Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors characterized by aggressive behavior, high risk of distant recurrence, and poor survival. Chemotherapy is still the main therapeutic approach for this subgroup of patients, therefore, progress in the treatment of TNBC remains an important challenge. Data derived from molecular technologies have identified TNBCs with different gene expression and mutation profiles that may help developing targeted therapies. So far, however, only a few of these have shown to improve the prognosis and outcomes of TNBC patients. Robust predictive biomarkers to accelerate clinical progress are needed. Herein, we review prognostic and predictive biomarkers in TNBC, discuss the current evidence supporting their use, and look at the future of this research field. Triple negative breast cancer (TNBC) is a subtype of breast cancer lacking expression of estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) . TNBC acHowever, sound clinical applications of this molecular classification are yet to appear .In the last years, a great effort has been spent to identify new biomarkers and relative therapies, but only few of these have proven useful in clinical trials. Beside poly ADP-ribose polymerase (PARP) inhibitors, that have successfully been incorporated in the clinical practice in the BRCA1/2 subgroup of patients ,10, and BRCA1/2) and hereditary breast and ovarian cancer syndrome (HBOC) revolutionized clinical cancer genetics. First, the identification of a germline BRCA1/2 mutations impacted cancer screening and prevention practices in this subgroup of patients and their relatives. Later, the knowledge of the pathological mechanisms of these mutations, led to the development of new therapeutic approaches, such as poly (ADP-ribose) polymerase (PARP) inhibitors, selectively directed on BRCA 1 or BRCA 2 deficient cells [The relationship between the tumor-suppressive genes BReast CAncer type 1 and Type 2  of DNA. The BRCA1 gene on chromosome 17q21 has a broader role than BRCA2 in responding to DNA damage; it controls the signal transduction pathway involved in HHR, including recognition of genomic damage, checkpoint activation, recruitment of DNA repairing proteins, and decision of whether DNA double strand breaks (DSBs) needs to be resected; in addition, it is also involved in chromatin remodeling and transcription control [BRCA2 gene on chromosome 13 plays the key role of recruiting the DNA recombinase RAD51 and localizing it to damaged DNA [BRCA1 or BRCA2 genes. However, compared to other subtypes of breast cancers, women with TNBC have a higher prevalence of germline BRCA mutations (gBRCAm), about 11–31% [BRCA1/2 genes (sBRCAm) were also found in primary ovarian and breast carcinomas [ control ,15. The aged DNA . HRR is rcinomas . BRCA1/2 genes are defective, DNA damage is repaired by non-conservative mechanisms of DNA repair, such as non-homologous end joining (NHEJ), in order to maintain cell viability. This process of repairing DSBs is simpler than HHR and consists in joining the two broken DNA ends without the homologous DNA sequence to guide the repair: it is, therefore, prone to joining errors with mutation of the original sequence. In BRCA1/2-deficient cells, DNA DSBs repair is dependent on PARP1 protein [BRCA1/2-deficient cells can lead to severe, highly selective toxicity in these cells [When  protein ,20. PARP protein . Therefose cells . This prse cells .® test , whose accuracy was established based on a retrospective/prospective analysis of the OlympiAD trial population. In the same year, FDA approved talazoparib for patients with deleterious or suspected deleterious gBRCAm, HER2 negative locally advanced or metastatic breast cancer, based on the phase III EMBRACA study results [In the past years, PARP inhibitors (PARPi) have been extensively studied as targeted therapy for gBRCAm ovarian and breast cancer patients. In 2014, the US Food and Drug Administration (FDA) approved the first PARPi, Olaparib, as monotherapy for patients with deleterious or suspected deleterious gBRCAm advanced ovarian cancer . Later,  results . Based o results . Promisi results .Other PARPis, such as Niraparib, Rucaparib, and Veliparib, are still under clinical evaluation in breast cancer both as monotherapy and in different combinations ,32,33.BRCA1/2 [BRCA1/2. The TNBC cell lines MDA-MB-436 (BRCA1-deficient), MDA-MB231, and MDA-MB-468 resulted sensitive to Talazoparib, suggesting that the benefit of Talazoparib might extend to TNBC without BRCA1/2 mutations [Several pre-clinical studies have shown that PARPis are able to inhibit cell growth and promote the death of breast cancer cells that are wild type for BRCA1/2 . A recenutations . This suBRCA1/2 genes. The term “BRCAness” has been used to describe a dysfunction in the BRCA-related DNA repair mechanism that is not due to mutations of the Deficiencies in a number of tumor-suppressor genes involved in HRR, such as ATM and ATR, may share the same therapeutic vulnerabilities with BRCAm tumors and confer sensitivity to PARP inhibition. Therefore, tumors with mutations in other HRR genes may also respond to a PARP inhibitor treatment .BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D), in 202 patients, including TNBC and hereditary non-TNBC patients. As a result, 22.2% of 158 patients in TNBC group carried mutations in genes involved in DNA repair by HR [PALB2, BARD1, BRIP1, RAD51, RAD51C, RAD51D, ATM, FAAP20, CHECK2, FAM1, FANCE, FANCM, POLQ [On 2015, Domagala et al. performed genetic testing for 36 common germline mutations in genes engaged in HRR, . AZD6738 is an ATP competitive, orally bioavailable inhibitor of the Serine/Threonine protein kinase Ataxia Telangiectasia and Rad3 related (ATR), while AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents in several trials [Based on these findings, some clinical trials are now testing the use of PARPis in patients with BRCAm or HRD to maximize the number of individuals who may benefit from PARP inhibition ,45,46. Tl trials . Both, Wl trials ,49.BRCA1/2, either germline or somatic, but also in wild-type BRCA1/2 [BRCA1/2 DDR-pathway genes (cohort 1) and on somatic mutations in these genes or BRCA1/2 (cohort 2). Results showed an ORR of 29.6% in Cohort 1 and 38.5% in Cohort 2, with sBRCA1/2 or gPALB2 mutations predictor of response in this last one [Somatic BRCA mutations (sBRCAm) were supposed to report a phenotype similar to tumors from patients with germline mutations and response to PARPi . A recen BRCA1/2 . In brealast one .BRCA1/2-deficient tumors have defects of DNA repair, the use of agents causing DNA damage have been extensively investigated as candidate therapy to promote mechanisms of cell cycle arrest and apoptosis in these tumors. Platinum drugs, causes platination of genomic and mitochondrial DNA, forming intrastrand crosslink adducts which cause double-strand breaks that culminate in the activation of apoptosis, especially when DNA lesions cannot be repaired [BRCA1/2 mutations, in advanced first and second line TNBC patients. In particular, ORR was 54.5% in patients with germline BRCA1/2 mutations, while, in patients without BRCA1/2 mutations, HRD-LOH/HRD-LST scores discriminated responding and nonresponding tumors . Five of the six long-term responders alive at a median of 4.5 years lacked germline BRCA1/2 mutations, and two of them had increased tumor HRD-LOH/HRD-LST scores [BRCA1/2 mutations, BRCA1 methylation, and non-BRCA1/2 HR germline mutations. Results showed that addition of Veliparib to cisplatin significantly improved PFS and numerically improved OS and ORR, of BRCA-like sub-group of patients, that included 76% of HRD ≥ 42, 17% of BRCA1 promoter methylation, and 7% of HRR genes mutations [Since repaired . This surepaired . RegardiT scores . At the utations .BRCA1, low BRCA1 mRNA expression or BRCA1 promoter methylation patients achieved pCR [BRCA1/2 mutation-associated breast cancer, revealed that combination of gemcitabine, carboplatin, and iniparib is active in the treatment of early-stage triple-negative and BRCA1/2 mutation-associated breast cancer. In particular, responder patients with sporadic TNBC, lacking BRCA1/2 mutation, had an elevated HRD-LOH score, suggesting that HRD assay could predict to platinum response [BRCA1/2 deficient samples [In early settings, several neo-adjuvant clinical trials have evaluated the impact of adding platinum to standard chemotherapy, however, its use remains controversial and it is not routinely recommended in unselected TNBC or BRCA mutations carriers . The neoeved pCR . The Gepeved pCR . This steved pCR . In thisresponse . In a reresponse . Recentl samples . Elevate samples .Preclinical data support a potential synergism between PARPi and platinum-based chemotherapy ,67. The Research is also focusing on biomarkers of resistance to PARPis. Acquired or innate resistance to single-agent PARPis has been frequently observed in both preclinical and clinical studies ,72,73,74The identification of biomarkers of resistance can drive the research of pharmacological strategies to delay or prevent the development of the drug resistant phenotype. Emerging data suggest that olaparib-resistant cancer models can be re-sensitized to olaparib when combined with AZD1775 or AZD6738 ,86,87, lIt is well-known that the immune system and cancer have a complex interplay: it is a multi-step process, named cancer immunoediting, largely mediated by CD8+ cytotoxic T lymphocytes, and in which both immune-stimulatory and inhibitory factors are involved. During the first phase, called elimination phase, the innate and adaptive immune system recognize and reject tumor cells, then the surviving tumor subclones can progress into a state of dormancy, the equilibrium phase, in which tumor growth is limited and tumor cells are gradually selected through upregulation of pro-survival pathways, changes in expression of molecules involved in immune suppression or angiogenesis. These immune-edited tumor cells can then enter into the escape phase, in which the tumor growth is uncontrolled .Breast tumors have been historically considered non-immunogenic diseases, with a relatively low mutation rate. However, among BC subtypes, TNBC is characterized by high mutation rate and greater tumor-infiltrating lymphocytes (TILs). TILs are present both intratumorally and in adjacent stromal tissues and are composed mainly of cytotoxic CD8+ lymphocytes, and, to a lesser extent, CD4+ T-helper cells, T-regulatory (Treg) cells, macrophages, mast cells, and plasma-cells. The presence of intra-tumoral and stromal TILs has predictive and prognostic role; in TNBC increased TILs at diagnosis have been associated with pathologic complete responses with neoadjuvant chemotherapy and improved survival after adjuvant chemotherapy ,90,91. TProgrammed cell death-1 (PD-1) is an immune checkpoint able to inhibit both adaptive and innate immune response, and is expressed on the surface of immune cells, such as T cells, B cells, natural killer (NK), macrophages, dendritic cells (DCs), and monocytes . PD-1 coPD-1/PD-L1 ligation acts as pro-tumorigenic pathway, leading to deactivation of T-cell function and resulting in the escape from immune surveillance ,101,102.PD-L1 is commonly expressed in 20% of TNBC and has been related to distinctive characteristics of BC, such as younger age, large tumor size, high grade, high proliferation, ER-negative status, and HER2-positive status. PD-L1 is expressed on about 10% of tumor cells (TC), while its expression on tumor infiltrating immune cells (IC) is higher (40–65%). The predictive role of PD-L1 positivity on IC has been validated in several clinical trials. The expression of both PD-1 and PD-L1 is associated with a good outcome and it is correlated with better overall survival and higher sensitivity to chemotherapy, confirming that the cytotoxic effect of chemotherapy is partially mediated by the immune response against tumor ,115,116.The introduction of immune checkpoint inhibitors (PD-1 inhibitors and PD-L1 inhibitors) as strategy to wake up the immune cells and reduce the tumor growth, is playing a critical role in improving treatment of