use different omics technologies to investigate disease molecular signatures, identify new biomarkers or build repositories.In the next sections, we will discuss these clinical trials mainly focusing on those completed with published results that permit the evaluation of the usefulness of the omics approaches utilized.Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease caused by the progressive loss of motor neurons (MNs) resulting in weakness and paralysis of voluntary muscles. The main clinical feature of ALS is the upper and lower MN involvement. The age of onset is about 60 years, and the incidence is 5 per 100,000 inhabitants [SOD1), FUSed in sarcoma (FUS), TAR DNA binding protein (TARDBP) and chromosome 9 open reading frame 72 (C9Orf72) [Although important research progress has been made, the etiopathology of ALS is mostly unknown. The mechanisms underlying the development of the disease are multiple, with the involvement of a complex interaction between genetic and molecular characteristics . The majC9Orf72) . Due to C9Orf72)  that decC9Orf72 gene helped to define a genetic basis to explain the spectrum ALS/FTD [Neuropathology and medical genetics have led to the discovery that ALS and Frontotemporal Dementia (FTD) are related diseases and form a broad neurodegenerative continuum . Both of ALS/FTD .The application of omic sciences has enabled the development of novel approaches to understand the molecular nature of ALS. As described in the following sections, their use has potential clinical implications to stratify patients and identify effective and safe treatments ,15.SOD1 expression in SOD1-fALS mutation carriers. The results revealed a successful strategy showing that the drug (ISIS 333611) was well tolerated [SOD1-fALS mutation carriers were used to test the safety, tolerability and efficacy of Arimoclomol, a drug promoting the correct folding of proteins. The study demonstrated drug tolerability and safety but did not show therapeutic efficacy [ATXN2 expansion were enrolled to assess the safety, tolerability and pharmacokinetic profile of BIIB105, an ASO designed to bind and degrade the ATXN2 mRNA.A genomic approach has been used in different clinical trials to select patients with specific drug-targetable gene mutations. Trial NCT01041222 was the first to use an intrathecally injected antisense oligonucleotide (ASO) designed to inhibit olerated . A similefficacy . In triaA transcriptomic approach has been used to evaluate pre- and post-treatment gene expression changes in ALS patients . In triaIn several clinical trials, the drug’s impact was determined by comparing the expression of different markers in patients exposed to drugs or placebo  or observing pre- and post-treatment clinical outcomes . Notably, the results of trial NCT01854294 showed that the master regulator peptide GM604 altered plasma expression levels of SOD1, TAU and TDP-43 proteins, slowing down disease progression . In triaAlthough discoveries related to the molecular basis of a disease may offer unprecedented opportunities to translate into new drugs, their development requires an enormous amount of time, money, and effort. For this reason, some trials have the objective to repurposing existing drugs to new disease areas . Based oClinical trial NCT04066244 proposed a multi-omics approach, the NCT04066244 to characterize the safety, tolerability and response of BLZ945, a Colony Stimulating Factor 1 (CSF-1) inhibitor. The outcomes of this trial will be based on genotyping cytochrome P4502C8 (CYP2C8), an enzyme involved in metabolism of xenobiotics.A genomic, metabolomic and transcriptomic approach has been used to select patients and monitoring therapy. In trials NCT03626012 and NCT04288856, C9Orf72 patients were enrolled to evaluate the safety, tolerability and pharmacokinetics of BIIB078, an ASO targeting C9Orf72 mRNA. A similar approach was applied in trials NCT05163886 and NCT05053035, where C9Orf72 patients were recruited to test the safety, tolerability and biological effect of LAM-002A, (apilimod) an inhibitor of the PIKfyve kinase that works by clearing toxic protein aggregates within lysosomes. The outcome of this trial was to evaluate LAM-002 levels, metabolites and neurofilament light chain (NfL) in plasma and CSF.Clinical trial NCT04993755 aimed to characterize the safety and tolerability of the reverse transcriptase enzyme inhibitor TPN-101 (censavudine) in C9Orf72 patients with ALS/FTD, ALS or FTD. A similar patient’s classification was used in trial NCT04931862 to test the effect of WVE-004, an ASO designed to mediate the degradation of C9Orf72 mRNA.SOD1 mRNA and to prevent protein synthesis. The results of this study confirmed drug safety and revealed drug-dependent SOD1 expression levels in CSF [SOD1 mutation carriers were used to test the antiparasitic drug, Pyrimethamine (Daraprim). The results of this study demonstrated a significant reduction of SOD1 protein concentrations in CSF [A genetic stratification was applied in trial NCT02623699, where SOD1-patients were enrolled to test BIIB067 (tofersen), an ASO designed to degrade s in CSF . The samFUS protein. This successful strategy showed a reduction in CSF of NfL concentration. Using a repurposing drug strategy, FUS patients were recruited in trial NCT03707795 to evaluate the effect of Betamethasone by assessing levels of different biomarkers in plasma. A similar strategy was exploited in trial NCT05189106, where the reduction of CSF inflammatory biomarkers was assessed in patients with Alzheimer’s disease and C9Orf72-ALS carriers following treatment with Baricitinib, a JAK inhibitor approved for rheumatoid arthritis and COVID-19 treatment [A genomic and metabolomic approach was used in trial NCT04768972 to evaluate the safety, pharmacokinetics and pharmacodynamics of ION363 in FUS-mutation carriers. This drug is an ASO designed to reduce the production of mutated neurotoxic form of reatment .Clinical trial NCT04220021 used a genomic and transcriptomic approach to evaluate safety and tolerability of Metformin in C9Orf72- patients. Since this drug, approved as a diabetes mellitus remedy, demonstrated beneficial effects through different signalling pathways , the triA multi-omics approach has been used in trail NCT02590276 to identify disease signatures in FTD/ALS C9Orf72-carriers . The resA transcriptomic approach has been used in several clinical studies. In trial NCT01984957, DNA microarray technology was used to identify dysregulated gene expression in muscle biopsies of ALS patients. Combined omics technologies were applied in trial NCT02670226 to identify novel biomarkers in ALS patients by exploration of blood, muscle and satellite cells metabolomes and transcriptomes. A similar approach was applied in clinical trial NCT03851302, where mRNA expression of metabolism genes was assessed in muscle of ALS patients following neuromuscular magnetic stimulation (NMMS).SMN1 gene, essential for the function and survival of motor neurons. Patients with SMA have a variable number of second gene copies, SMN2, which encodes a shortened form of the SMN protein, with reduced functionality compared to the full SMN protein encoded by the healthy SMN1 gene. The number of copies of the SMN2 gene is therefore the basis of the great variability of the disease, with more or less severe forms and a very wide range of symptoms [SMA is a disease characterized by motor neuron degeneration of the spinal cord anterior horns resulting in muscle atrophy and weakness of the trunk and limbs. Based on the age of onset and the severity of symptoms, four different SMA forms can be distinguished: type 1 SMA (SMA1) is the most severe and appears from 6 months of age; type 2 SMA (SMA2) become visible indicatively, between 6 and 18 months of life; type 3 SMA (SMA3) starts after 12 months of age, while type 4 SMA (SMA4) begins in adulthood and is the least severe form. In 95% of cases, the disease is caused by specific mutations in the symptoms . Due to symptoms .SMN gene in SMA1 patients [SMN2 and SMN1 genes. [A genomic approach for patient stratification has been used in several studies to evaluate the safety, tolerability and pharmacokinetics of a SMN gene-targeted therapy. Trail NCT02122952 was the first to test a replacement gene therapy, based on a self-complementary adeno-associated viral vector-9 (AAV9), to provide a functional copy of the patients . Encourapatients . In a glpatients ; Europe,patients ; Pacificpatients ). A simi1 genes. .SMN2 gene to produce the full-length and functionally normal protein. This drug has recently been approved by over 40 countries as a treatment for SMA.In trial NCT04576494, adults with genetic SMA type 2 and type 3 were enrolled to study the functional effect of Nusinersen (Spinraza), an ASO designed to allow the A transcriptomic approach has been used in trial NCT00439569 to evaluate the effect of sodium phenylbutyrate (a histone deacetylase inhibitor) on SMN-mRNA and -protein levels in children with SMA type 2 and type 3. The study was closed prematurely due to poor compliance with drug administration.A genomic and transcriptomic strategy has been applied