With the development of sequencing technology, a large number of long non-coding RNAs (lncRNAs) have been identified in addition to coding genes. LncRNAs, originally considered as junk RNA, are dysregulated in various types of cancer. Although protein-coding signaling pathways underlie various biological activities, and abnormal signal transduction is a key trigger and indicator for tumorigenesis and cancer progression, lncRNAs are sparking keen interest due to their versatile roles in fine-tuning signaling pathways. We are just beginning to scratch the surface of lncRNAs. Therefore, despite the fact that lncRNAs drive malignant phenotypes from multiple perspectives, in this review, we focus on important signaling pathways modulated by lncRNAs in cancer to demonstrate an up-to-date understanding of this emerging field. Long noncoding RNAs are >200 nt in length and have almost no protein-encoding function, but they can exert regulatory effects on transcriptional, post-transcriptional, and post-translational modifications of coding genes.Signaling pathways are a series of enzymatic reactions in response to intracellular and extracellular stimuli and underlie almost every disease, including cancer.LncRNAs participate in tumorigenesis and cancer progression by fine-tuning various cancer signaling pathways.Whether disparate signaling pathways regulated by an individual lncRNA converge on similar cancer hallmarks?What are the mechanisms through which the crosstalk among diverse signaling pathways is established?How can we target lncRNAs to fine-tune cancer signaling pathways?cis or trans [Recently, many noncoding RNAs have been discovered using high-throughput sequencing technology. Although they do not encode proteins, they have powerful functions, participating in transcriptional, post-transcriptional, and post-translational regulation of coding genes. Long noncoding RNAs (lncRNAs) are >200 nt in length and have almost no protein-encoding function . LncRNAsor trans . They plor trans , 5. Theyor trans , 7.Protein is the executor of biological function. Protein-coding signaling pathways underlie almost every biological process, especially carcinogenesis and progression. Signaling pathways are a series of enzymatic reactions in response to intracellular and extracellular stimuli. They participate in the transmission of cellular signals and are important for biological reactions and gene expression. Signaling pathways are extremely complex, and many signaling molecules are involved in cascade reactions, which are often regulated, directly or indirectly, by various types of molecules . EmerginWnt/β-catenin signaling pathway is the canonical pathway of the Wnt signaling cascade. Its dysregulation is implicated in tumorigenesis, including proliferation, invasion, metastasis, and apoptosis, and has been the focus of cancer research –17. The LncRNAs play a tumor-suppressive or tumor-promoting role via interacting with key factors in the Wnt pathway. For instance, in lung adenocarcinoma, lncRNA LINC00673 enhances the interaction between DDX3 and CK1ε to phosphorylate DVL, thereby activating the Wnt /β-catenin signaling pathway and promoting cancer progression . LncRNAsCollectively, lncRNAs can regulate multiple molecules of the Wnt/β-catenin signaling pathway, ultimately facilitating malignant phenotypes Fig. .Fig. 1ScHippo signaling is an evolutionarily highly conserved pathway that regulates multiple biological processes including cell proliferation and differentiation. Disruption of the Hippo pathway may trigger tumorigenesis. However, mutations in Hippo pathway genes are not common. Molecular events that interfere with the Hippo pathway might be an important mechanism in cancer initiation . The HipAs mentioned above, molecular events might play an important role in regulating the Hippo pathway. Many lncRNAs are involved in regulating this pathway. LncRNAs can interact with the core kinase, thus affecting the downstream cascade. LncRNA MIR100HG binds to the histone methylation regulator EZH2, acting as a scaffold that epigenetically silences LATS1/2. MIR100HG plays an oncogenic role in osteosarcoma through inactivating the Hippo pathway . LncRNA Thus, lncRNAs modulate the Hippo signaling pathway in diverse manners, eventually leading to the activation or suppression of downstream cancer-related genes Fig. .Fig. 2ScNotch signaling pathway is involved in various biological processes, including cell differentiation, proliferation, and apoptosis. Notch signaling