TNBC. Atezolizumab (anti-PD-L1) is the first in class receiving the FDA accelerated approval on March 2019, based on results from IMpassion 130 study that showed a significant OS improvement (25 months vs. 18 months) in patients with IC PD-L1 expression (PD-L1+ IC ≥ 1%), treated with Atezolizumab plus nab-paclitaxel versus nab-placlitaxel alone as first line therapy for metastatic TNBC ,120. FDAIn the same setting, results from Keynote-355 trial, evaluating the combination of Pembrolizumab (anti-PD-1) plus chemotherapy as first line treatment of metastatic TNBC, have lately been presented at ASCO meeting. This study confirms the importance of evaluating PDL1 expression as biomarker. PDL1 has here been evaluated with the 22C3 (DAKO PharmaDx) assay using the combined positive score (CPS) defined as which is the number of PD-L1 staining cells  divided by the total number of viable tumor cells, multiplied by 100. Indeed, pembrolizumab has shown to improve progression-free survival (PFS) only in patients with a CPS score ≥10 . Of noteFinally, in neo-adjuvant setting, conflicting results from Keynote-522 and NeoTRIPaPDL1 studies, investigating the combination of standard chemotherapy with Pembrolizumab and Atezolizumab respectively, showed that the role of PD-L1 expression as predictive marker of response to immune checkpoint inhibitors is still controversial ,125. ParEvaluating other predictive biomarkers, in addition to PD-L1 testing, may help to select patients who could benefit from ICI. Microsatellite instability (MSI) is a hypermutable phenotype generally deriving from a deficit in the DNA mismatch repair mechanism . MSI is evaluated by identifying mutations involving microsatellites located throughout much of the genome as short DNA sequences repeated in tandem. High levels of microsatellite instability (MSI-H), corresponding to dMMR, were found across several cancers, such as endometrial and gastrointestinal cancers (20%–30%). MSI-H is correlated to a high neoantigen burden and, therefore, to a high immunogenic potential and sensitivity to immuno-checkpoint inhibitors, irrespective of the tumor histologic type. MSI-H/dMMR was the first biomarker to grant a “site-agnostic” FDA approval to an anticancer drug: on October 2016, based on the results of the Keynote 158 trial, Pembrolizumab was approved for use as monotherapy on solid tumors with MSI-H or dMMR and without satisfactory therapeutic alternatives . While t® CDx test to identify patients with solid tumors with TMB score who may benefit from immunotherapy treatment with Pembrolizumab monotherapy. FoundationOne CDx is a next-generation sequencing-based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens. The accelerated approval was based on data from a prospectively planned retrospective analysis of 10 cohorts of patients with various previously treated unresectable or metastatic solid tumors, who were enrolled in KEYNOTE-158 (NCT02628067), a multicenter, non-randomized, open-label trial evaluating Pembrolizumab (200 mg every three weeks). TMB status was assessed using the FoundationOne CDx assay and TMB-High (TMB-H) was defined as TMB ≥10 mut/Mb. The results showed that patients with TMB-H in solid tumors who were treated with Pembrolizumab had a higher overall response rate (29%) compared to patients with TMB [Tumor mutational burden (TMB), calculated as the total number of mutations in a sample divided by the length of the genomic target region (mut/Mb), is a good marker of tumor antigenicity. Since the greater number of somatic mutations, it is probable that these mutations will yield to misfolded proteins (neoantigens) capable of being immunogenic and providing targets for T-cell response. In several tumors, such as lung cancer, melanoma, and colorectal cancers, TMB, easily evaluated by NGS techniques, represents a good predictive biomarker for ICI response, since high TMB is associated with high neoantigen burden, high T-cell infiltration and high response rate to ICI, regardless the PD-L1 status ,131. In with TMB .Despite the promising results of PD-1/PD-L1 axis blockade, additional strategies to improve the response rate and to overcome resistance to immunotherapy are ongoing, such as testing novel immunomodulatory compounds, which might increase the activity of immunotherapy treatment and contribute to convert “cold tumors” into “hot tumors.” This novel combinations are focusing on the dual immune checkpoint blockade, which includes the introduction of antibodies against co-inhibitory, such as anti-LAG-3 antibodies.Lymphocyte-associated gene 3 (LAG3) is a transmembrane protein with structural homology to the CD4 co-receptor and mainly expressed in activated CD4+ T cells, T-regulator cell, Tr1 cells, activated CD8+ T cells, natural killer cells, dendritic cells, B cells, and exhausted effector T cells. LAG3 negatively regulates the proliferation, activation, and effector function of T cells . The rolThe suggestion that other molecules can stimulate and increase the number of immune cells, enhancing the anti-tumor immune function, is leading to new combination approaches of immune checkpoint inhibitors and novel agents for the treatment of TNBC. The InCITe trial (NCT03971409) is evaluating the combination therapy of Avelumab (anti-PD-L1) and Binimetinib (MEK 1/2 inhibitor), Utomilumab (anti-4-1BB), or PF-04518600 (anti-OX40) in a multi-arm study for treatment of first or second line in metastatic TNBC patients.gain-of-function mutations of phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit, alpha (PIK3CA), loss-of-function alterations of the tumor suppressor phosphatase and tensin homolog (PTEN), deregulation of receptor tyrosine kinase signaling, and amplification and mutations of receptor tyrosine kinases [PIK3CA, PIK3CB, and PIK3CD encode three highly homologous class IA catalytic isoforms: p110α, p110β, and p110δ, respectively [PIK3CA), overall mutated in ~9% of primary TNBC. PIK3CA mutations result in activated alpha PI3K, leading to an activating downstream pathway. PTEN alterations are also frequent in TNBC, with genetic loss of function occurring in 15% [PTEN, including truncating and frameshift mutations or homozygous deletion, cause loss of function with consequent hyperactivation of AKT activity. Single nucleotide variants hotspots mutations such as R130X, R233X, and R335X, allelic loss in loci of the10q23 region were also reported [mTOR were found in 1.8% of breast cancer [PIK3CA mutations have proven to have a predictive value for treatment with α-selective and β-sparing PI3K inhibitors, Alpelisib and Taselisib respectively, in the advanced setting [PIK3CA-altered, ER/PgR-positive, and HER2-negative breast cancer, indicating that the integration of genomic testing for PIK3CA mutation may be useful in the selection of therapy [The phosphatidylinositol 3-kinase (PI3K) pathway is a key regulator of survival, growth, proliferation, angiogenesis, metabolism, and migration. It comprises a family of intracellular signal transducer enzymes with three key regulatory nodes, PI3K, AKT, and mammalian target of rapamycin (mTOR). PI3K activation phosphorylates and activates AKT, that regulates the functions of numerous cellular proteins, including the FoxO proteins, mTOR complex 1 (mTORC1), and S6 kinase ,141. In  kinases ,143. The kinases . PI3Ks aectively . In TNBCg in 15% ,147. Theg in 15% ,149. Inareported ,151. A sreported . Finallyt cancer . The hig setting ,157,158. therapy ,155.PIK3CA mutations may also have future relevancy for TNBC. Recently, Yuan et al. showed that combination of CDK4/6 inhibitor Ribociclib and Alpelisib caused the reduction of p-RB and p-S6, of MCL-1, induction of apoptosis, and an enhanced reduction of tumor growth in a TNBC PDX model [The predictive effect of DX model . Other fDX model . These sPIK3CA mutations [LAR TNBC, in particular, are sensitive to endocrine manipulations with AR antagonists and are enriched (approximately 40%–50%) in activating utations ,162: thiutations . PTEN-loss could enhance activation of AKT signaling. The randomized Phase II study  compared activity of Ipatasertib [PIK3CA/AKT1/PTEN genomic alterations  characterized by NGS. Results have shown that the increase in median PFS was quite modest in the Intention-to-treat population and PTEN-low subgroup but more pronounced in predefined analyses of the patient population with PIK3CA/AKT1/PTEN-altered tumors, suggesting that a complete assessment of PI3K pathway could have a predictive value rather than a single alteration [Loss of PTEN tumor suppressor activity has been investigated as biomarker of response to AKT inhibitors, based on finding that tasertib ,166,167,teration . PIK3CA/AKT1/PTEN-Altered, locally advanced or metastatic, TNBC or ER/PgR-positive, HER2-negative patients. Genetic alterations will be evaluated in relevant genes in the PI3K/Akt pathway, both in tissues and in blood samples, by NGS assay. At present, Phase III Ipatunity 130 trial (NCT03337724) is aiming to confirm data from LOTUS trial, evaluating Ipatasertib in combination with Paclitaxel in PIK3CA/AKT1/PTEN alterations; there was, indeed, in the trial, a numerically but non-significant increase in pCR rates, with more pronounced results in patients with PTEN-low tumors (32% versus 6%) and PIK3CA/AKT1/PTEN-altered tumors (39% versus 9%) [In the neoadjuvant setting, the phase II FAIRLANE study (NCT02301988), evaluating Paclitaxel plus Ipatasertib or placebo in TNBC, partially supports the potential utility as biomarker for the rsus 9%) . In contrsus 9%) ,171. A cPIK3CA/AKT1/PTEN alteration may is also supported by other AKT inhibitors trials. In the PAKT trial, Capivasertib (AZD5363), a potent and selective oral inhibitor of all three isoforms of the serine/threonine kinase AKT [PIK3CA/AKT1/PTEN-altered tumors [The predictive potential of nase AKT  has beend tumors .In summary, alteration of PIK3CA/AKT1/PTEN pathway have not yet satisfied criteria for the clinical use in TNBC, but diverse evidences support further research on this topic.Antibody drug conjugates (ADCs) are a new class of anticancer drugs that share the same general mechanism of action; they are designed as a monoclonal antibody that are conjugated with a potent cytotoxin . The monoclonal antibody is directed against an antigen on the surface of the target cancer cell, and upon binding to the target antigen, they are internalized and release the payload inside the cell, leading to selective cytotoxicity. This allows selective intracellular delivery of very potent payload that, because of their inherent toxicity, could not be infused as free molecules to the patient. As an additional mechanism of action, ADCs can elicit a potent immune response by inducing dendritic cell maturation and CD8 and CD4+ T-cell infiltration ,175.For an ADCs to be effective, a critical factor is the target antigen, that has to be selectively expressed  on the intended cancer cell. Therefore, the presence (or the overexpression) of the target antigen can be tested as biomarker to identify potentially sensitive patients. Several molecules have been identified in TNBC cells that meet these characteristics. The most promising ones are: (1) the glycoprotein non-metastatic b (GPNMB); (2) trophoblast cell-surface antigen 2 (Trop-2); (3) LIV-1; (4) the mucin 1-attached sialoglycotope CA6.GPNMB was found highly overexpressed in aggressive tumors like TNBC, or in advanced setting, where it is involved in processes like cell migration, invasion, angiogenesis, or epithelial-mesenchymal transition ,177,178;Trop-2 is a type I transmembrane glycoprotein, with a relevant role in migration, cell proliferation, cell cycle progression, and metastasis ,186,187.LIV-1 is a zinc transporter protein downstream target of STAT3, implicated in cell adhesion and epithelial-to-mesenchymal transition ,193,194.CA6 is selectively expressed on solid tumors and is, therefore, an ideal target for ADC therapy. SAR566658 is an ADC directed against CA6 which carries DM4, a maytansine-derived anti-microtubule agent as payload. Based on promising results from a phase I trial, a phase II study in CA6-positive TNBC (NCT02984683) is currently ongoing .Detection