in trials NCT03032172, NCT02908685 and NCT02913482 to characterize the safety, tolerability and response of the splice modifier RO7034067 (Risdiplam) in adult, paediatric or infant SMA patients. In these trials, drug effects were assessed by evaluating changes in SMN mRNA and protein levels in the blood following RO7034067 treatment.SMN1-SMN2 mutations. By reducing the need for blood transfusions in patients with sickle cell anaemia, this drug is used for the treatment of chronic myeloid leukaemia as well as in the prevention of painful episodes [A repurposing drug strategy was exploited in trial NCT00485511 where the efficacy and safety of Hydroxyurea (HU) was tested in children with episodes .SMN1 in SMA families and, in trial NCT04833348, to quantify motor function in infants treated with innovative therapies (gene therapy or pharmacogenomics).A genomic approach has been applied in trial NCT02550691 to identify specific cis duplication biomarker of SBMA, or Kennedy disease, is a genetic late onset MND characterized by slowly progressive weakness of the bulbar muscles and limbs with fasciculations, muscle atrophy and gynecomastia . The disA genomic approach has been used in clinical trial NCT00303446 to evaluate the pharmacological properties of dutasteride in SBMA patients. By acting as a 5-alpha-reductase I and II inhibitor, this treatment is used against benign prostatic hyperplasia . The resOmics sciences are quickly modifying clinical investigations of MNDs to achieve a more comprehensive view of underlying molecular pathways and allow patients stratification into diagnostic, prognostic and therapeutic subgroups. Today we are at a critical point in the development of personalized medicines for MNDs. The landscape of clinical trials using omics approaches and their surge in recent years are expected to produce an important transformation of clinical management of MNDs. The omics-based development of newly launched therapies is speeding solutions to the clinic and have a profound impact in both diagnostics and therapy. However, the road to translate these solutions to the clinic can be challenging. There is still a wide gap between discovery research and clinical practice. Bridging this gap and allowing an effective and sustainable implementation of personalized medicine will require new investments, technologies as well as the training of health professionals. The lack of these may result in inequities in the access or delivering of person-centred care.


Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB) represent the second most common type of degenerative dementia in patients aged 65 years and older, leading to progressive cognitive dysfunction and impaired quality of life. This study aims to provide a consensus based on a systematic Brazilian literature review and a comprehensive international review concerning PDD and DLB. Moreover, we sought to report on and give recommendations about the best diagnostic approaches focusing on primary and secondary care. Based on the available data, we recommend clinicians to apply at least one brief global cognitive instrument to assess PDD, such as the Mini-Mental State Examination and preferably the Montreal Cognitive Assessment and the Addenbrooke’s Cognitive Examination-Revised. Validated instruments to accurately assess functional abilities in Brazilian PD patients are still incipient. Further studies should focus on biomarkers with Brazilian cohorts. The umbrella term Lewy Body Dementia (LBD) includes both of these syndromes, representing the second most common type of degenerative dementia in patients aged 65 years and older and leading to progressive cognitive dysfunction, motor deterioration, and impaired quality of lifeDespite having the same pathological substrate, Parkinson’s disease (PD) and DLB are classified as different diseases based on the temporal relationship of cognitive and motor symptoms. Current criteria recommend diagnosing a patient with PDD when dementia develops in the context of well-established PD), using English and Portuguese and human studies filters. Inclusion criteria were 1) Brazilian studies referring to PD and DLB 2) investigating cognitive or associated features 3) in older adults or those over 18 years old. Exclusion criteria were 1) studies focusing on other cognitive primary diagnoses.The consensus group was later divided into three subgroups concerning epidemiology and risk factors, clinical features and neuropsychological assessment, or biomarkers and management, each subgroup in charge of critically reviewing the literature selected based on predefined selection criteria. A comprehensive literature search was also performed to add current information and knowledge for insufficient data acquired from the systematic review or if the specialists wanted to enrich the consensus study.The search gathered 732 records identified by database searching. After an initial exclusion, 106 articles published in Brazilian cohorts were selected, of which 60 were later excluded due to the reasons explained in In total, 46 articles were included in this consensus. For topics without specific articles in the Brazilian literature, 17 studies, including research articles, review articles, and international consensus, were added to the 46 previously selected Brazilian articles, in a sum of 63 studies.The PRISMA flow diagram summarizes the search .) and 56.7% had it in the Pietá study),--. This concept changed in 2007 when a task force of the Movement Disorder Society (MDS) developed specific diagnostic criteria for PDDThe fourth consensus report of the DLB Consortium, in turn, has recently refined the recommendations for the clinical and pathologic diagnosis of DLBWe suggest that the diagnosis of PD should use validated diagnostic criteria, which can be either the United Kingdom Parkinson’s Disease Society Brain Bank diagnostic criteriaWe also suggest maintaining the “1-year rule” for clinical and research purposes, despite the controversy over whether PDD and DLB are the clinical spectra of the same disease. In turn, specialists from the MDS have recently proposed not using the “1-year rule” to distinguish PDD from DLBWe recommend adopting the formal criteria for PD-mild cognitive impairment (MCI) published in 2012 ), sleep behavior disorder (RBD). These may precede the motor signs whereas dementia and psychosis are common in the late stages of the illness),,,,15, has a high specificity for suggesting an alpha-synucleinopathy. RBD is a parasomnia characterized by loss of atony during REM sleep. The current DLB),,,1,. This indicates that level I procedures had lower sensitivity but greater specificity than level II for the diagnosis of dementiaThe MDS proposed practical procedures for level I diagnosis of PDD based on an algorithm or checklist. Both of these procedures are advantageous, either for clinical routine or clinical research. However, they require some adaptations that we suggest in ),, the Disability Assessment for Dementia (DAD), the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE), Pill questionnaire, the phone call test, and the Direct Assessment of Functional Abilities (DAFA) 15,,,,. The MDS has proposed the MMSE as a first-line assessment tool for global cognitive efficiency in PD because of the instrument’s simplicity and wide use in dementia. However, other studies, including some with Brazilian populations, showed that the MMSE had lower sensitivity than other scales to detect cognitive abnormalities in PD patients),35, to screen PD patients for dementia)-,,,, and the Addenbrooke’s Cognitive Examination-Revised (ACE-R) are other brief global cognitive instruments that have been used in PD and DLB. They have already been translated and validated for Brazilian populations)-35,,, Scales for Outcomes in Parkinson’s Disease-Cognition (SCOPA-COG), Wisconsin Card Sorting Test (WCST), Frontal Assessment Battery (FAB), Verbal Fluency TestsBased on the available data, we recommend clinicians to apply at least one brief cognitive instrument, such as MMSE and preferably the MoCA or ACE-R, to investigate PDD and DLB - especially since we found no Brazilian studies or samples concerning neuropsychological profiles of the latter. However, for a tertiary center and other treatment strategies such as DBS, a comprehensive neuropsychological evaluation is more appropriate if suitable.Besides analyzing the clinical history and conducting neurological examination with brief cognitive instruments, we also recommend applying ancillary tests to confirm the PDD and DLB diagnoses. Routine screening tests are recommended to exclude other causes of cognitive impairment. We suggest conducting a complete blood count, as suggested in article one, and using structural imaging such as magnetic resonance imaging (MRI) or computed tomography (CT) scans to exclude other causes for dementia syndromes, such as vascular dementia, stroke, and brain tumors.The PDD and DLB biomarkers do not define the presence of alpha-synuclein in vivo, but seek to reflect the repercussions of the pathophysiological process. Specifically for PDD, the current diagnostic criteria do not recommend structural, functional, or electrophysiological studies for routine diagnostic purposes in differentiating PD from PDD due to the lack of specificity123I-MIBG) is a method that provides a functional analysis of the sympathetic postganglionic pathway, evaluating in vivo the cardiac noradrenergic neurotransmission. 