also plays pleiotropic roles in carcinogenesis and tumor progression. Four receptors  and five ligands  have been identified in Notch signaling . The recLINC01152 is overexpressed in glioblastoma multiforme, functioning as an oncogene. It activates the Notch signaling pathway by upregulating the expression of transcriptional co-activator 2 (MAML2). LINC01152 serves as a competing endogenous RNA via sponging microRNA miR-466. Moreover, LINC01152 can prevent MAML2 from shearing via recruiting SRSF1, thus maintaining the level of MAML2. Besides, MAML2 could, in turn, promote the expression of LINC01152 via regulating the Notch pathway . LncRNA Although the Notch signaling pathway has a relatively simple framework, it plays an important role in cell proliferation, differentiation, and apoptosis Fig. . As mentSince the NF-κB subunits were recognized >20 years ago as being homologous to the viral oncogene v-Rel, the role of these proteins in tumorigenesis has been accepted. However, early research on NF-κB mainly focused on the regulation of the immune response. With the deepening of the research on NF-κB, the role of the inflammatory response in tumor development gradually became prominent , 45. NF-In mammals, NF-κB complexes comprise five proteins, including RelA (p65), RelB, c-Rel, p50 (p105 precursor), and p52 (p100 precursor), which all share an N-terminal Rel homology domain that is required for dimerization, DNA binding, and inhibitory IκB protein interaction . There aAs an important signaling pathway, NF-κB signaling is modulated by multiple regulatory mechanisms. Increasing evidence shows that lncRNAs are involved in the regulation of the NF-κB signaling pathway. LncRNAs can directly interact with functional signaling proteins involved in NF-κB signaling, serving as modulators. LncRNA NKILA inhibits IKK-induced IκB phosphorylation by masking the phosphorylation motifs of IκB and impeding NF-κB activation. NKILA prevents breast cancer metastasis by inhibiting over-activation of the NF-κB pathway . In gastLncRNAs are emerging as important modulators in the management of NF-κB signaling, gradually unraveling the mystery of cancer development and progression Fig. .Fig. 4ScThe HH signaling pathway is essential for many fundamental processes, including embryogenesis, stem cell maintenance, and tissue regeneration . Some stLncRNAs, as small-molecule regulators, may be involved in modulating the HH signaling pathway at different levels and might be related to tumorigenesis and development. LncRNA HOTAIR expression is dysregulated in various cancers and affects cancer development through multiple mechanisms . In renaThe TGF-β signaling pathway consists of TGF-β ligands, receptors, and downstream signaling molecules. The pathway participates in organismic homeostasis, controlling cell proliferation, motility, and differentiation , 70. In Many studies have confirmed that lncRNAs are involved in the regulation of the TGF-β signaling pathway, thus affecting tumorigenesis and cancer progression. Lnc-TSI exerts a tumor-suppressor role in clear cell renal cell carcinoma by inhibiting metastasis. Lnc-TSI impedes the phosphorylation of SMAD3 by blocking the interaction between SMAD3 and TGFBR1 at the MH2 domain. Consequently, the TGF-β/SMADs signaling pathway is inhibited, which suppresses epithelial–mesenchymal transition . LncRNAsAlthough a large number of lncRNAs have been reported to be related to TGF-β signaling pathway, the exact mechanisms of their mutual interactions are mostly unknown. Further research into the lncRNA–TGF-β signaling network will expand our understanding of cancer pathogenesis and provide novel insight into therapeutic strategies.In addition to the signaling pathways mentioned above, lncRNAs also play regulatory roles in other signaling pathways during tumorigenesis. One of these is the JAK-STAT signaling pathway, which is necessary for homeostasis and development, mainly mediating inflammation and immunity . DisturbAnother potent pathway is the PI3K/AKT signaling pathway, which consists of two main proteins, PI3K and AKT. Activated by growth factors through GPCRs or receptor tyrosine kinase receptors, PI3K activates AKT and downstream effectors through the lipid second messenger phosphatidylinsoitol-3,4,5-trisphosphate. The pathway is important for cell growth and differentiation. Disturbance of the PI3K/AKT pathway might be a critical event in the process of tumor development . It has Deregulation of protein-coding signaling pathways