of circulating tumor cells (CTCs) has a promising potential as minimally invasive “liquid biopsies” that can facilitate prognosis, target therapy, or monitoring therapeutic response to drugs in several cancers ,199. CTCOver the past decade, several strategies were developed to capture CTCs based on biological properties of the cells, like the expression of cell surface proteins, or biophysical properties using filtration, microfluidics, and dielectrophoresis, or by applying high throughput imaging to unpurified blood cell preparations . DespiteA consistent number of prospective studies have demonstrated that CTC counts in cancer patients can be used as a dynamic prognostic biomarker in metastatic disease. More than a decade ago, Cristofanilli et al. in a study involving 177 metastatic breast cancer (MBC) patients, demonstrated that CTC count detected using the CellSearch was an independent prognostic factor for PFS and OS in metastatic Breast Cancer. The cut-off of 5 CTCs/7.5 mL was identified to classify patients with good or poor clinical outcome  and subsSeveral groups reported that the use of CellSearch platform for CTCs counts has limited prognostic power in TNBC, because of the fact that in these tumors, cells tend to switch from epithelial to mesenchymal phenotype, loosing EpCAM expression and exhibiting more stem cell-like properties ,225,226.CTC analysis of the tnAcity trial reported better outcomes among patients with CTC levels at baseline that were reduced or eliminated in subsequent cycles of chemotherapy, compared with CTC levels that persisted post-baseline, suggesting that CTC clearance may predict the chemosensitivity of metastatic TNBC tumors. However, as reported by the authors, this study has some limitations because of the use of only CellSerch platform that can exclude populations of low- or non-EpCAM-expressing cells, the possibility that during chemotherapy cells underwent an epithelial to mesenchymal transition change, and the low number of patient samples (N = 126) . FinallyVIM, SNAIL1, TIMP1, CRIPTO1, CD49F, ALDH2, CD44, and BCL11A [Single CTCs have been extensively studied in recent years because its detection could be particularly useful for certain types of cancers, however, also actively growing, aggressive tumors tend to release relatively low numbers of detectable CTCs into the circulation, and technical improvements in their isolation are needed . Factorsd BCL11A . Razmarad BCL11A . While, d BCL11A . In partd BCL11A ,246. Altd BCL11A ,238. Sevd BCL11A ,248,249 d BCL11A . Moreoved BCL11A ,252,253.In summary, CTCs, CTC clusters detection and their relative molecular characterization represent a significant source of biomarkers. However, in TNBC, detection methods need to be improved in order to not lose a consistent number of cells with mesenchymal phenotypes. Moreover, large trials are needed to confirm their prognostic and predictive role.PIK3CA and AKT1 respectively, was also found in the analysis of comparison between plasma-based and tissue-based DNA sequencing of LOTUS trial [BRCA somatic mutations in ctDNA not detected in primary tumors materials [Cancer cells can also disseminate their contents as free DNA fragments and exosomes into the bloodstream via different mechanisms ,255. TheUS trial . While, aterials . In earlaterials . In a staterials . Furtheraterials . A phaseIn the past years, numerous evidences highlighted the contribution of cancer stem cells (CSCs) in tumorigenic potential, high risk of metastasis, and drugs resistance of TNBC . CSCs reIn particular, it seems that over-expression of EMT pathways promote the generation of mammary CSCs and is responsible of capacity of CSCs to survive in hard metabolic conditions because of reduction of nutrients or hypoxia ,285,286.Several evidences have reported that self-renewal activity of CSCs could be ascribed to the alteration of different signal transduction pathways such as STAT signaling, SRC signaling, Wnt/β-catenin signaling. In particular, STAT3 signaling is involved in the mechanism for self-renewal regulation of CSCs, and conversion of non-CSCs into CSCs, through the regulation of IL-6-Jak1-STAT3-OCT3  or in tuIn TNBC patients, STAT3 activation is a biomarker of poor prognosis. The phosphorylated isoform is preferentially expressed in TNBC cell lines , and it TNBC is an intrinsically heterogeneous group of breast cancer and there is a need for effective biomarkers that can help physicians in selecting the most appropriate treatment.BRCA1/2 genes turned out to be factors predicting the efficacy of PARPis and alterations of other genes involved in homologous recombination seem promising in this setting. Several proposed biomarkers for TNBC have been studied in clinical trials demonstrating, so far, modest clinical benefits. Mutations of PD-L1 protein expression either in IC, tumor cells or both can be used as a predictive biomarker for response to immunocheckpoint inhibitors. There are different commercially available assays that use different antibodies as long as different scoring systems. However, there still is uncertainty as to what is the best assay in TNBC and if the results apply to all immunocheckpoint inhibitors. At present, however, data clearly support the use of the VENTANA SP142 assay to predict the efficacy of Atezolizumab in metastatic TNBC.Despite many research efforts, only a few useful biomarkers have been identified so far in TNBC. Some of these are already in the clinical practice. As new therapeutic agents are developed, parallel preclinical and clinical research is needed to identify biomarkers for the responsive, or conversely, the resistant patient population.


The rs139753473 from RFTN1 and six other suggestively significant loci from CUB and sushi multiple domains 1 (CSMD1) gene were also associated with cigarettes per day (CPD) in an independent Chinese sample consisting of 1,329 subjects (805 smokers and 524 nonsmokers). When treating males separately, associations between smoking initiation and PCAT5/ANKRD30A, two genes involved in cancer development, were identified and replicated. Within RFTN1, two haplotypes  formed by rs796812630-rs796584733-rs796349027-rs879511366 and three haplotypes  formed by rs879401109-rs879453873-rs75180423-rs541378415-rs796757175 were strongly associated with smoking initiation. In addition, we also revealed two haplotypes  in the CSMD1 gene showing a significant association with smoking initiation. Further bioinformatics functional assessment suggested that RFTN1 may participate in smoking behavior through modulating immune responses or interactions with the glucocorticoid receptor alpha and the androgen receptor. Together, our results may help understand the mechanisms underlying smoking behavior in the Chinese Han population.Tobacco use is one of the leading causes of preventable disease worldwide. Genetic studies have elucidated numerous smoking-associated risk loci in American and European populations. However, genetic determinants for cigarette smoking in Chinese populations are under investigated. In this study, a whole-genome sequencing (WGS)-based genome-wide association study (GWAS) was performed in a Chinese Han population comprising 620 smokers and 564 nonsmokers. Thirteen single-nucleotide polymorphisms (SNPs) of the raftlin lipid linker 1 (RFTN1) gene achieved genome-wide significance levels (P < 5 x 10 Although many programs and regulatory policies of tobacco control have been introduced, reducing the smoking prevalence to a satisfactory level remains an unsolved issue in many countries, especially low- and middle-income countries . It has CHRNA3 (CHRNA4 (CYP2A6), dopamine receptors and transporters , and neuregulin signaling pathway proteins  are also considered to have high impacts on nicotine addiction  , 9. BothCHRNA3 , 12 and  . Clean paired-end reads were mapped against the human reference genome (GRCh37/hg19) by BWA (v.0.7.15) (https://gatk.broadinstitute.org/hc/en-us). The discovered SNPs/indels were annotated in the National Center for Biotechnology Information (NCBI) dbSNP database (https://www.ncbi.nlm.nih.gov/snp/).For each participant, 5 ml of peripheral blood was collected. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s recommendation. Approximately 2 μg of genomic DNA  was prepared for DNA library construction. WGS was performed using the BGISEQ-500 platform (average depth >=30X). Quality control of raw sequences (FASTQ file reads) was conducted by FastQC  . VariantWe applied the following exclusion criteria to filter the samples: 1) mean sequencing depth <10X; 2) 10X coverage <90%; 3) GC content outliers; 4) relative duplication; 5) absolute inbreeding coefficient >1; 6) principal components analysis (PCA) outliers; and 7) sex mismatch.−4. In addition, variants without dbSNP IDs [also with minor allele frequency (MAF) <0.005] were excluded.Quality control of variants was applied by the standard recommended GATK filters, including variant quality score recalibration (VQSR), largest contiguous homopolymer run of the variant allele (HomopolymerRun), binomial test (GetHetCoverage), root mean square of mapping quality , and strand bias (FisherStrand). To further reduce bias, the following exclusion criteria were adopted: 1) minor allele average depth <4X; 2) average depth in case or control <8; 3) eightfold rate for case or control <0.9; 4) and P-value of Hardy-Weinberg equilibrium test <10Individual SNP-based association tests were performed by PLINK (v.1.9) using a logistic regression model . AdjusteTo replicate the GWAS associations, 1,329 participants including 805 smokers and 524 nonsmokers were recruited from local hospitals in Jincheng and Taiyuan of Shanxi Province in China during 2012–2013 , African (YRU), American (AMR), South Asian (SAS), and East Asian (EAS) obtained from the phase 3 release of 1000 Genomes Project . The disAfter mapping all clean reads against the human hg19 reference genome, we identified a total of 46,835,008 raw variants . Of them, 7,985,057 SNPs (19.3%) and 3,660,574 indels (67.1%) were eliminated in the variant quality control steps. Finally, 35,189,377 variants remained and were used for genome-wide association analysis . Forty-seven variants displayed statistical significance at a P-value of 10−5, including 2 SNPs without a dbSNP ID, −8). Of them, the SNP rs139753473 showed the strongest signal [P = 2.53 x 10−10, MAF = 0.0574, odds ratio (OR) = 4.664; RFTN1) gene , phosphatidylinositol transfer protein cytoplasmic 1 , and family with sequence similarity 162 member B . In addition, 98 variants from 19 previously reported smoking-associated genes were also identified , rs572691375 from CYP2C19 (cytochrome P450 family 2 subfamily C member 19), and rs4875371 from CSMD1 (CUB and sushi multiple domains 1) had P-values <10−4. Of these, the rs140333915 and rs572691375 were low-frequency and rare variants . When treating males separately, we found one genome-wide significant signal, rs11010435 (P = 4.72 x 10−8) located between PCAT5 (prostate cancer associated transcript 5) and ANKRD30A (ankyrin repeat domain 30A) and 98 variants from 31 known smoking-associated genes achieved P values of 10−3  gene Figure 3−5, with the rs78955061 from the intergenic region of ACKR3  and LOC93463 had the smallest P value of 7.92 x 10−7. With a cutoff of p <10−3, 27 variants from 19 known smoking-associated genes were determined. When the study sample was restricted to males, 22 SNPs yielded a P value of <10−5, of which the rs143124048 from PALLD  had the smallest P value of 1.70 x 10−6 . As a result, none of the smoking initiation or CPD associated loci from the total discovery sample (with men and women combined) were significant. However, 18 male-specific smoking initiation associated loci  yielded significant results , was an intergenic SNP between LOC101928283 and GRM8. For male-specific CPD- associated loci in the primary analysis, no evidence of replication was observed. Intriguingly, in testing for an association with CPD, the rs139753473 within RFTN1 and six SNPs within CSMD1 showed a P-value of <0.05 , and four SNPs from CSMD1  achieved a nominally marginal significance level (P < 0.10) for