123I-MIBG scintigraphy documenting cardiac sympathetic denervation is a supportive criterion for PD diagnosis, having over 80% specificity to distinguish PD from other parkinsonian conditions123I-MIBG as an indicative biomarker, showing good sensitivity (69%) and specificity (87%) values for discriminating probable DLB from probable AD123I-Meta-iodo-benzylguanidine scintigraphy Moreover, normal functional neuroimaging of the presynaptic dopaminergic system is currently considered an absolute exclusion criterion for the MDS PD criteria. However, the task force mentioned that PD diagnosis does not require dopaminergic functional imagingPSG is the third indicative biomarker for DLB diagnosis. It aims to confirm the clinical suspicion of RBD, showing the loss of atony in the REM sleep stageSupportive biomarkers for DLB include: the relative preservation of medial temporal lobe structures on CT/MRI scans since patients with AD show greater atrophy of medial temporal lobe structures; the generalized low uptake on SPECT/PET perfusion/metabolism scan, which shows reduced occipital activity; and the posterior cingulate island sign on FDG-PET imaging. ),,,59,(


Connexins are assembled into dodecamer intercellular channels, a collection of which is termed a gap junction, and their canonical function allowing direct exchange of ions and metabolites has been unequivocally established. When initially assembled into undocked cell surface connexin hemichannels, healthy cells may also engage in cell signaling via a regulated small-molecule release. Recent advances in the field have led to an expanded view of the functional roles of intercellular channels and hemichannels in both physiology and pathology. As more of the 21-member human connexin family is intensely interrogated, mounting evidence points to the biological uniqueness of each member, and no longer can we confidently refer to all connexins engaging in the same cellular processes. Innovations in high-resolution cryo-electron microscopy have revealed important insights into the structure of functionally important domains of both hemichannels and channels. These and other studies have established a foundation of knowledge that should allow inhibitory smart drug design for situations where enhanced intercellular or hemichannel activity is at the root of a connexin-linked disease. Assessment of the connexin interactome, which varies widely for each connexin subtype, continues to provide regulatory insights into the assembly and function of connexins that exhibit a short half-life. As the most intensely studied, Cx43 is found in about 50% of all human cell types and is extensively regulated by multiple inhibitory and enhancing phosphorylation events that have direct implications on tissue function and outcomes of disease, including cancer. Here, we briefly discuss these advances and give our thoughts on where the field is headed. Gap junctions play both structural and intercellular communication roles to help regulate many cell processes, including cell migration, cell proliferation, embryonic development, differentiation, wound repair, and the coordinated contraction of heart and smooth muscle2. There is unequivocal evidence that, in humans, the channel-lining proteins of gap junctions are composed of proteins from the 21-member connexin gene family3  accelerated the structural revolution as high-resolution images of native lens gap junctions composed of Cx46 and Cx50 were obtained17. Here, the authors definitively proved the transmembrane domain arrangements and found open conformational states that were distinct from Cx2617, possibly revealing differences between alpha and beta connexin family members. This same team further resolved the structure of Cx46/50 channels in a dynamic aqueous-lipid context at near atomic resolution (1.9 Å), where the connexin proteins were found to stabilize the local lipid microenvironment18. This level of molecular detail is now sufficiently informative to serve as a model for rational drug design for the possible treatment of any number of inherited connexin-linked diseases8 and the countless pathologies linked to connexin regulation in diseased tissue and cancer19.Interrogation by X-ray diffraction, freeze-fracture electron microscopy, nuclear magnetic resonance, and atomic force microscopy, together with sophisticated image analysis, revealed that the dodecameric arrangement of connexins was needed to form a complete intercellular channel20. In the years that followed, the community had to consider that connexins may, in fact, have a second canonical function in forming cell surface channels capable of releasing  metabolites or a variety of signaling molecules or both. While this core cellular function has molecular rivals, most notably pannexin channels22, there was no doubt that connexin hemichannel functions needed to be considered in both physiological and pathological contexts. The question continues to arise as to where and when connexin hemichannels play a role in normal healthy physiology and where and when aberrant hemichannel activity would lead to cell death or disease. No fewer than 1500 peer-reviewed papers have featured the existence and functional role of connexin hemichannels in virtually every organ of the human body. These studies range from Cx43 hemichannels engaging in a ventricular arrhythmogenic mechanism within microdomains of cardiac intercalated discs23 to Cx43 hemichannels in astrocytes serving as a therapeutic target in spinal cord injury24 and to Cx43 hemichannels in osteocytes playing a critical role in mechanical load-induced bone formation25. Intuitively, connexin intercellular channels tend to favor an open state, as supported by recent structural experimental evidence, but our view is that hemichannels must have a resting closed state under homeostatic conditions in order to protect the integrity of the cytosol30.While connexins form intercellular channels that permit the direct passage of metabolomes, the 1991 discovery that macrophages could release ATP, via what the authors termed “half-gap junctions” or what today we would call hemichannels, expanded our view of connexin function31. Likewise, the evidence for functional Cx46 and Cx50 hemichannels in highly specialized lens tissue in the presence and absence of disease is compelling33. Still, other less well-studied connexins like Cx62 may have dual roles in platelets, functioning as both hemichannels and intercellular channels34. One must also consider that connexin hemichannels may only partially open, permitting the smallest of channel permeants to pass. Insight into this notion was provided when cryo-EM was used to solve the hemichannel structure of a connexin that appears unable to form functional gap junction channels, Cx31.331. Resolution analysis that exceeded 2.6 Å revealed that Cx31.3 hemichannels adapt to a partially closed state that would allow the passage of chloride ions through the 8 Å pore while preventing the passage of other cell metabolites27. Cryo-EM was also used to examine the open conformation of Cx26-N176Y mutant hemichannels within dynamic lipid bilayer nanodiscs28. Here, the alpha-helical structures found within the mutant Cx26 hemichannels were found to be the same as reported for Cx26 intercellular channels while the flexibility identified within the extracellular loops would probably serve to facilitate hemichannel docking in cases where complete gap junction channels are formed28. However, likely owing to their intrinsic disorder, we still know little about the structure of intracellular loop and C-terminal tail regions. Thus, there is no doubt that further high-resolution analysis of different connexin hemichannels and intercellular channels will continue to inform on their open and closed states.However, evidence continues to mount that hemichannels play a more universal and canonical functional role in non-diseased tissues. As an example, considerable evidence suggests that Cx31.3 may not even have the capacity to form traditional gap junction intercellular channels, raising the notion that their main role in cells may be to act as functional hemichannels36. Mutations in this class often lead to the loss of cell integrity and cell death, but other mechanisms of action need to be considered36. Blocking connexin hemichannels as a means of regulating the inflammatory response has given credence to the notion that this may be an effective strategy to treat chronic inflammatory eye diseases and eye injuries9. In fact, hyperactive or leaky hemichannels in disease or injury are somewhat ideal targets when considering commercialization and deployment of connexin hemichannel blockers37. Preferably, such hemichannel blockers should be specific to the aberrant hemichannel in question, a consideration that aligns