is fundamental to tumorigenesis and cancer progression. However, lncRNAs, hidden in the signaling pathways, cannot be neglected for their fine-tuning functions. Notably, one signaling pathway can be fine-tuned by multiple lncRNAs. As mentioned previously, Linc01354, Linc00210, Linc01197, LincTCF7, and LINC00673 participate in activating or suppressing the Wnt/β-catenin signaling pathway. In another example, lncRNAs, as upstream mediators, have a dual function in the STAT3 signaling pathway in different cancers. NEAT1, SNHG3, and H19 induce STAT3 expression, while MEG3, PTCSC3, and NKILA inhibit STAT3 expression . Not surThe regulation of signaling pathways by lncRNAs is complex and diverse. Studies have confirmed the importance of lncRNAs in the regulatory network, functioning to make the cascades operate more smoothly or with greater flexibility. LncRNAs may interact directly or indirectly with signaling molecules to regulate signaling pathways. Despite the support of advanced technologies, such as RNA precipitation, RNA immunoprecipitation, crosslinking immunoprecipitation, and RNA interference or CRISPR  technologies, the complexity of signaling pathways makes research on the exact interaction mechanism between lncRNAs and signaling molecules a challenge. With the further development of experimental techniques, a deeper understanding of lncRNA-mediated signaling transduction will be gained.Although numerous studies have shown that lncRNAs play significant roles in the regulation of signal networks and even in the development of tumors, the human body is a complex system, and no molecule can achieve the effect of affecting the whole body by one action; therefore, it would make sense to investigate intermolecular synergy. However, in the cascades of signaling pathways, a “butterfly effect” might also be a feature. In this complex network, many fields remain unexplored. Thus, further effort on the influence of lncRNAs on signaling pathways and even the whole biological process will shed light on the mystery of cancer. However, even our current limited knowledge has provided a novel and promising avenue for the fast-emerging field.


Quorum sensing is a cell-cell communication system that bacteria use to express social phenotypes, such as the production of extracellular enzymes or toxins, at high cell densities when these phenotypes are most beneficial. However, many bacterial strains are known to lack a sensing mechanism for quorum signals, despite having the gene responsible for releasing the signals to the environment. The aim of this article is 2-fold. First, we utilize mathematical modeling and signaling theory to elucidate the advantage that a bacterial species can gain by releasing quorum signals, while not being able to sense them, in the context of ecological competition with a focal quorum sensing species, by reducing the focal species' ability to optimize the timing of expression of the quorum sensing regulated phenotype. Additionally, the consequences of such “dishonest signaling,” signaling that has evolved to harm the signal's receiver, on the focal quorum sensing species are investigated. It is found that quorum sensing bacteria would have to incur an additional, strategic, signaling cost in order to not suffer a reduction in fitness against dishonest signaling strains. Also, the concept of the Least Expensive Reliable Signal is introduced and applied to study how the properties of the regulated phenotype affect the metabolic investment in signaling needed by the quorum sensing bacteria to withstand dishonest signaling. Bacterial cells lead a surprisingly social life, commonly growing in surface-attached communities consisting of multiple species, called biofilms  and S (mg bacteria/l) as the densities of the QS toxin producer and the sensitive strain respectively in the system. T, N, and Q (mg/l) are the concentrations of the toxin, nutrient and the quorum signals. Qth is the threshold quorum concentration at which toxin production is initiated. f is the fraction of metabolic energy invested by the producer strain for toxin production. qP and qS are the fractions of energy utilized for the production of quorum signals by the producer strain and the sensitive strain respectively. KT (l/mg toxin/hr) is the toxin's killing rate, while βT (1/hr) is the toxin decay rate in the environment and α (mg toxin/mg bacteria) is the stoichiometric coefficient for toxin production. C (mg bacteria /mg quorum molecules) is the cost of a single signaling