an association with CPD.Due to the fact that very few females smoke in China, only males were included in the replication sample. Variants which had a P-value lower than 10of <0.05 Table 3.RFTN1 with a P-value of <10−3 according to the results from individual SNP-based association analysis for smoking initiation, two LD blocks were identified (D’ > 0.97)  Figure 4CSMD1 had the largest number of SNPs with P <10−3, of the reported smoking-associated genes, haplotype-based association analysis was also performed on the 29 SNPs in CSMD1. One LD block exhibited a D’ larger than 0.97 .Given that in the individual SNP-based association analysis of smoking initiation, the In silico functional analyses based on the RegulomeDB (https://regulomedb.org/) and HaploReg  databases were performed for SNPs with P <10−8, i.e., rs139753473, rs200713609, rs116358832, rs796950514, rs796881087, rs796687837, rs796068970, and rs796689769. All of these SNPs were intron variants within RFTN1. The male-specific smoking associated rs11010435 was omitted because the GWAS did not have adequate power and this locus was not replicated. Although evidence of regulatory potential was weak for rs139753473 (RegulomeDB score = 0.008), this SNP alters E2F, Egr-1, MOVO, Nrf1, UF1H3BETA, YY1, and SP1 transcription factor binding motifs according to HaploReg  data could be found. Furthermore, according to the expression pattern retrieved from GTEX PORTAL , RFTN1 had the highest expression in lymphocytes and was also expressed in various brain tissues, such as the cortex, the frontal cortex (BA9), and cerebellum , using another Chinese Han sample containing 1,329 male subjects. Although variants associated with smoking initiation and CPD in the total discovery sample were not replicated, we replicated 18 loci for their association with smoking initiation in men, which including rs10128115, rs10128145, rs72795203, rs12241402, rs10128497, rs16936694, rs7072685, rs12261634, rs11010478, rs11010482, rs112089093, rs12248963, rs1480525, rs10128398, rs12256178, rs10128169, rs1122458, and rs7071386 from the intergenic region between PCAT5 and ANKRD30A (P < 0.05). For male-specific associations with CPD, no evidence of replication was found. Furthermore, although RFTN1 and CSMD1 were originally identified in the test of smoking initiation, in the replication test of CPD, the RFTN1 gene’s rs139753473 and the CSMD1 gene’s rs76965088, rs117740219, rs78094590, rs138695620, rs76195425, and rs148939406 reached a P-value of <0.05. Given that smoking initiation and CPD are moderately correlated smoking behavior traits   from the former and three  from the latter were significantly associated with smoking initiation. Additionally, haplotype-based association analysis also showed that two CSMD1-derived haplotypes, C-A-G-G and T-C-T-T formed by rs4875371-rs4875372-rs17070935-rs11991366 were strongly correlated with smoking initiation.Within the 24 SNPs observed in the in silico and need experimental validation. Although RFTN1 had the highest expression in lymphocytes, its expression can also be found in brain tissues, e.g., the cortex, frontal cortex (BA9), and cerebellum. These regions are believed to be involved in the brain’s reward and inhibitory control processes  signaling, toll-like receptor (TLR) 3 signaling, and interleukin-17 (IL17) production (RFTN1 could play a role in smoking initiation by regulating immune responsiveness.The lead SNP rs139753473) associated with smoking initiation is located within the intron region of the RFTN1 gene. It may bind to two transcription factors, i.e., the glucocorticoid receptor alpha and the androgen receptor, which have been proposed to play a role in smoking. Nonetheless, the functional studies were carried out rocesses , 36. In oduction –40. It hoduction , 42. In oduction . In partoduction –47, and 3 associaoduction . Moreoveoduction , but is oduction .It is plCSMD1 is a complement-regulatory protein that is highly expressed in the central nervous system, contributing to addiction vulnerability  is a widely used instrument to estimate nicotine dependence. It is possible that GWAS of this phenotype may produce more robust results. Additionally, as a major metabolite of nicotine, cotinine represents a direct biomarker of smoking quantity. Measuring the cotinine level in serum, urine, or saliva could benefit further validation studies, by providing more objective information on smoking quantity.One major limitation of this study is the sample size. Due to the relatively high sequencing cost per sample and difficulty in recruiting participants, our sample size was not large enough to provide various phenotypes and adequate statistical power. Although the application of WGS in genome-wide association analysis allowed for successful detection of a few novel or low-frequency variants associated with cigarette smoking, these SNPs are infrequently reported in previous smoking or other psychiatric‐related traits, preventing further functional assessments. This also raised the question of whether the observed loci, especially those from RFTN1 and CSMD1 are involved in smoking. Associations between smoking initiation and PCAT5/ANKRD30A were also detected and replicated in a male-specific manner. RFTN1 might function in smoking initiation through interactions with the immune system, the glucocorticoid receptor alpha and androgen receptor signaling. These findings provide extensive insight into the biological mechanisms of smoking behavior in the Chinese Han population.In summary, to the best of our knowledge, we have reported the first WGS-based GWAS of smoking phenotypes in a Chinese Han cohort. We provided exploratory evidence that zhuzhouhai@gmail.com.The datasets presented in this article are not readily available because due to the restriction of “Regulation of the People’s Republic of China on the Administration of Human Genetic Resources”, sequencing data of this study can not be shared publicly. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Biomedical Ethics Committee of Joint Institute of Tobacco and Health. The patients/participants provided their written informed consent to participate in this study.ML, JY, and ZZ conceived the study. YC, ML, and QL performed the data analysis. ML wrote the manuscript. All authors contributed to the article and approved the submitted version.The Joint Institute of Tobacco and Health received research grants from China Tobacco Yunnan Industrial CO. LTD. Author QL was employed by the Hangzhou Global Biotechnology and Bioinformatics CO. LTD. However, this study was carried out independently from either the funding agency or employer.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The concentrations of choline, creatine, lactate, alanine, fumarate and succinate varied remarkably among different cell types. The significantly higher intracellular concentrations of glutathione, myo-inositol, and phosphocholine were found in the SKOV3 cell line relative to other cell lines. The concentration of glutamate was higher in both SKOV3 and RCC cells compared with other cell lines. For cell culture media analysis, isopropanol was found to be the highest in RCC media, followed by A549 and SKOV3 media, while acetone was the highest in A549, followed by RCC and SKOV3. These results demonstrated that 1H-NMR-based metabolic phenotyping approach allows us to characterize specific metabolic signatures of cancer cell lines and provides phenotypical information of cellular metabolism.Cancer cell lines are often used for cancer research. However, continuous genetic instability-induced heterogeneity of cell lines can hinder the reproducibility of cancer research. Molecular profiling approaches including transcriptomics, chromatin modification profiling, and proteomics are used to evaluate the phenotypic characteristics of cell lines. However, these do not reflect the metabolic function at the molecular level. Metabolic phenotyping is a powerful tool to profile the biochemical composition of cell lines. In the present study,  Cancer is the second leading cause of global mortality with approximately 9.6 million deaths in 2018. Some types of cancers including lung and colorectal cancer are among the most common causes of the mortality, accounting for 18% and 8.9% of the total cancer death, respectively. It was reported in 2018 that lung, colorectal, kidney, brain, and ovarian cancer contributed to 12.3%, 10.6%, 2.4%, 1.7%, and 1.7% of the total number of cancer cases, respectively . AlthougTo explore cellular or molecular mechanisms, responses to therapies and drug discovery and development of cancers, cancer cell lines have been widely used and served as the workhorse for cancer research. However, it has been recently reported that single cell-derived clones showed continuous instabilities, leading to the heterogeneity of the cell lines, altering the drug responses, and dysregulation of xenobiotic metabolism . This su1H-NMR spectroscopy-based approach has previously been applied to study the metabolic responses of cancer cell lines, such as lung (A549) and colon (Caco2), to xenobiotics and mycotoxins -propionic acid sodium salt (TSP) for the spectral calibration, 0.075 M KH2PO4, and 0.1 mM sodium azide (NaN3). The resulting mixture was centrifuged at 20,817 g for 10 min, and 180 μl supernatant was transferred to an NMR tube  with an outer diameter of 3 mm pending 1H-NMR spectral acquisition.The dry cell extract samples were resuspended in 210 μl of potassium phosphate buffer (pH = 7.4) containing deuterium oxide  at the operating 1H frequency of 600.13 MHz at a temperature of 300 K. A standard NMR pulse sequence (recycle delay-90°-t1-90°-tm-90° acquisition) was applied to acquire 1H-NMR spectral data . The water peak suppression was achieved using selective irradiation during a recycle delay of 4 s and tm. A 90° pulse was adjusted to ∼10 μs. A total of 64 scans for cell extracts and 32 scans for cell media were collected into 64 k data points with a spectral width of 20 ppm. Two-dimensional (2-D) 1H-1H correlation spectroscopy (COSY), 1H-1H total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence spectroscopy (HSQC) were acquired on the selected cell and media samples for to aid in metabolite identification.1H-NMR spectra obtained from cell extracts and media samples were phased, referenced to TSP at δ1H 0.00 and baseline-corrected in TopSpin 4.0.3 . MATLAB software R2018a  programming language was used to import and process the NMR spectral data. Water peak regions of the cell extract (δ1H 4.74–4.85) and cell media (δ1H 4.7–5) spectra were deleted to minimize the effect of the disordered baseline. Regions containing only noise in the cell extract  and cell media (δ1H 0–0.3) spectra were removed. Two cellular extract samples from 5 million H4 cell group and one media sample from 5 million RCC cell group were excluded due to extremely low intensities of signals. The remaining spectra data from 1, 5, and 10 million cells were normalized using a probabilistic quotient normalization method separately [2X and R2Y represent the variation explained by the model in X and Y matrices, respectively. Q2Y represents predictability of the model and a good model usually has a Q2Y > 0.5 [P value. Models with P < 0.05 are considered as valid OPLS-DA models.ct δ1H 4.4–4.85 an1H-NMR spectra of the cell extracts obtained from 1, 5, and 10 million cells per cell type are shown in Supplementary Figure S1. By visualizing the spectra, peak intensities increase proportionally as the number of cells increases from 1 to 10 million. The metabolite assignment from cell extracts and media samples are listed in the Supplementary Table S1. Five million cells produced a better quality of the spectra with 64 scans than one million cells. A total of 34 metabolites were identified from these cellular extracts and confirmed using 2-D 1H-1H COSY and 1H-1H TOCSY NMR spectra ; these metabolites included acetate, alanine, asparagine, aspartate, choline, creatine, formate, fumarate, glutamate, glutamine, glutathione, glycerol