with smart small-molecule design modeled from high-resolution connexin hemichannel structures, peptide mimetics that take into account both structure and domain flexibilities, and high-avidity antibodies. As an example, an antagonist antibody was shown to have efficacy in blocking leaky mutant Cx30 hemichannels in the treatment of Clouston syndrome38. Similarly, the hemichannel blocker flufenamic acid was found to inhibit aberrant Cx26-G45E hemichannel function, reducing the symptoms of keratitis ichthyosis deafness found in mutant mice39. As noted by others, several connexin-based therapeutics that have entered clinical trials need to consider the specificity of the connexin blocking agent and whether the pathological features of hemichannels can be selectively and effectively targeted40.To our minds, there remains little doubt that aberrant connexin hemichannel function is at the root of several inherited connexin-linked diseases and cases of tissue injury as well as in chronic and acute diseases. Many missense and truncating connexin-gene mutations lead to hyperactive or leaky hemichannels that appear especially prevalent in connexin-linked skin diseases41. In the case of Cx43, the interactome includes proteins involved in protein trafficking, connexin turnover, connexin assembly, connexin post-translational modification, scaffolding, and other functions that have been extensively reviewed elsewhere42. The gap junction scaffold is also functionally important for other connexins, as has been shown for Cx30 in the cochlea, where ephrin-B2 interacts at the periphery of the gap junction regulating gap junction turnover43 in a manner perhaps analogous to ZO-1 interaction with Cx4344. We believe the scaffold function of gap junctions will be found to play critical regulatory roles as more research is performed on other connexins, and we look forward to new revelations.Finally, while connexins are foundational molecules needed to assemble both hemichannels and gap junction channels, we would be remiss if we did not draw attention to the connexin interactome. The connexin interactome is highly dependent on the connexin family member as some interactomes are small , while others are in excess of 50 proteins 45. It is now clear that more than eight protein kinases phosphorylate Cx43 at most of the 21 serine residues and at least three of the six tyrosine residues found within the cytoplasmic exposed carboxy terminus  or to mimic a constitutively phosphorylated residue . Via this approach, casein kinase 1 phosphorylation of Cx43 was found to regulate several critical physiological events, including (a) proper cardiac beat rhythm58 and response to ischemia56, (b) efficient epidermal wound healing57, and (c) the effects of stromal fibroblasts on promoting pancreas cancer progression59, while (d) MAPK phosphorylation was shown to regulate neuroprotection during stroke60.Our current understanding of the regulation of gap junctions is heavily biased toward Cx43. Cx43 is expressed in most cell lines even if they originated from cells that do not typically express Cx43 . Furthermore, our analysis of the literature suggests that Cx43 is natively expressed in nearly half of the more than 200 cell types found in the human body. Many Cx43 post-translational modifications, including ubiquitination, acylation, hydroxylation, carboxylation, methylation, sumoylation, and nitrosylation, have been reported, but we know the most about the functional consequences of Cx43 phosphorylationterminus . We also45, but in some cases the specific residue or direct linkage of the phosphorylation event to an effect on connexin-linked physiology is lacking. We conclude that many of the alpha subfamily of connexins that have been studied have shown at least some evidence of regulation by phosphorylation. However, some connexins, particularly those of the beta subfamily that have short C-terminal regions, have not been shown to be specifically regulated by phosphorylation , although caution should be exercised as the effects of phosphorylation might be apparent only under very specific conditions. In any case, we believe new interest in the less common connexins that are expressed in diverse tissues will more clearly delineate the roles connexin phosphorylation plays in gap junction and hemichannel regulation.Our knowledge of phosphorylation of connexins other than Cx43 and the resulting physiological effects is in general less well-defined. There are reports that Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx46, Cx47, and Cx50 are phosphorylatedin situ. While connexin family members share considerable sequence homology, it is now clear that connexin members are remarkably diverse with unique structures, regulatory motifs, post-translational modifications, interactomes, cell expression profiles, and subcellular distributions that all contribute to diverse intercellular channel and hemichannel functions in cells and tissues.After an exhaustive review of over 180 peer-reviewed papers related to endogenous connexin expression, we conclude that the 21-member connexin family can be mapped to over 110 distinct cell types found within all 12 human body systems. On one end of this spectrum, it is unclear what human cells express Cx23, while at the other end, Cx43 has been convincingly shown to be expressed in 92 cell types, reflecting its dominance as the most widely distributed human connexin. After decades of intense investigation, the gap junction community not surprisingly is best equipped to describe where and when Cx43 functions regulate cell and tissue physiology. That said, the functional roles of lesser studied connexins  remain poorly understood, a situation made more difficult by the lack of reliable antibodies to specifically detect their expression 61, connexin expression at non-canonical sites such as exosomes62 and mitochondrial outer membranes63, as well as the role that connexins play in cancer invasion, metastasis, and resistance to treatment64. There remains no doubt that connexins regulate a variety of cellular functions in almost every human organ. However, the challenge remains to sort out the tissue-context roles played by hemichannels/channels, a task made more complicated by our relatively rudimentary knowledge of what metabolites pass through each channel subtype in situ and the fact that connexin subtypes can intermix to form heteromeric and heterotypic channels. As more and more connexins get linked to diseases, a broad spectrum of organ-specific investigators from around the world are being attracted to the gap junction field in search of new therapeutic targets. We wholeheartedly welcome these new investigators to the field and believe they will pave the way to a better understanding of the functional importance of connexin-based cellular communication in all tissues.New advances in understanding hemichannel functions, intercellular channel and hemichannel structures, genetic linkages to disease, and the connexin interactome all point to critical and canonical roles for connexin hemichannel and channel functions in both disease and normal physiology. Some new areas of intensive study include the role of hemichannels in cardiac conduction


Using a robust policy evaluation framework is critical to inform policymakers about the relevance of a policy in a structured way, clarifying its implications and providing arguments and criteria to compare competing options and decide which policy should be prioritised. In the current context of complex societal, environmental and public health challenges, decision-makers demand tools that allow a comparative assessment of the effectiveness of multisectoral policies and their comparison under different scenarios.This presentation will detail a socio-technical “desirability-doability” framework (2xD). In 2xD, an additive value model is constructed to measure the desirability of public policies, and their doability is appraised under two contrasted scenarios. For these purposes, the MACBETH approach is used in developing the three group modelling phases of 2xD: (I) Structuring facilitated workshops, (II) Evaluation decision conference, and (III) Desirability-doability decision conference. Tailor-made interactive protocols or questioning procedures are used to elicit group judgements, based on which objective-specific value scores are assigned to the policies, and the objectives are weighted (in II). Finally, we elicit doability scores for the policies under each scenario (in III).We present how the 2xD framework was applied in Lisbon's urban health policymaking setting and discuss case insights regarding the role of scenario analysis in the appraisal and selection of policies.The 2xD framework advances knowledge on how to assist policymakers in evaluating and selecting policies and contributes to the literature on overall policy formulation and evaluation. Specifically, the framework enabled a group of policymakers to balance (multicriteria) desirability versus doability of policies under the light of two contrasting scenarios, therefore incorporating uncertainty in their decision-making process.