molecule in terms of bacterial biomass. μ (1/hr), μmax (1/hr) and KN (mg/l) are the growth rate of a bacterial species, the maximum specific growth rate and the half saturation constant respectively. In the first equation, a heaviside step function controls the production of the toxin, where H(Q−Qth) = 1, when Q > Qth and H(Q−Qth) = 0, otherwise. The nominal values of the parameters  is the corresponding threshold concentration at which the expression of the regulated trait occurs.where γ (mg bacteria/l) is the quorum sensing molecules concentration, expressed in terms of its equivalent bacterial mass, per unit volume of the system, γ = m grid, seeded with uniformly distributed 80 cells of each strain and carried out till the biofilm height reached 150 μm.The well-mixed system model simulations are carried out using the ode45 solver in Matlab. All the simulations are seeded with a concentration of 1 mg/l of each species. The individual-based modeling has been conducted using MICRODIMS, an in-house IbM platform for simulating microbial dynamics in colonies and biofilms. MICRODIMS has been developed in BioTeC+ and has been applied to model bacterial growth either as surface colonies or biofilms  is introduced. Bacteria can ensure the activation of density dependent phenotypes at optimal timing using signaling systems with different metabolic costs, by adjusting the rate of signal production and the threshold concentration at which the response occurs. A QS system is considered to be reliable if the fitness of the focal QS strain when subjected to dishonest signals producing competitor is higher than or equal its fitness in a dishonest signals free competition. And when this property is satisfied using the least amount of metabolic investment in the QS system on the behalf of the QS strain, the QS system is said to be operating at LERS. Hence, for signals cheaper than LERS, the fitness of the focal QS toxin producing strain will get lower when exposed to a dishonest signals producing competitor, as seen in f, affect the metabolic investment needed for ensuring the reliability of the QS system to regulate it, qP. It is noticed in viaWhile the value of LERS could be convenient in analyzing the reliability of QS systems, one should not expect that bacteria in nature operate exactly at LERS. First, because it is competition dependent, a QS system could be reliable when facing ecological competitors which have no or limited capacity of producing dishonest signals. However, the same system will be less reliable when facing a competitor that is efficient in producing such signals. In addition, bacteria can occur in such a wide variety of environments and sometimes there will be no need to protect the QS system against dishonest signaling at all. Hence, bacteria in nature are expected to have sufficiently reliable QS systems against the most frequently encountered competitors but not toward all possible competitors. The LERS concept is implemented here to study how the parameters of the model affect the reliability of a QS system. We started by studying how changes in the fraction of investment in toxin production, KT, the toxin's killing rate, of the toxin regulated and searched for the LERS value corresponding to different toxin killing rates. It was found that there was no effect of having a more efficient toxin on the LERS value of the QS system of the focal strain regulating it, shown in KT, the fitness of the QS toxin producing strain increases over the whole range of the timing of toxin production, hence the gain for the sensitive strain from a premature triggering event remains roughly the same across different toxicities.But does a QS system need to be more expensive when the function regulated plays more critical role at the outcome of the competition, such as when regulating the expression of highly effective extracellular enzymes or highly lethal toxins? To study that, we varied the Materials and Methods. First, we studied the effect of the timing of toxin production on the fate of competition. In So far, all the simulations were carried out for the competition between a focal QS toxin producing strain and a dishonest signals producing sensitive strain in a well-mixed environment. To further test the validity of our model, the reliability of a QS regulation system was investigated in the context of spatial competition between the focal strain and the sensitive strain growing together in a biofilm. An individual-based model for the competition