phosphocholine, glycine, histidine, hypoxanthine, isoleucine, lactate, leucine, lysine, methanol, methionine, myo-inositol, phenylalanine, phosphocholine, serine, succinate, taurine, threonine, trehalose, tryptophan, tyrosine, uracil, uridine, and valine.The median The metabolic profiles obtained from 5 and 10 million cells were analyzed using unsupervised PCA analysis with three principal components (PC). The PCA scores plots of PC1 versus PC2 derived from both 5 and 10 million cells  show a g2X, Q2X, Q2Y, and permutation P values of these OPLS-DA models are summarized in Pair-wise comparisons between different cell types were carried out using OPLS-DA analysis with one predictive component and one orthogonal component. The RStatistically significant models were observed in the vast majority of pair-wise comparisons, except for A549 versus Caco2 (10 million) and A549 versus H4 (5 million). The model of A549 versus Caco2 from 5 million cell extracts was statistically significant, which was contributed by higher concentrations of phosphocholine, and decreased concentrations of glycine in Caco2 cells. However, the model of A549 versus RCC from 10 million rather than 5 million cell extracts was significant, corresponding to higher concentrations of formate, phosphocholine, and choline in RCC. The biochemical composition of A549 cells was also significantly different from SKOV3 cells. In the 5 million cell extracts model, the concentrations of phosphocholine, myo-inositol, glutathione, and glutamate were higher in SKOV3 compared with A549 cells. Additional metabolic differences, including higher concentrations of formate, uridine, lactate, and creatine, and lower concentrations of choline, acetate, and isoleucine, were observed in SKOV3 with 10 million cells compared with A549. The model of Caco2 versus H4 from 10 million cell extracts was statistically significant, with higher concentrations of threonine, valine and glycine, and lower concentrations of succinate in H4. The concentrations of formate, lactate, glutamate, glycine, myo-inositol, taurine, and glycerol phosphocholine was higher in both 5 and 10 million cell extracts of Caco2 compared with RCC. However, lower levels of uridine and phosphocholine and a higher level of choline were only observed in 5 and 10 million cell models, respectively. Higher concentrations of glutathione, myo-inositol, creatine, and lactate, and lower concentrations of choline in SKOV3 cells were observed to distinguish from Caco2 cells (both 5 and 10 million cells). While 5 million RCC cells showed higher concentrations of lactate, taurine, and glutamate in contrast with H4 cells, additional metabolites such as glycerol phosphocholine, phosphocholine, choline, succinate, threonine, serine, valine, and isoleucine were found to be different in concentrations in 10 million cells. Higher concentrations of uridine, glutathione, phosphocholine, lactate, myo-inositol, creatine, and succinate were observed in SKOV3 cells compared with H4 and RCC, whereas choline was found to be higher in RCC in comparison with SKOV3 .High-intensity peaks present in the media samples were assigned based on 2D NMR spectra. These include acetate, acetone, alanine, citrate, formate, glucose, glutamate, glutamine, glycine, histidine, isoleucine, isopropanol, lactate, leucine, lysine, phenylalanine, pyroglutamate, pyruvate, succinate, threonine, tryptophan, tyrosine, and valine .1H-NMR spectral data from the cell culture media of these cancer cell lines were analyzed using PCA. As expected, the grouping pattern observed in the scores plots of media samples is based on the cell types , unlike the cell extracts where PC1 is dominated by the number of cells . Similar grouping patterns were observed from the PCA scores plots of all media samples, 5 or 10 million cell culture media samples. There is a clear separation along the PC1 between H4 and the other cell types, while a separation between Caco2 and the rest was observed along the PC2 . Given that both H4 and Caco2 were cultured using DMEM and the other three cell lines were cultured using RPMI, the major variation revealed by PCA was likely due to metabolic behavior of the H4 cells rather than the compositional differences between the two media. Additional PCA analyses were carried out for each type of media to compare the metabolic contribution of the cells to the media biochemical composition. The PCA scores plots based on the 5 million cell culture samples , glucose, and tricarboxylic acid (TCA) cycle intermediates (e.g. succinate and pyruvate) were observed in A549 compared with RCC or SKOV3, together with higher concentrations of lactate. Additionally, a higher concentration of alanine and a lower concentration of acetone were also found in a 10 million cell culture media (RPMI) of SKOV3, in contrast with A549. Metabolites present in the SKOV3 media that distinguish it from RCC include higher concentrations of glucose, glutamine, and lower concentrations of lactate, isopropanol, pyro glucose, succinate, acetone, acetate, and alanine.Lower concentration of glucose was only observed in 5 million H4 cultured in DMEM compared with Caco2, whereas the increased concentrations of several metabolites were found in both 5 and 10 million models; these included formate, phenylalanine, tyrosine, threonine, lactate, isopropanol, alanine, glutamine, glycine, pyruvate, acetone, acetate, valine, isoleucine, and leucine .1H-NMR spectroscopy-based metabolic phenotyping and demonstrated the significant metabolic differences among five cell lines, namely, lung (A549), colonic (Caco2), brain (H4), renal (RCC), and ovarian (SKOV3) cancer cells. The intra-group variation was higher in the H4 cell line in contrast with the others, particularly when cultured as 10 million cells. It is likely that H4 cell growth are more sensitive to environmental conditions, and the cell growth rate and metabolic behavior may likely be affected with subtle changes in culture condition. One of the most profound findings with regards to metabolic changes was the significantly higher intracellular concentrations of glutathione, myo-inositol, and phosphocholine in SKOV3, compared with other cell lines. Glutathione (GSH) is the most abundant non-protein thiol, which functions as an antioxidant and a redox regulator. It has been found that stem cells required high levels of GSH to maintain stem cell function and migration capabilities in vitro [The present study was based on  lung A54, colonicin vitro .Glutamate was found to be higher in SKOV3 and RCC cells compared with other cell lines. Glutamate is an amino acid that plays a key role in energy and carbon metabolism and synthesis of amino acids and nucleotides for all cells. In cancer cells, glucose-based glycolysis and glutamate-based glutaminolysis are major two ways for ATP production. With the high levels of glutamate, glioma cells can be rescued from death .Another key finding was the high abundance of amino acids in the H4 cells; amino acids presented in higher levels in H4 versus other cell lines include serine, methionine, threonine, valine, glycine, and acetate. The function of mitochondria is largely dependant on the pathway of serine to formate, which is then released into the cytoplasm to contribute to nucleotide synthesis . As the It has been found in our study that the concentrations of choline, creatine, lactate, alanine, fumarate, and succinate varied significantly among different cell types. Ovarian cancer cell line exhibited the highest levels of alanine, lactate, and TCA cycle intermediates  and methanol, and the lowest levels of choline, whereas lung cancer cell line showed the lowest levels of lactate and alanine. Lactate has been reported to be elevated in the cancerous cells due to lactic acidosis and glucose deprivation , while gMedia concentrations of isopropanol was found to be the highest in RCC, followed by A549 and SKOV3, while acetone was the highest in A549, followed by RCC and SKOV3, together with a higher level in H4 compared with Caco2. Interestingly, isopropanol and acetone were deemed as a potential biomarker in a series of diseases including cancer ,21. TherALDH6A1 gene [ALDH6A1 gene may encode methylmalonate semialdehyde dehydrogenase, was deficient and hence the level of alanine was increased.The concentrations of alanine in the culture media were the highest in the RCC, followed by SKOV3 and A549. A previous study showed that renal cell carcinoma results in an increased level of alanine in cells, which might be due to the downregulated expression of 6A1 gene , thus de1H-NMR-based metabolic phenotyping analysis can detect the cellular metabolic profile in five different cancer cell types, including lung, colonic, brain, renal, and ovarian cancers. Similarly, their metabolic profiles can also be measured in culture media. It may be concluded that 1H-NMR-based metabolic phenotyping can be used to detect cellular metabolisms of different cancerous cells and improve our understanding of the metabolism of certain cancers.Our study showed that Click here for additional data file.


On March 13, 2020, the United States declared a national emergency in response to the coronavirus disease 2019 (COVID-19) pandemic. Subsequently, states enacted stay-at-home orders to slow the spread of SARS-CoV-2, the virus that causes COVID-19, and reduce the burden on the U.S. health care system. CDCInternational Classification of Diseases, Tenth Revision (ICD-10) codes: MI = I21–I22; stroke = I60–I61 (hemorrhagic stroke) or I63 (ischemic stroke); and hyperglycemic crisis = E10.1, E11.1, or E13.1 (diabetic ketoacidosis) or E11.0, E13.0, or E10.65 and E10.69 (hyperosmolar hyperglycemic syndrome). Weekly numbers of ED visits for each of the three conditions were compared for two 10-week periods: January 5–March 14, 2020  and March 15–May 23, 2020 . The absolute differences and percentage change in number of visits from pre- to early pandemic periods were tabulated, overall and within age-sex strata. Analyses were conducted using SAS .CDC used data from its National Syndromic Surveillance Program (NSSP) to assess trends in ED visits from week 1, 2019 through week 21, 2020 for three life-threatening health conditions: MI, stroke, and hyperglycemic crisis. NSSP is a collaboration among CDC, federal partners, local and state health departments, and academic and private sector partners to collect, analyze, and share electronic patient encounter data received from emergency departments, urgent and ambulatory care centers, inpatient health care settings, and laboratories for public health action.Trends in number of ED visits for MI and stroke were relatively stable during the first half of 2019, increased slightly in the second half of 2019, and then stabilized during the first few weeks of 2020, remaining stable throughout the prepandemic period . The numThe relative decline in the number of ED visits between the prepandemic and early pandemic periods was similar across age groups for MI and stroke, whereas the decline in ED visits for hyperglycemic crisis tended to be larger among younger age groups, particularly for females . The absIn the weeks following the declaration of COVID-19 as a national emergency on March 13, 2020, NSSP identified substantial reductions in numbers of ED visits by males and females in all age groups for three potentially life-threatening conditions: MI (23% decrease), stroke (20%), and hyperglycemic crisis (10%). These estimates are consistent with, but smaller in relative magnitude than, the 42% overall decline in ED visits observed during the early pandemic period  visits during the COVID-19 pandemic.In the 10 weeks following declaration of the COVID-19 national emergency, ED visits declined 23% for heart attack, 20% for stroke, and 10% for hyperglycemic crisis.Persons experiencing chest pain, loss of motor function, altered mental status, or other life-threatening issues should seek immediate emergency care, regardless of the pandemic. Communication from public health and health care professionals should reinforce the importance of timely care for acute health conditions and assure the public that EDs are implementing infection prevention and control guidelines to ensure the safety of patients and health care personnel.