Previous research suggests associations between low systemic levels of vitamin D and poor breast cancer prognosis and between expression of the vitamin D receptor (VDR) in breast cancers and survival. This study aimed to study associations between pre-diagnostic systemic levels of vitamin D and expression of VDR in subsequent breast tumors, and interactions between vitamin D and VDR on breast cancer mortality. Systemic vitamin D levels were measured in women within the Malmö Diet and Cancer Study. The expression of VDR was evaluated immunohistochemically in a tissue microarray of subsequent breast cancers. Statistical analyses followed. Women with high levels of vitamin D had a smaller proportion of VDR negative breast tumors compared to women with low levels of vitamin D . Vitamin D levels were not found to modify the association between low VDR expression and high breast cancer mortality. To conclude, there was no statistical evidence for an association between pre-diagnostic levels of vitamin D and the expression of VDRs in breast cancer, nor did vitamin D levels influence the association between VDR expression and breast cancer mortality. Further studies are needed in order to establish the effects of vitamin D on breast cancer. Over the past few years, there has been an increasing interest in studying the potential associations between vitamin D and breast cancer. Results from studies on vitamin D and breast cancer risk have been inconclusive, although several recent studies have reported such an association to be unlikely ,2,3. In Previous studies in our research group performed within the Malmö Diet and Cancer Study (MDCS) suggest no association between pre-diagnostic levels of vitamin D and breast cancer risk . HoweverA possible explanation for the reported association between pre-diagnostic vitamin D levels and breast cancer mortality may be that the levels of vitamin D affect the expression of VDR in subsequent breast tumors. Moreover, pre-diagnostic vitamin D levels and VDR interaction may influence breast cancer prognosis. The primary aim of the present study was to analyze if pre-diagnostic systemic levels of vitamin D were associated with VDR expression in subsequent breast tumors, as we have found no previous study investigating this relationship. Secondly, this study aimed to investigate whether an association found between VDR expression and breast cancer mortality was influenced by pre-diagnostic systemic levels of vitamin D.n = 17,035) completed baseline examinations; anthropometric measurements, blood sampling, and a self-administered questionnaire investigating lifestyle, reproductive factors, previous diseases, and treatments  levels, within the previously reported case-control study . Levels Information about lifestyle and reproductive factors was retrieved from the questionnaire included in the MDCS baseline examination. Information on the type of surgery, axillary surgery, and planned adjuvant treatment (as suggested by a multidisciplinary treatment conference following surgery) was retrieved from medical records and clinical notes.Medical records and pathological reports were used to retrieve information on the size of the tumor and lymph node status. Women included in this study had been diagnosed with breast cancer between 9 December 1991 and 31 December 2010 and the histopathological reports have changed considerably during this time. Hence, information on histological grade, hormone receptor status, proliferation index factor (Ki67), and human epidermal growth factor receptor 2 (HER2) status has been retrieved from different sources during different time periods, as described previously ,15,16.Estrogen receptor (ER) and progesterone receptor (PR) status were classified as positive when a fraction exceeding 10% was reported. HER2 status was defined by in situ hybridizations (ISH) when available. When immunohistochemistry was used, a reported value of 3+ was considered positive, 0 or 1+ was negative, and 2+ was defined as missing if ISH could not be used to confirm the result. On the evaluation of Ki67 expression, it was noted that the distribution differed considerably between different periods of diagnosis. Expression of the Ki67 proliferation marker was therefore set as high, intermediate, or low based on tertiles during different time periods .In the South Swedish Health Care Region, molecular subtypes are used for prognostic evaluation . AccordiThe TMA had been constructed previously and included breast tumors diagnosed before 31 December 2010. Each of the 718 tumors contributed with two 1-mm cores to the recipient block . In thisA previous study within the MDCS identified ten SNPs that were associated with levels of vitamin D . Minor aWomen in the MDCS were followed using the Swedish Cause of Death Registry. This registry covers all deaths in Sweden and provided information on the date of death as well as the cause of death. In this study, death from breast cancer was defined as breast cancer registered as the underlying cause of death or contributing cause of death. Other women included in the survival analyses were either alive, dead from another cause, or had emigrated. The end of follow-up was the date of death, date of emigration, or 31 December 2016.Cases and controls were compared to observed differences in the distribution of lifestyle and reproductive factors. All factors differing at least three percentage points between cases and controls were potential confounders and included in the following statistical analyses.To study the risk of breast cancer, a binary logistic regression analysis, yielding odds ratios (OR) and 95% confidence intervals (CI), was used to analyze associations between tertiles of pre-diagnostic systemic vitamin D-levels and odds of invasive breast cancer overall, and odds of either VDR-negative breast cancer or VDR-positive breast cancer. Crude analyses were followed by analyses adjusted for age and year of inclusion in the MDCS, type of occupation, age at first childbirth, exposure to oral contraceptives, hormonal replacement therapy, and alcohol consumption. As a sensitivity analysis, an additional analysis was performed including body mass index (BMI).The analyses were run first with only cases and controls with complete data on all variables included , and thereafter including all cases and controls after multiple imputations of missing data. The imputation model is described in detail in Tertiles of pre-diagnostic serum levels of vitamin D were compared to note differences in breast tumor characteristics and breast cancer treatment. To investigate associations between tertiles of vitamin D levels and breast cancer mortality, a Cox proportional hazards analysis yielding hazard ratios (HR) and 95% CI:s was performed. The HR of breast cancer death was tested with medium levels of vitamin D (2nd tertile) as a reference group. A multivariable-adjusted model was subsequently constructed, where covariates known to affect levels of vitamin D, such as age and season of diagnosis were included, as well as factors known to influence breast cancer prognosis; the size of the tumor, lymph node status, and molecular subtypes.Another Cox proportional hazard analysis was used to confirm the previously shown association between VDR expression and breast cancer mortality. Following this, all analyses were repeated, stratified by tertiles of vitamin D, in order to examine whether vitamin D levels influence the association between VDR expression and breast cancer mortality . These mKaplan-Meier estimates were plotted to visualize unadjusted survival associations and to confirm the association of proportional hazards.All statistical analyses were performed using SPSS .Levels of vitamin D ranged between 18 and 90 nmol/L within the 1st tertile, between 64 and 116 nmol/L within the 2nd tertile, and between 89 and 288 nmol/L within the 3rd tertile. There is an overlap of levels between tertiles, due to the seasonal variation of vitamin D levels, as tertiles were ranged within the month of collected blood samples. After multiple imputations, a pooled analysis of results from the imputation of vitamin D levels showed ranges from 18 to 90 nmol/L within the 1st tertile, 64 to 111 nmol/L within the 2nd tertile, and from 88 to 288 nmol/L within the 3rd tertile of vitamin D.As a description of the cohort, the