between the two strains has been built using MICRODIMS, an in-house individual based modeling platform for bacterial growth . This is because, as the biofilm mode of growth is an open system, signals can diffuse across the boundary to the surrounding environment of the cells, while in the well-mixed system signals can only accumulate in the system, thus causing a discrepancy in the level of metabolic investment in signaling needed to reach the threshold concentration.A notable difference observed between the spatial model and the well-mixed one is that the LERS value is an order of magnitude higher in the spatial model  is studying how a focal QS strain that uses multiple QS circuits in parallel to regulate the expression of a social trait fares in the face of dishonest signaling. The first bacterial species known to use multiple QS circuits to regulate its traits is qP1 and qP2 in producing two different signaling molecules for two parallel circuits. Three ways of integrating information from both circuits have been modeled. Information could be combined in logic gates; an “AND” gate signifies that both signals would need to cross the threshold concentrations, γ1 > γth1 and γ2 > γth2, for the toxin to be expressed, where γ1 and γ2 are the concentrations of the two signaling molecules. γth1 and γth2 are their corresponding threshold concentrations. The two circuits are assumed to have identical parameter values and to operate at lower than LERS; th1 = γth2 = 0.002mg/l.Here, the model was extended such that the focal species uses two parallel QS circuits to regulate the expression of its toxin, while being subjected to dishonest signaling on only one of the two signals. The focal species invests 1 > γth1 or γ2 > γth2. Finally, a third method by which the information from the two circuits can be combined is through the weighted sum of both signals. Long et al. γ2 > γth, with w = 0.5.Alternatively, in an “OR” gate the release of the toxin happens when any of the two signaling molecules crosses the threshold concentration: γg et al.  have shoThe three methods of integrating information were tested against increasing levels of dishonest signaling on only one of the circuits. The results are shown in In this work, the reliability of QS systems of bacterial species in an environment where they are subjected to dishonest signaling has been investigated. The model used is the competition between a focal QS toxin producing strain against a sensitive strain that is able to produce quorum signals, but not able to sense their concentration. It has been shown that even without sensing the signals, the sensitive strain can still benefit from an early triggering of toxin production by the focal QS strain. Additionally, this may ultimately lead to the evolution of a more expensive QS signaling system for the focal strain.The concept of LERS has been put forward to indicate the mathematically optimal signaling cost for a QS focal strain given a certain opponent(s). Hence, LERS constitutes the Evolutionary Stable Strategy (ESS) for the QS strain given a specific dishonest signaling opponent. A QS strain operating lower than LERS would suffer a fitness reduction in the presence of a dishonest signaling opponent. On the other hand, when a QS strain invests higher than LERS in its signaling system, it will have lower growth rate compared to operating at LERS, while still being reliable against opposition. The reliability of a QS system can only be assessed in the context of competition with a specific dishonest signaling opponent. If the fitness of the focal QS strain decreases when competing with a dishonest signaling strain, and this reduction in fitness can be directly attributed to the effect of dishonest signaling, then the focal strain is said to be operating at lower than LERS. On the other hand, when the fitness of a focal strain stays the same or increases in the existence of a dishonest signaling, then the focal QS strain is said to operate at higher than or equal to LERS. This increase in fitness would occur despite the effect of dishonest signaling; while the expression of the regulated trait would occur suboptimally, the cost incurred by the dishonest signaling opponent to elicit this response would be too high that it will suffer a fitness disadvantage. Hence, assessing whether a QS system is operating at higher than or lower than LERS would give an indication about its reliability.The optimal ESS levels for the expression of beneficial traits can exhibit different dynamics from spiteful traits, for example as a function of population relatedness , the ERA-NET FACCESurPlus FLEXIBI Project, co-funded by VLAIO project HBC.2017.0176, the Fund for Scientific Research-Flanders (projects G.0863.18 and G.0B41.21N), and the European Union's Horizon 2020 Research and Innovation Programme (Marie Sklodowska-Curie grant agreement numbers 813329 and 956126).