Tolypocladium cylindrosporum is assembled and annotated. This circular mitogenome is 34,698 bp in length and consists of two rRNA genes (rnl and rns), 26 tRNA genes, 14 standard protein-coding genes of the oxidative phosphorylation system, and four intergenic free-standing ORFs. A total of six introns  were identified in atp9, cob, cox1, and rnl, and they may encode ribosomal protein S3, LAGLIDADG or GIY-YIG endonucleases. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirms T. cylindrosporum in Ophiocordycipitaceae.In this study, the complete mitogenome of an entomopathogenic fungus  Tolypocladium cylindrosporum W. Gams 1971 is a fungus that has been isolated from soil, from asymptomatic plants as an endophyte, and has been shown to be pathogenic to several species of arthropods   information is not yet available for this fungus. Herein, we present the complete mitogenome of T. cylindrosporum ARSEF 963, which was isolated from soil in a spruce-fir forest (elev. 5500-6000 ft) in north Luscar, Alberta, Canada . The strain is obtained from USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF).de novo assembled from clean reads using MITObim  is a circular molecule of 34,698 bp with an AT content of 73.0%. It is rather compact with genic regions  accounting for 85.5% of the total bases. This mitogenome encodes two ribosomal RNAs (rnl and rns), 26 tRNAs, 14 conserved proteins of the oxidative phosphorylation system , and four free-standing ORFs at intergenic regionsf. These tRNA genes code for all 20 standard amino acids. Among them, there are three tRNA genes for methionine with the same anticodon, two tRNA genes for arginine, glycine, leucine, and serine with different anticodons. The majority of tRNA genes are clustered upstream  and downstream  of the rnl gene, and downstream  of the rns gene. For the two commonly-found neighboring gene pairs, nad3 follows immediately nad2, whereas nad5 overlaps one nucleotide with its upstream gene nad4L. The four intergenic free-standing ORFs encode either GIY-YIG endonucleases (orf221 and orf296) or hypothetical proteins (orf194 and orf142). It should be noted that nad6 overlaps 50 bp at the 3’ end with trnV. Sequences at rns/trnY intergenic region showed weak similarity  to orf221, but not a complete ORF larger than 300 bp could be identified. This region possibly contains a degenerating GIY-YIG endonuclease.The mitogenome of atp9, cob, cox1, and rnl, respectively. They are all group I introns but belong to several specific types, namely IA (mL2450 and atp9P181), IB (cox1P212 and cox1P731), IC1 (mL965), and ID (cobP393). Intronic ORFs encode ribosomal protein S3 (in mL2450), LAGLIDADG endonuclease (in cox1P731), or GIY-YIG endonucleases (in remaining introns). Intronic regions (including intronic ORFs) have a total length of 7480 bp.A total of six introns were identified, including one, one, two, and two in T. cylindrosporum as a member of Ophiocordycipitaceae (Tolypocladium inflatum and Tolypocladium ophioglossoides, and their clustering receives 100% support.Phylogenetic analysis based on mitochondrial protein sequences confirms pitaceae . The fun


Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749.Quinolones are commonly used for treatment of infections by bacteria of the E. coli J53 strain as a recipient. The quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were PCR-amplified.MICs of ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 and transconjugant c749 were determined by the Etest method. Conjugation was performed using sodium azide-resistant aac (6′)-Ib-cr, qnrS2, and oqxAB) and ten acquired resistance genes. One of the genes, qnrS2, was shown for the first time to be flanked by two IS26s. Three IS26-mediated circular molecules carrying the PMQR genes were detected.RJ749 was highly resistant to quinolones, while c749 showed low-level resistance. S1-nuclease pulsed-field gel electrophoresis revealed that RJ749 and c749 both harboured a plasmid. PCR presented chromosomal mutation sites of the quinolone resistance-determining region, which mediated quinolone resistance. The c749 genome comprised a single plasmid, pRJ749, with a multiple resistance region, including three plasmid-mediated quinolone resistance (PMQR) genes (qnrS2 flanked by IS26 elements. IS26 plays an important role in horizontal spread of quinolone resistance.We revealed the coexistence of three PMQR genes on a multiple resistance plasmid and a new surrounding genetic structure of  Enterobacteriaceae family [Escherichia coli to ciprofloxacin was 57.0% in China [Quinolones are widely used for empirical and directed therapy in infections caused by members of the e family . The rise family . In 2017in China .gyrA and gyrB) and DNA topoisomerase IV (parC and parE) and, to a lesser extent, changes in outer membrane proteins or efflux pumps [aac (6′)-Ib-cr, and efflux pumps qepA and oqxAB [Mechanisms of fluoroquinolone resistance occur principally through chromosomal mutations in the quinolone resistance-determining region (QRDR) of DNA gyrase  that reduce susceptibility to fluoroquinolones have been identified [Escherichia coli clinical isolates from Japan and Belgium in 2007 and encodes an MFS-type efflux pump, which is able to excrete quinolone into the extracellular space [oqxA and oqxB genes. It confers resistance to quinoxalines, chloramphenicol, trimethoprim, and quinolones [Qnr proteins bind to DNA gyrase and topoisomerase IV, protecting them from quinolone inhibition. The escribed . Subsequentified , 11. Theentified , 12–14. ar space . The effqnrS2, aac (6′)-Ib-cr, and oqxAB).In the current study, we discovered a novel plasmid with a multidrug resistance region (MRR), which comprised three kinds of plasmid-mediated quinolone resistance genes .E. coli J53 (sodium azide-resistant) as the recipient. Selection was performed with 100 μg/mL concentrations of sodium azide and 2 μg/mL concentrations of cefotaxime. RJ749 and the transconjugant, c749, were both digested with S1 nuclease and subjected to S1-PFGE, as described previously [Conjugal transfer experiments were performed in broth culture using the strain RJ749 as the donor and the strain eviously .gyrA, gyrB, parC, and parE genes with primer pairs as previously reported [PCR assays were conducted targeting QRDRs of the reported . The genreported . Open rereported . Proteinreported . PlasmidBecause IS26-flanked structures can generate circular DNA intermediates, reverse PCR was performed to detect potential circular forms. A series of PCR primers at sites flanking IS26 was used to determine the order of the circular structures carrying quinolone resistance determinants Table .Table 1The MICs of cefotaxime, ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 were ≥ 256, ≥ 256, ≥ 256, ≥ 32, and ≥ 32 mg/L, respectively. The MICs of cefotaxime, ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for c749 were ≥ 256, ≥ 256, 24, 12, 2, and 1 mg/L, respectively.Through mating experiments, cephalosporin and quinolone resistance was transferred from the donor, RJ749, to the recipient, J53 (sodium azide-resistant). S1-PFGE revealed that RJ749 and c749 both harboured a plasmid, about 250 kbp , which mediated resistance to quinolone.In the process of amplifying the QRDRs of the aadA16, aac (3)-IIa, aac (6′)-Ib-cr); β-lactams ; quinolones (aac (6′)-Ib-cr, qnrS2, oqxAB); macrolide-lincosamide-streptavidin B (MLS) (mph(A)); chloramphenicol (catB3); rifampicin class (ARR-3); sulfonamides (sul1); tetracyclines (tet(A)); and trimethoprim (dfrA27). Results of PlasmidFinder and pMLST revealed the IncHI2/−IncN multireplicon plasmid, in which IncHI2 belonged to ST3.Whole genome sequencing was carried out for the transconjugant c749. The genome of c749 comprised a single plasmid, pRJ749, which was 253,002 bp in length and with a GC content of 47%, comprising 769 putative ORFs. pRJ749 had a multiple resistance region at positions 192,481–244,472, including for aminoglycosides -Ib-cr-blaOXA-1-catB-ARR3-qacEΔ1-Δsul1, IS26-qnrS2 and IS26-oqxAB) were amplified by reverse PCR and sequenced, respectively. These circular structures and sequences were identical to those of the corresponding region in plasmid pRJ749. Sequence analysis of the three circular molecules revealed the structures of a single IS26 and three PMQR genes, respectively  isolated from Hong Kong, China, also carried the same three PMQR genes as we found . oqxAB was mainly reported in IncHI2-ST1/2 plasmids in Asia [oqxAB-blaCTX-M-9 plasmids in E. coli belong to IncHI2-ST3 [oqxAB in our study was located on the IncHI2-ST3 plasmid in E. coli, which suggested that IncHI2 plasmids undergo constant evolvement and dissemination.The  by IS26 , 27–31.  in Asia . NeverthcHI2-ST3 . oqxAB iaac (6′)-Ib-cr gene is usually found in a cassette as part of an integron in a multi-resistance plasmid, which may contain other PMQR genes [aac (6′)-Ib-cr was located at a cluster of resistance genes flanked by IS26, which consisted of the aac (6′)-Ib-cr, blaOXA-1, catB3, arr-3, qacEΔ1, and Δsul1 elements as previously reported [Enterobacteriaceae in China, as demonstrated by BLAST.The QR genes , 35, andQR genes . In pRJ7reported . This caqnrS2 is mainly found on the IncQ [Aeromonas spp.). Two different surrounding genetic structures have been described for the qnrS2 genes. Two repC- and repA-like genes were located immediately downstream of the qnrS2 gene in two plasmids [mpR gene encoding a putative zinc-metalloprotease (MpR) [E. coli detection rate of qnrS2 in faeces of normal people was 3.7% [qnrS2 flanked by two IS26s  isolated from Hong Kong, China, was also flanked by IS26s. This suggested that IS26 played an important role in the transfer of qnrS2 genes in plasmids among Enterobacteriaceae in China.the IncQ  and IncUthe IncQ  plasmidsplasmids , 40. Anose (MpR) . In 2016was 3.7% . Our stuEnterobacteriaceae [IS26 was observed to be predominant in the plasmids of eriaceae . Harmer eriaceae  proposederiaceae . To inveeriaceae  reportedE. coli in China, and a new surrounding genetic structure of qnrS2 flanked by IS26 elements was identified for the first time. IS26 mediates the formation of circular intermediates, which results in various forms and horizontal spread of quinolone resistance.In conclusion, a plasmid carrying three PMQR genes has been emerging in