distribution of differences in lifestyle factors and levels of vitamin D between cases and controls are presented in There was no statistical evidence for an association between levels of vitamin D and the risk of breast cancer, although there was a tendency toward a lower risk of invasive breast cancer among women with medium levels of vitamin D, compared to low levels, in both crude  and adjusted  analyses using data from multiple imputations .Compared to low levels of vitamin D, a statistically non-significant association between high levels of vitamin D and a lower risk of VDR negative tumors was found in the crude  and the adjusted  analysis . When teThe distribution of tumor characteristics in relation to vital status is presented in Distributions of tumor characteristics between different tertiles of vitamin D are presented in The The mean follow-up time for breast cancer mortality was 13.1 years with a standard deviation of 5.8.p = 0.02, Differences in breast cancer survival according to the tertile of vitamin D showed that women with medium levels of vitamin D had a survival rate of 88% compared to women with low or high levels of vitamin D with survival rates of 78% and 81%, respectively  when imputed data were used. Moreover, an association between high levels of vitamin D and a relatively higher risk of breast cancer death in both crude and adjusted analyses was seen; however, this result was not statistically significant .Kaplan-Meier curves comparing survival for positive vs. negative VDR, stratified by tertiles of vitamin D are shown in The results from this study show no statistically significant evidence that pre-diagnostic vitamin D levels are associated with breast cancer risk. Nevertheless, there were indications that women with high levels of vitamin D were less likely to develop a breast tumor with low VDR expression, compared to women with low levels of vitamin D. Low pre-diagnostic levels of vitamin D were associated with an increased breast cancer mortality. Pre-diagnostic levels of vitamin D did not alter the association between VDR expression in a breast tumor and a favorable breast cancer prognosis.Recent research from large cohorts indicates that there is no association between vitamin D levels and risk of breast cancer ,3. The rWomen with high pre-diagnostic levels of vitamin D had a lower percentage of VDR negative tumors compared to women with low or medium levels of vitamin D. This, together with results from Cox analyses showing a lower risk of a VDR negative tumor with increasing levels of vitamin D, indicate that vitamin D levels may influence whether or not a breast tumor express VDR, even though these results did not reach statistical significance. VDR negative tumors were less common than VDR positive tumors, and therefore statistical power was limited in the analysis of VDR negative tumors. As there are no publications from previous studies on associations between vitamin D levels and VDR expression, it is not possible to compare our results with others. Our results need to be confirmed in a cohort containing a larger number of tumors.In this study, we found that women within the lowest and highest tertiles of pre-diagnostic vitamin D levels had a relatively higher breast cancer mortality compared to women within the medium tertile of vitamin D. Similar results have been reported previously by our group . The curThe present analysis also found an association between low expression of VDR and a relatively higher risk of breast cancer death in the crude analysis, but when adjustments for factors known to be associated with prognosis were made, hazard ratios were attenuated, and confidence intervals widened which weakened the statistical evidence. A previous study by our group, using the same population showed similar but statistically stronger results . This inOur previous study showed a stronger statistical association in the molecular subtype group of Luminal B-like tumors, compared to other subtypes , and othAnother aim of this study was to analyze if the association between VDR expression and breast cancer mortality in any way was affected by pre-diagnostic vitamin D levels. When the analysis was stratified on different tertiles of vitamin D, a pattern with a relatively higher risk of breast cancer death for women with VDR-negative tumors could be seen within all the tertiles, with the strongest association within the lowest tertile of vitamin D. The association did not differ between tertiles to an extent that suggested any effect modification by pre-diagnostic vitamin D levels. In order to investigate if vitamin D levels modify the association between VDR status and prognosis, it would probably be more accurate to compare vitamin D levels from blood samples taken at the time of diagnosis, which we, unfortunately, do not have access to.Vitamin D ranges used in this study are based on tertiles, as this method gives similarly sized groups which are preferred in statistical analyses. Since vitamin D levels vary with seasons in northern latitudes the groups were based on which month the blood sample was taken, and therefore there were overlaps of vitamin D levels between tertiles. There is still an ongoing debate on recommendations for adequate vitamin D levels. Swedish cut-offs are based on recommendations from the Endocrine Society, <25 nmol/L—deficiency, 25–50 nmol/L—insufficiency, >50 nmol/L—sufficient, 75 nmol/L—optimal level, >125 nmol/L—potentially unhealthy and >250 nmol/L—potentially intoxication ,25. A reIn the present study, test results from two different studies on women within one large cohort were combined. As a consequence, values for vitamin D levels were missing for 12% of subjects and VDR expression was missing for 26% of women in the study population. Still, a study population of 497 women with breast cancer remained, but since there is a probable pattern of missingness at random of both levels of vitamin D and VDR expression we choose to perform multiple imputations to also include women with missing values in the statistical analyses. For instance, when a TMA is used for the analysis of protein expression, small tumors contribute with tissue less often than large tumors. Using multiply imputed data on VDR status diminishes this selection bias to some extent since values on VDR status can be imputed on small tumors, taking tumor size into account. Since data on other predicting variables, such as age and BMI at inclusion and age at the time of diagnosis and size of tumor are complete, the multiple imputation model is considered stable and reliable to impute large numbers of missing values.In the Cox regression analyses, we chose to adjust for known prognostic factors of breast cancer. These factors are also used when patients are evaluated in the post-surgical treatment conference which suggests adjuvant treatment. We decided not to adjust analyses for treatment factors, which could have led to over-adjusting the statistical model. There is a known association between low levels of vitamin D and a high BMI , and alsMost previous studies investigating associations between levels of vitamin D and breast cancer have not had access to pre-diagnostic levels of vitamin D but have instead measured vitamin D from blood samples drawn at the time of diagnosis ,29. SincWe hypothesize that women with habitually higher levels of vitamin D could develop breast cancers that are more often VDR positive. They might also have a larger proportion of unliganded VDR present, which in a previous study has been found to have tumor proliferative effects , and henFuture studies should be performed in order to identify subgroups of breast cancer patients that could benefit from vitamin D supplementation along with conventional breast cancer treatment. Further studies on associations between tumor characteristics and host factors with breast cancer prognosis are needed.The hypothesis that pre-diagnostic vitamin D levels would associate with VDR expression in subsequent breast cancer could not be confirmed with statistical evidence in this study. The association between VDR expression and breast cancer prognosis was not modified by pre-diagnostic levels of vitamin D. It would be preferable to investigate whether vitamin D levels at diagnosis and VDR expression in a tumor coincide to influence breast cancer prognosis.


In this review, we discussed mitochondrial function in cancer cell metabolism and specially explored the unique role of mitochondria in cancer stem cells and the tumor microenvironment. Targeting the OXPHOS pathway and mitochondria-related metabolism emerging as a potential therapeutic strategy for various cancers.Mitochondria are intracellular organelles involved in energy production, cell metabolism and cell signaling. They are essential not only in the process of ATP synthesis, lipid metabolism and nucleic acid metabolism, but also in tumor development and metastasis. Mutations in mtDNA are commonly found in cancer cells to promote the rewiring of bioenergetics and biosynthesis, various metabolites especially oncometabolites in mitochondria regulate tumor metabolism and progression. And mutation of enzymes in the TCA cycle leads to the unusual accumulation of certain metabolites and oncometabolites. Mitochondria have been demonstrated as the target for cancer treatment. Cancer cells rely on two main energy resources: oxidative phosphorylation (OXPHOS) and glycolysis. By manipulating OXPHOS genes or adjusting the metabolites production in mitochondria, tumor growth can be restrained. For example, enhanced complex I activity increases NAD Mitochondria, known as the “powerhouse of the cell”, are one of the most crucial organelles inside the cell. They evolved from bacteria and participated in the eukaryotic cell via the process named endosymbiosis, thus improving cell energy production and generating adenosine triphosphate (ATP) via the respiratory chain. Mitochondria have their translation system and genetic material mitochondrial DNA (mtDNA) without introns which are similar to bacterial DNA. These maternally inherited mtDNAs present in multiple copies which are easily mutated due to replication errors, oxidative stress or inefficient DNA repair. Usually, cancer cells harbor somatic mutations in mtDNA and mutations of mtDNA showed advantages of tumor growth. Now the somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing Mitochondria are the metabolic hub of tumor cell proliferation, survival and metastasis due to their bioenergetic and biosynthetic function This review covers several fundamental functions of mitochondria, intending to introduce non-experts to understand the unique role of mitochondria and exploring the possible therapeutic target related to mitochondria.Mitochondria are the major cellular source of NADH and house parts of the pyrimidine and lipid biosynthetic pathways, it plays various roles in cancer cell metabolism including but not limited to ATP metabolism, lipid metabolism and nucleic acid metabolism. Many different metabolites or intermediates in the tricarboxylic acid (TCA) cycle are critical in cancer cell proliferation, metastasis and apoptosis.ATP is the central energy source in the cell, it can be generated via two pathways: glycolysis in cytostome and OXPHOS in mitochondria 2 (known as the Warburg effect), slow-cycling tumor cells may prefer mitochondrial respiration as a primary source of energy Due to the heterogeneity, tumors exhibit different metabolic phenotypes. In gliomas, there are both OXPHOS-dependent cells and glycolytic-dependent cells, the mechanism lies in the lactate dehydrogenase (LDH), the former expressed both LDH-A and -B isoforms whereas the latter only expressed LDH-B and LDH-B would be expected to be essential for the use of extracellular lactate to fuel cell activities Compared with glycolysis, OXPHOS produces ATP in a higher yield but the lower rate (30 or 32 mol ATP versus 2 mol per mole of glucose) Mitochondria generate ATP through OXPHOS mainly by using pyruvate derived from glycolysis. In the absence of glucose, ATP will be produced via the degradation of fatty acids and proteins. Ketone bodies  will be produced from fatty acid oxidation (FAO) during fasting or prolonged exercise when the glucose is insufficient. Ketone bodies are generated in the mitochondrial matrix of liver cells and are subsequently exported via the blood to other organs to cover the energy demands Mitochondria consist of around 1000 proteins and more than 400 lipids The history of the relation between mitochondria and lipid synthesis and metabolism can be dated back to 1959 Mitochondria associate with lipid droplets (LDs) in fat-oxidizing tissues with fatty acid storage and oxidation capacity In addition, side products of respiratory chain such as ROS attack unsaturated lipids thus lead to lipid peroxidation (LPO), which will initiate secondary cellular responses. LPO is one of the markers for oxidative stress de novo and mainly from glucose, glutamine and CO2Purine and pyrimidine nucleotides are required for the synthesis of RNA and DNA. The purine and pyrimidine bases are from various nonessential amino acids and methyl groups donated from the one-carbon or folate pool. DNA replication and RNA production are essential processes in cell division and proliferation, their biosynthesis is upregulated in the late G1 phase Small metabolites or molecules are transported to the matrix of mitochondria by the mitochondrial carrier family (MCF) Cancer cells exhibit heterogeneity in energy resource, some rely more on OXPHOS and some others rely on more on glycolysis. The energy resource of cancer cells may shift their reliance from one to the other. For example, loss of LUC7L2 and U1 snRNP subunits shifts the energy metabolism from glycolysis to OXPHOS Mitochondrial metabolism plays an essential role in a variety of physiological and pathological processes Figure . It is c2+ and nuclear factor of activated T called signaling in vitro and in vivoAcetyl-coenzyme A (Acetyl-CoA), the most important substrates for acetylation modification, can be derived from glucose (pyruvate oxidation), fatty acid, and amino acid catabolism, it is the key indicator of cell metabolism and links metabolism, signaling, and epigenetics Citrate in mitochondria is oxidized in the TCA cycle to produce ATP, it is important for lipid synthesis and epigenetic regulation; cancer cells import extracellular citrate to support their growth in vivo in normoxia, indicating isocitrate is helpful in avoiding the failure of gasping generation and autoresuscitation in pathological conditions. The application of isocitrate showed a significant and persistent attenuation for anemia of inflammation in a mouse acute and severe anemia of inflammation (AI) and in a rat arthritis model of moderate chronic AI Isocitrate is the product of aconitase and the substrate of isocitrate dehydrogenase, it is an intermediate metabolite in the citric acid cycle found both inside the mitochondria and outside in the cytosolic shunt. Treatment of isocitrate may enhance breathing and render it more resistant to hypoxic insult and increase the amplitude of ventilation +-dependent manner and nicotinamide adenine dinucleotide phosphate (NADP)+-dependent manner, it can also be formed in the cytosol in an NADP+-dependent manner + and promote 2OG to D-2-hydroxyglutarate (D-2HG) IDH1 is the most frequently mutated gene in cancer. In low-grade glioblastoma (LGG), more than 80% of IDH mutations occur in the IDH1 gene, being dominated by R132H IDH1Isocitrate is oxidized by the enzyme IDH, where carbon dioxide is liberated and 2-oxoglutarate  is the oxidized product, it converts isocitrate to α-KG. IDH can be inside mitochondria in a nicotinamide adenine dinucleotide (NAD)IDH1 and IDH2 induce epigenetic and transcriptional reprogramming, and differentiation bias and promote the development of a number of malignancies Moreover, IDH mutations are associated with the altered IL-1β responses in acute myeloid leukemia (AML) α-KG is a membrane-impermeable and key endogenous metabolite in the TCA cycle, its level changes upon fasting, exercise and aging α-KG is an important cofactor for histone demethylase including Jumonji C (JmjC) and TET, it plays important roles in methylation modification α-KG is catalyzed by α-ketoglutarate dehydrogenase (α-KGDH) and metabolized into succinyl-CoA, this GDH-mediated α-KG can inhibit IKKβ activation and block nuclear factor κB (NF-κB) activation, promote glucose update and cell survival by upregulating GLUT1, thereby accelerating gliomagenesis Succinate is a key modulator of cell metabolism in hypoxic response, an important player in tumorigenesis, protein succinylation and inflammatory signal + T cells Succinate also acts as an inflammation signal Succinate and other metabolites with similar structures, such as D-2HG and fumarate, are considered oncometabolites SDH, also known as mitochondrial Complex II, is a mitochondrial enzyme participating in both the citric acid cycle and the electron transport chain. It oxidizes succinate to fumarate in the TCA cycle and oxidizes ubiquinone to ubiquinol in the mitochondrial ETC SDH has now been defined as a tumor suppressor and succinate an oncometabolite Fumarate is generated in the TCA cycle by the SDH-catalyzed dehydrogenation of succinate, acting as a bona fide oncogenic molecule and a key activator of a variety of oncogenic cascades FH is distributed in both cytosol and mitochondria; the mitochondrial FH is part of the TCA cycle and it catalyzes the reversible hydration of fumarate to malate. The mutation of FH has been identified in a variety of cancers. FH-deficient cells respond to mitochondrial impairment by a series of compensatory metabolic changes: they increase their glycolytic pathway with the increase of glycolytic-related gene transcription and the inhibition of pyruvate dehydrogenase (PDH); the glutamine is converted to