NRAS, AXL and PIM1 that bypass or reinforce FLT3 signaling. Off-target mutations can occur just in the primary FLT3-mutated clone or be gained by the evolution of other clones. A small number of cases show primary resistance by an FL-dependent, FGF2-dependent, and stromal CYP3A4-mediated manner. To overcome these mechanisms, the development of novel agents such as covalently-coupling FLT3 inhibitor FF-10101 and the investigation of combination therapy with different class agents are now ongoing. Along with novel agents, gene sequencing may improve clinical approaches by detecting additional targetable mutations and determining individual patterns of clonal evolution.FLT3-ITD and FLT3-TKD mutations were observed in approximately 20 and 10% of acute myeloid leukemia (AML) cases, respectively. FLT3 inhibitors such as midostaurin, gilteritinib and quizartinib show excellent response rates in patients with FLT3-mutated AML, but its duration of response may not be sufficient yet. The majority of cases gain secondary resistance either by on-target and off-target abnormalities. On-target mutations  such as D835Y keep the TK domain in its active form, abrogating pharmacodynamics of type II FLT3 inhibitors . Second generation type I inhibitors such as gilteritinib are consistently active against FLT3-TKD as well as FLT3-ITD. However, a “gatekeeper” mutation F691L shows universal resistance to all currently available FLT3 inhibitors. Off-target abnormalities are consisted with a variety of somatic mutations such as  FLT3 (FLT3-ITD) was identified in acute myeloid leukemia (AML) cells . In. In96]. 40. Among the 50 resistant patients treated with crenolanib, five FLT3  mutations were observed in six patients; the D200N and L601F mutations did not result in leukemia [FLT3, although the existence of off-target resistance mechanisms was considered in this patient. These findings suggest the existence of a polyclonal resistance mechanism in patients with AML that relapses after quizartinib treatment.Although the FLT3-F837K and FLT-C35S mutations occurred after the gilteritinib treatment in one patient each, both were considered silent mutations as these did not induce self-proliferation in Ba/F3 cellsleukemia . In addiNRAS, KRAS, PTPN11, CBL, and BRAF mutations, were acquired. Of note, the mutations in the RAS/MAPK pathway and FLT-F691L mutations were mutually exclusive. In vitro experiments conducted in MOLM-14, an AML cell line with FLT3-ITD, where either mutant RAS or FLT3-F691L was transduced into the parental cells and gilteritinib was administered at low/high-dose (25 and 250 nmol/L), suggested that the RAS-mutant clones were more likely selected by the high concentration of the inhibitor, besides the FLT3-F691L which was more likely to be selected by a low one. Similar to RAS mutations [NRAS, STAG2, CEBPA, ASXL1, and IDH2 mutations were observed in FLT3-WT clones [TET2, IDH1, and TP53 mutations occurred simultaneously in FLT3-mutated clones during crenolanib treatment. These results suggest that the off-target resistance mechanism is more frequent when using type I inhibitors, such as gilteritinib or crenolanib, than type II inhibitors. Besides, IDH1 inhibitor ivosidenib [Resistant clones formed after treatment with gilteritinib and crenolanib, and a type I inhibitor that exerts an inhibitory effect on FLT3-TKD, have characteristics that are different from those observed after treatment with type II inhibitors. In a comparative genetic analysis before and after relapse in patients treated with gilteritinib, several distinct patterns of clonal selection were observed during the treatment period with gilteritinib . In fiveutations ,98, the utations . In anotutations . The surT clones . These fosidenib  and IDH2osidenib ,103, botosidenib . Pim-1 iosidenib . Its oveosidenib . Additioosidenib . Pim-1 wosidenib ,109,110.FLT3, which confers resistance to all existing FLT3 inhibitors, it is necessary to develop a novel FLT3 inhibitor. As described above, while type I inhibitors can also inhibit FLT3-TKD, they exhibit low selectivity, whereas although type II inhibitors cannot inhibit FLT3-TKD, they exhibit high selectivity. FLT3-TKD inhibitory activity and FLT3 selectivity share a trade-off relationship. To resolve these issues, a novel FLT inhibitor known as FF-10101 was designed, which would form covalent bonds with the C695 residues of FLT3. The creation of covalent bonds by FF-10101 enables the selective and irreversible inhibition of FLT3 in either the active or the inactive form [FLT3 mutations, including F691L. Currently, phase 1/2 trials are underway to evaluate its safety, tolerability, pharmacokinetics, and efficacy against recurrent refractory AML (NCT03194685). In addition, several agents that may overcome or prevent resistance are currently under investigation. A pan-PIM/FLT3 inhibitor SEL24 [Previous reports suggest that on-target resistance tends to occur in patients after type II inhibitor treatment, while off-target resistance is likely to occur after type I inhibitor treatment. Since these reports are currently limited to patients recruited during clinical trials, for a better understanding of the mechanism underlying the resistance to each FLT3 inhibitor, it is necessary to determine the characteristics of patients with resistance in real-word settings. In addition, to counter the gatekeeper mutation (F691L) in ive form . Furtheror SEL24 , a type or SEL24 , a MERTKor SEL24 , a BCR-Aor SEL24 , and a mor SEL24  have exhFLT3 and PIM1, respectively [ATM or its downstream effector G6PD also induced synthetic lethality along with FLT3 inhibition by enhancing mitochondrial oxidative stress, which eventually resulted in tumor apoptosis [Existing FLT3 inhibitors are now being tested in combination with HMAs, standard chemotherapy, bortezomib (proteasome inhibitor), atezolizumab (anti-PD-L1 antibody), venetoclax (BCL-2 inhibitor), milademetan (MDM2 inhibitor) and homoharringtonine (STAT inhibitor). Ongoing trails of combination strategy are summarized in ectively ,120,121.poptosis .FLT3 has become an important determinant of the treatment methods in AML. Currently, a companion diagnostic tool LeukoStratCDx  is widely used for the clinical use of FLT3 inhibitors in Japan, the United States, and Europe, among others. However, LeukoStratCDx is only able to detect D835 and I836 mutations and cannot detect any other FLT-TKD mutations, including F691L. Therefore, the instrument might incorrectly analyze the condition in patients with FLT3-TKD that is potentially treatable by FLT3 inhibitors. Although intensive chemotherapy has ensured substantial clinical benefit in AML patients, several patients eventually require targeted therapy, particularly young patients. In addition, CEBPA and NPM1, and recently TP53, ASXL1 and RUNX1, have been determined to be important markers prognosis [DNMT3A, TET2, SRSF2, and ASXL1 continues to be associated with high relapse rates and poor prognosis [SPRY3 and GSK3 confers resistance to quizartinib by inducing the reactivation of the FGF/RAS/ERK pathway and Wnt signaling [As described, the presence or absence of mutations in rognosis ,31,123, rognosis . Althougrognosis , FLT3 inignaling . Likewisignaling .RAS and PIM1 pathways as well as metabolic modifications  are subject to preclinical investigations. Recent studies have suggested the non-negligible importance of clone-evolutional patterns in terms of acquiring resistance, which possibly affects clinical strategy in managing FLT3-mutated AML. Simply, when you find two distinct targetable mutations and the corresponding agents are available , you can choose either one agent if both mutations are limited in a single leukemic clone, but if each mutations are found in different clones, it is worth considering combination or sequential therapy, if allowed. Routine and successive gene sequencing will help detecting additional targetable mutations and determining individual patterns of clone evolution, which would improve our clinical approaches along with the development of novel agents and combination strategies.This paper described the principal mechanisms of resistance to FLT3 inhibition and the current investigations to overcome it. Secondary on-target mutations  can be managed by choosing type I inhibitors such as gilteritinib that are consistently active against FLT3-TKD as well as FLT3-ITD, except for a “gatekeeper” F691L mutation. Covalently-coupling FF-10101 and other novel FLT3 inhibitors are now under investigation and have shown promising data on FLT3 F691L. Strategies for secondary off-target abnormalities and a part of primary resistance cannot be simple, regarding the diverse relating genomic abnormalities and complex patterns of clonal evolution. Nevertheless, some genetic abnormalities are/will be clinically targetable, expecting a synergistic anti-tumor effect with FLT3 inhibition. For example, several agents targeting BCL-2, MDM2 and STAT as well as conventional chemotherapy are being evaluated in combination with FLT3 inhibitors. Similarly, abnormal Several new agents such as FLT3 inhibitors can create overlapping treatment options, especially in the elderly, unfit AML patients as well as in relapse/refractory AML patients. A lot of clinical trials evaluating the efficacy of promising investigational drugs in AML are ongoing and more drugs will go to the market than ever before. Based on the resistant mechanisms during treatment, how to use these new agents properly is one of the issues with the treatment of AML. Physicians should select an optimal treatment depending on factors such as age, performance status, comorbidities, and genome profiling analysis upon new diagnoses and during treatment.