α-KG and eventually to citrate via reductive carboxylation. FH-deficient cells harbor lower fumarate and preserve Aconitase 2 (ACO2) function to promote tumor progression.The FH deficiency also affects epigenetics. The loss of FH causes hypermethylation in the promoter of the tumor suppressor cyclin-dependent kinase inhibitor 2A (CDKN2A), the hypermethylation of CDKN2A is a predictive factor for unfavorable prognosis of various cancers +/NADH homeostasis to maintain the activity of mitochondrial MDH and promote L-lactate oxidation in mitochondria Malate is synthesized from fumarate by fumarase and further oxidized to oxaloacetate by malate dehydrogenase (MDH) with the accompanying reduction of NAD+. The metabolic imbalance results in the impairment of either mitochondrial and/or cytoplasmic metabolism. The oxidation of cytosolic NADH by the mitochondria requires malate-aspartate shuttle MDH reversibly converts malate to oxaloacetate using NADH and serves as important oxidoreductase in several metabolic pathways. MDH has two isoenzymes, MDH1 in cytosol and MDH2 in mitochondria. Both are closely associated with cancers. The mutation of MDH2 destroys the TCA cycle and affects energy metabolism, leading to mitochondria dysfunctional diseases such as epilepsy, hypotension etc. The upregulation of MDH2 increase energy metabolism and the deficiency of MDH2 reduces the production of ATP and the augment of ROS. MDH2 is highly expressed in many types of cancers, MDH2 concentration was higher in early-stage NSCLC patients compared with that in controls, therefore, MDH2 can be a potential biomarker for early detection of NSCLC de novo synthesis of OA and inhibit LDHA in cancer cells Oxaloacetate can be generated from malate, pyruvate, phosphoenolpyruvate and aspartate. It plays important roles in cancers and other diseases by modulating cell metabolism. OAA is a competitive inhibitor of LDHA, high pyruvate kinase M2 (PKM2) activity reduces LDHA activity via upregulating cytosolic OAA in cancer cells, the elevated PKM2 increase the Cancer stem cells (CSC) or tumor-initiating cells, characterized by the ability to self-renew, are a small subset of cancer cells responsible for tumor initiation, maintenance, growth, metastasis, recurrence and drug resistance. Mitochondria play crucial roles in cancer stem cells in different aspects such as cell metabolism, drug resistance, apoptosis etc. Metabolic reprogramming through the regulation of mitochondrial activities is a key feature of CSCs Increasing evidence had demonstrated that a myriad of CSCs relies highly on OXPHOS for obtaining energy in vitro and in a xenotransplantation model of human CML Inhibition of OXPHOS in CSCs induces cell death, in chronic myeloid leukemia (CML), the combination treatment with imatinib (a tyrosine kinase inhibitor) and tigecycline (an antibiotic) that inhibits mitochondrial protein translation, selectively eradicates CSCs both de novo synthesis, and regulates different energy-producing metabolic processes including OXPHOS. It has been demonstrated to be overexpressed in circulating tumor cells. High expression of PGC-1α enhances mitochondrial biogenesis, strong antioxidant activity and fewer ROS, increase OXPHOS and promote cancer cells' invasion and metastasis PGC1-α, the transcription co-activator peroxisome proliferator-activated receptor gamma co-activator 1α, acts as a master regulator of mitochondria ROS as the side product of the respiratory chain has been applied in radiation therapy, which causes DNA damage directly by ionization or indirectly through the generation of ROS to destroy cancer cells Mitochondria are dynamic organelles frequently undergoing fission and fusion to maintain their morphology and regulate their function. Mitochondrial morphology and function contribute to a stem-like phenotype of cancer cells The exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate the AML resistance to OXPHOS inhibition By asymmetric division, stem cells generate two daughter cells in different traits. Daughter cells that received fewer old mitochondria maintain stem cell traits. The failure to asymmetrically apportion old mitochondria in a single division can cause a persistent loss of stemness in stem cells and cancer stem cells 6-methyladenosine (m6A) accumulation Mitophagy is an evolutionarily conserved cellular process, it is critical in maintaining mitochondrial integrity and cell homeostasis by removing damaged or surplus mitochondrial fragments to the lysosome for degradation TME comprises various cell types including tumor cells, immune cells and the surrounding environment of tumors. TME plays pivotal roles in tumor imitation, progression and metastasis Mitochondria generate ROS from complex I and complex III at the inner mitochondrial membrane during oxidative phosphorylation, this can be identified by inhibitors of the mitochondrial respiratory chain such as Rotenone and Antimycin A as well as the substrates for various complexes such as pyruvate or succinate Mitochondrial ROS also oxidizes proteins in the ETC and leads to a decrease in mitochondrial respiration and finally causes related signaling pathway activation The interaction of ROS and autophagy play important roles in both cell damage and cell survival. ROS may regulate autophagy at transcriptional and post-transcriptional levels via various signal pathways such as OS-FOXO3-LC3/BNIP3-autophagy, ROS-NRF2-P62-autophagy, ROS-HIF1-BNIP3/NIX-autophagy and ROS-TIGAR-autophagy ROS can modify cellular functions via interacting with HIF-1α and NFκB Hypoxia has been regarded as a hallmark of the tumor microenvironment, it happened due to increased oxygen consumption and inadequate oxygen supply in the majority of tumors Hypoxia activates mitophagy 2O2 treatment. The mitochondrial HIF-1α protects against oxidative stress induced apoptosis and reduces ROS production to maintain mitochondrial membrane potential and reduce the expression of mitochondrial DNA-encoded mRNA in response to hypoxia HIF is a master regulator for adaptation to low oxygen content and HIF proteins are key determinants of cellular response to hypoxia Hypoxia also impacts the mitochondrial morphology including cristae structure. More mitochondria are in the fission process to promote mitophagy and keep ROS production in a comparatively low level and maintain decreased respiratory activity Mitochondria dysfunction and oxidative stresses have been found in many diseases including cancers, neurodegenerative disorders, pathogen infections, etc. The mtDNA decrease, OXPHOS dysfunction, and the upregulation of ROS are found in various tumors. The OXPHOS downregulation is associated with poor clinical outcome across most of the cancer types and correlates with a gene signature characteristic of invasive and metastatic tumors. Targeting mitochondria has been reviewed a lot and various inhibitors have been developed Table . Some ofMetformin has been used as a treatment for diabetes for the past 100 years IACS-010759 is a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain tumors and AML reliant on OXPHOS. IACS-010759 is currently being evaluated in phase 2 clinical trials in relapsed/refractory AML and solid tumors Gboxin, a small molecule, emerges as a novel OXPHOS inhibitor that targeting the activity of F0F1 ATP synthase in mitochondria Atovaquone, an approved antimicrobial drug, has demonstrated anti-cancer potential and ability in clinical trial in treating ovarian cancer recently, it inhibits tumor cell proliferation by reducing ATP production by inhibiting mitochondrial complex III and increasing ROS levels Dichloroacetic acid, a novel anticancer drug, has been proved to have synergistic and inhibitory effects on liver cancer cells in clinical studies CPI-613 is a potent mitochondria metabolism inhibitor + have been demonstrated to possess signaling capacity and to influence immunity Mitochondrial metabolism is necessary for cancer cell proliferation, tumorigenesis and metastasis. Mitochondria execute mitophagy to clear damaged mitochondria and their fusion and fission dynamics may support tumor stemness and growth. Mutations of mtDNA are commonly found in various tumors, eliminated mutated mtDNA limits tumorigenesis. mtDNA escaped from stressed mitochondria by mPTP- and VDAC-dependent channels to activate the cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA In multiple cancers, the majority of tumor cells especially slow-dividing cancer cells generate ATP via mitochondrial OXPHOS. CSCs are resistant to regular chemo- and radiotherapy, but a great number of them rely on OXPHOS for energy supply, plenty of data highlight the existing connection between CSCs fate and mitochondrial metabolism and lots of inhibitors have been developed to target the complexes or proteins in OXPHOS to reduce CSC viability.Targeting specific players including both TCA enzymes and OXPHOS-related-genes in mitochondria or mitochondria-related pathways may serve as novel and promising therapeutic approaches for cancers due to the multiple functions of mitochondria in cancer cells. However, the obstacles are there too, mitochondria are not only working in cancer cells but also in normal cells, how to selectively eliminate cancer cells without affecting healthy cells is the biggest challenge. The novel inhibitor Gboxin is one of the trials since its function relies on the high gradient in mitochondria of CSCs. Further investigations are required on the difference of mitochondria between cancer cells and normal ones so that targeting this difference will be a potential selective therapy.