Development of new, safe, and effective microbicides to prevent human immunodeficiency virus HIV sexual transmission is needed. Unfortunately, most microbicides proved ineffective to prevent the risk of HIV-infection in clinical trials. We are working with G2-S16 polyanionic carbosilane dendrimer (PCD) as a new possible vaginal topical microbicide, based on its short reaction times, wide availability, high reproducibility, and quantitative yields of reaction. G2-S16 PCD exerts anti-HIV activity at an early stage of viral replication, by blocking gp120/CD4/CCR5 interaction, and providing a barrier against infection for long periods of time. G2-S16 PCD was stable at different pH values, as well as in the presence of seminal fluids. It maintained the anti-HIV activity against R5/X4 HIV over time, did not generate any type of drug resistance, and retained the anti-HIV effect when exposed to semen-enhanced viral infection. Importantly, G2-S16 PCD did not modify vaginal microbiota neither in vitro or in vivo. Histopathological examination did not show vaginal irritation, inflammation, lesions, or damage in the vaginal mucosa, after administration of G2-S16 PCD at different concentrations and times in female mice and rabbit animal models. Based on these promising data, G2-S16 PCD could become a good, safe, and readily available candidate to use as a topical vaginal microbicide against HIV. Joint United Nations Program on HIV/AIDS (UNAIDS) estimates that 37.9 million people were living with HIV worldwide at end of 2018 and the fastest growing phase of this pandemic is currently related to heterosexual transmission in women. HIV sexual transmission infection (STI) is held accountable for around 80% of all infections, with roughly half of the affected individuals being women ,5,6. AltLactobacilli spp. that preserve an acidic environment in vaginal vault [Previous publications showed that microbicides are classified as semi-solid  and solid  formulations that could be applied to vaginal or rectal mucosa to attack viral or cell targets, and prevent, or at least reduce, HIV transmission and infection . Brieflyal vault . HIV potal vault ,20. Theral vault ,22.As previously described in the available bibliography, microbicide prevention is rooted in the use of drugs or compounds that directly inactivate HIV in vaginal fluids or in semen, or alternatively, impede HIV integration target cells found in vagina or rectum . An effeThis review analyzes the advances in the state of the art of G2-S16 PCD  as a posProgress of microbicides is based on the use of antiretrovirals (ARV) that precisely act against the HIV lifecycle. Dapivirine (DPV), which binds allosterically to HIV reverse transcriptase, prevents HIV replication and is one of the leading drug candidates in the field. It is currently being tested in various dosage forms and several possible presentations such as gel, film, or intravaginal ring were analyzed ,33,34,35Gardenella vaginalis, supplies a biological mechanism included in a multifactorial process, causing increased vaginal inflammation and adherence [Reverse transcriptase inhibitor, tenofovir (TFV), is one of the most studied microbicides for topical vaginal application against HIV and HSV-2. During bacterial vaginosis, the rapid depletion of TFV by various anaerobic bacteria, such as dherence ,34,38. Tdherence ,40, propdherence .One of the main barriers for microbicide success, as shown by Zirafi et al. is based on the fact that semen decreases sensitivity of HIV to the nucleotide, NNRTI, or integrase inhibitors acting on intracellular targets . This isIn summary, the main weaknesses of microbicides are—variability of their features, adherence needs, emergence of virus resistance against them, need for a high concentration so that they are effective, side effects derived from their toxicity , failure to prevent semen-exposed HIV infection, high daily cost, need to use them as a lifelong medication, and possible drug–drug interactions that might arise.Nanotechnology provides new and suitable approaches to acquire novel, potent, and safer antiviral agents, such as PCD ,51,52,53PCDs with functionalized groups at their periphery can bind to their target, providing a polyvalent strategy for the development of strong viral entry inhibitors. The only topical nano-microbicide that entered a phase I clinical trial for intravaginal microbicides against HIV is poly(L-lysine) (PLL)-dendrimer SPL7013, the active compound of VivaGel™ . HoweverMore sophisticated PCDs acting as HIV entry inhibitors that could prevent non-specific HIV binding to host cells are under development as vaginal microbicides. These PCDs act by blocking different molecules, such as heparan sulfate proteoglycans (HSPG), glycosphingolipids, or CD4/gp120 interactions, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), among other mechanisms . Further50) and toxicity (CC50) indices. From this initial selection, we selected G1-S4, G2-S16, G2-S24P, and G3-S16 dendrimers, as they showed the lowest EC50 values and highest selectivity indices. The selected dendrimers displayed noteworthy antiviral activity (EC50 < 300 nM) with practically no cytotoxicity associated, as toxicity started appearing at significantly higher concentrations. Although, we are currently working with 4 selected PCD, G2-S16 dendrimer showed the most potent EC50 among all PCDs. The potent anti-HIV activity of G2-S16 PCD  was previously reported [Our group performed an impartial, cell-based screening assay for 74 antiviral nanocompounds to determine their potency and cytotoxicity against HIV, HSV-2, and other STI. These nanocompounds included 28 stable PCDs, 27 polyanionic carbosilane dendrons, 11 “bowtie”, 6 gold nanoparticles functionalized with polyanionic carbosilane dendrons, and 2 iron oxide nanoparticles. Primary screening at 1–100 µM concentration determined that 21 of these compounds were highly active and non-toxic, while two compounds were ruled out for further studies, due to their significant cytotoxicity. Given the number of primary hits, we set out the counter screen for these 19 candidates to prioritize them by calculating their potency , human vagina (VK2/E6E7), ectocervix (Ect1/E6/E7) and endocervix cells (End1/E6E7), and in primary human cells  ,66,67. IGenital epithelial cells (GEC) are the first cells in FGT that HIV encounters during sexual transmission. GEC are specialized in recognizing and responding to incoming pathogens, immediately initiating the innate immune responses that constitute the second line of defense in the FGT ,69,70. TPrevious studies propose that dissimilarities in the capacity to answer to microbial products can reach various levels of TLR-mediated activation. TLR trans-membrane proteins are a highly conserved family of pathogen-associated molecular pattern (PAMP) receptors, specialized in detecting foreign material ,72. ThesThe cytokine profiles VK2/E6E7 epithelial cell line and PBMC treated with G2-S16 PCD at early and late times, were assessed with a Th1/Th2 inflammation assay . G2-S16 PCD did not activate this cellular inflammatory pathway and did not induce changes in the expression levels of a wide array of inflammatory-related genes, which is another factor to take into account when designing and developing a safe topical microbicide . These dpH of healthy FGT is acid (pH: 4.0–5.8), while pH of normal semen is alkaline (pH: 8–8.5). Thus, ensuring that G2-S16 PCD could be effective in an alkalinized environment after sexual intercourse is crucial. G2-S16 PCD is stable in a wide pH range of 3–8.5 and maintains a stable anti-HIV activity against different R5 and X4 HIV strains, overtime in seminal fluids . Sperm cLactobacillus acidofillus, which is an abundant beneficial species in the intestinal tract and vagina. Lactobacilli contribute to immunity in FGT by providing a non-specific protection against a broad, large range of pathogens [Vaginal microflora regulates epithelial innate immunity in a species- and strain-specific manner, and topically applied microbicides should not alter the delicate homeostatic balance between epithelial components and microbiota. Normal vaginal microbiota contains a wide variety of species that maintain an acidic pH, by producing hydrogen peroxide and lactic acid . Changesathogens . Bacteriathogens . G2-S16 NL(AD8) laboratory strain. G2-S16 PCD clearly showed inhibitory results on HIV infection at non-toxic concentrations in TZM.bl cells and PBMC. This dendrimer reduced the rates of HIV infection, not only by neutralizing the negative charges of the virus, but also by creating a competitive binding to viral targets (gp120 complex) and to cellular targets (CD4 receptor in areas relevant to CD4/gp120 and CCR5/gp120 or CXCR4/gp120 interactions). Antiviral activity of its sulfonate end-groups was not modified by semen components, by the alkaline pH of semen [G2-S16 PCD has multifactorial and non-specific function and acts as a virucidal agent inhibiting viral entry, providing a barrier against infection for a long time and blocking the spread of HIV from cell-to-cell (CTC) ,55,56,66of semen ,82,83,84of semen .R23 (clade A1), R5-HIVX-1936 (clade C), R5-HIVX2160-2 (clade G), R5-HIVESX-3016-2 (clade F1), R5-HIVESP-2392-3 (clade CRF02_AG), and R5-HIVESX-2457-2 (clade CRF47_BF) isolates [NL(AD8), R5 HIVWT/BAL, R5-HIV-2HSM2.03, R5-HIV-2HCC12.3, X4 HIVNL4.3, X4-HIV-2XT03, X4-HIV-2X4-CC10.3, and dual tropic HIV89.6 laboratory strains [The env-glycoproteins of HIV are the crucial elements involved in HIV entry. The establishment of HIV infection in a naïve host most often results from transmission and successive propagation of a single virus strain, termed as T/F virus . T/F virisolates , as well strains . SummariThe capacity of mucosal epithelial cells to maintain an intact, polarized monolayer when treated with a microbicide, is one of the crucial steps to differentiate safe nanoparticles. Cell-free and cell-associated viruses are prevalent infectious forms of HIV present in semen and cervico-vaginal secretions ,93. G2-SAs aforementioned, adult cervical and foreskin epithelia serve as an entry site for sexual transmission of HIV ,95,96. TDevelopment of resistance to nanocompounds and ARV entry inhibitors is one of the main problems when testing microbicides in human clinical trials. Treatment with G2-S16 PCD against HIV infection for thirty successive cells passages in MT-2 cells, did not generate any type of drug resistance. Even more, efficacy and thigh genetic barrier of G2-S16 PCD was also demonstrated, thus, reinforcing the evidences for its security and effectiveness  Table 1Table 1.One of the most promising targets within HIV lifecycle is the viral entry/fusion process , which iIn essence, G2-S16 PCD interacts non-specifically with gp120 and the cell receptors, in a dose-dependent manner, although it does so predominantly with the CD4 receptors of the host cells. Previous studies using molecular dynamics simulations, support this idea, because the mechanism of action of dendrimer is related to electrostatic interactions between the anionic functional groups located at dendrimer periphery and glycoproteins of viral envelope or proteins at host cell surface that are involved in HIV infection. Our data clearly suggest that G2-S16 PCD could protect urogenital epithelial cells from the tight junction disruption induced by HIV infection.NLAD8 and X4-HIVNL4.3 isolates and its HIV virucidal activity were evaluated [SPL7013 dendrimer, the active product of VivaGel™, is not a virucidal agent, as it lacks a broad antiviral activity against R5-HIV isolates, due to the fact that V3 loops vary in positive charges between the R5- and X4-HIV isolates. We obtained several lines of evidence that other effective mechanisms of inactivation can be involved. G2-S16 PCD not only binds to the V3 loop and co-receptor binding sites , but canvaluated .A safe microbicide should remain effective for many hours after gel application, even after the gel is washed away. First-generation microbicides are only effective for a few hours, and consequently, require administration shortly before sexual intercourse . In contIn order to show the antiviral target of G2-S16 PCD, a time-of-addition assay was performed to delimit the stage(s) of HIV lifecycle that get blocked by G2-S16 PCD, compared to inhibitor T-20 (that decline after 2 h), AZT (that decline after 5 h), and raltegravir (RAL) (that decline after 7 h post-infection). G2-S16 PCD retained its antiviral activity for up to 2–3 h after HIV infection, showing a way of action similar to that of T-20. These findings, consistent with those obtained for other polyanionic compounds , confirmG2-S16 PCD prevents HIV entry in a manner that is independent of the number of viral particles or the presence of semen, and it also acts as an inhibitor of CTC transmission ,57. CTC Topical effect of G2-S16 PCD on mucosa was first studied in CD1 (ICR) mice for in vivo evaluation, analyzing the integrity of mucosal tissue . No mortw/v) of G2-S16 PCD was mixed in 2% (w/v) of HEC, which is biocomplatible with normal human vagina  geland and the active pharmaceutical ingredient is G2-S16 PCD. A total of 3% weight/volume  mice validated this mechanism .+ h-BLT mice  [JR-CSF, G2-S16 PCD was found to efficiently prevent vaginal HIV transmission at 84% level  models are used in pre-clinical studies, it is very important to take into account their high costs, limited availability of female animals, inability to employ HIV for challenge studies and tests against HIV drug-resistant, as well as ethical problems related to their use. It is unclear whether NHP models exactly predict what would occur in humans. The best small-animal models for HIV are humanized mice, specifically, the ones that are genetically immune-compromised and engrafted with human tissues to reconstitute their immune system . Human-BC alone) . In vivoC alone) ,124,125.4% level . These rIn conclusion, a vaginal/rectal microbicide must display activity against most HIV strains, and other sexually transmitted pathogens that act as a direct virucidal, retain the activity for several hours in the presence of vaginal fluids over a broad pH range, must not fail to prevent semen-exposed HIV infection, should not leak immediately after application, should not accumulate in order to avoid toxicity effects, should not affect normal vaginal microflora, and the structural integrity of vaginal  mucosal epithelium must be odorless, colorless, tasteless, stable at higher temperatures, and compatible for use with the male latex condom. Microbicides must have an excellent stability, should be easy to use, show a long shelf-life and be inexpensive and readily accessible. G2-S16 dendrimer could be a new innovative and promising microbicide, meeting all the aforementioned criteria and very close to a clinical trial that could contribute to the development of multi-purpose interventions to prevent HIV infection in women.