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve’s construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies.Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV-1/-2 (mqPCR_HTLV) assays, targeting the  Human T-lymphotropic virus 1 and 2 (HTLV-1 and HTLV-2) are endemic to Brazil and their distribution varies according to the ethnic background of individuals and geographic regions . Brazil The laboratory diagnosis of HTLV-1 and -2 is performed on blood samples from patients using screening tests followed by confirmatory tests. The screening tests detect antibodies against HTLV-1/2 in plasma/serum samples; they have high sensitivities, and negative results exclude HTLV-1/2 infection . The mosThe HTLV Brazilian Protocol for Sexually Transmitted Infections 2020 recommends performing WB or LIA as the first confirmatory assay, and in case of inconclusive results conducting a PCR for confirmation . Howeverenv, tax, LTR), PCR-RFLP , and qPCR (pol) as confirmatory assays  Central Public Health Laboratory in São Paulo, Brazil, has been conducting studies on HTLV-1 and -2 infections, and has performed HTLV serology routinely since 1998. At this laboratory, we searched for the best assays and algorithms that could be used for testing in São Paulo and Brazil in general, based on performance and cost-effectiveness. During these years we had the opportunity to use WB , LIA, n-PCR  for the standard curve construction. We used two platforms and seven commercial master mix formulations simulating the various core facility laboratories in Brazil and their different conditions and supplies.In light of this scenario, and considering the exorbitant expenses on reagents, the shortage of clinical specimens, the different qPCR platforms, and the supplies available in each Central Public Health Laboratory in Brazil, we decided to develop and validate two in-house multiplex quantitative real-time PCR assays (mqPCR_HTLV), targeting the Blood samples obtained from patients tested for HTLV-1/-2 specific antibodies which were infected and not infected with HIV-1, HBV and HCV were distributed in the following groups: Group 1, 37 patients infected with HTLV-1 alone confirmed by WB; Group 2, 152 patients infected with HIV-1 and reactive of HTLV-1/2 infection by screening assays; Group 3, 30 patients not infected with HTLV but infected with HIV-1 and/or HBV and/or HCV.env and tax sequencing) using DNA extracted from peripheral blood mononuclear cells (PBMCs) isolated by Ficoll-Paque Plus density gradient centrifugation . The HTLV-1 sequences from Recife were deposited in GenBank , and the remaining DNA samples were tested for mqPCR_HTLV assays.Briefly, samples of Group 1 belonged to blood donors from Recife, Pernambuco (Northeast Brazil), tested positive for HTLV-1 during the years 2012–2017, and sent to IAL for HTLV-1 subtype characterization  for HTLV-1/2 diagnosis. The samples were used to detect the prevalence of HTLV-1 and HTLV-2 in HIV-1 infected patients from São Paulo, for HTLV-1/2 subtype surveillance, and for searching markers of diagnostic and prognostic values in retroviruses coinfection. The data of such research were published elsewhere , PBMCs were isolated by Ficoll-Paque Plus density gradient centrifugation , washed twice in phosphate buffered saline, and 106 cells pellets stored at −20°C for further qPCR quantitative analysis.In some of the HTLV-1/-2-infected patients (Group 2) in clinical and laboratory follow-up tests .® LC Robot Instrument, , with the LC MagNA Pure Nucleic Acid Isolation kit I from Roche Diagnostics, , according to the manufacturer’s instructions, and eluted with 100 μL of PCR-grade water. The extracted DNA was aliquoted into 4 vials for subsequent analysis and thawed only once.Genomic DNA was extracted and purified from PBL and PBMC samples using Roche MagNA Purepol and tax) assays for HTLV-1 and HTLV-2 detection, their position in the genomic prototypes , MoT , human albumin (GenBank accession number M12523.1), amplicon size, and the authors’ reference. The oligonucleotide concentrations were optimized using different concentrations of primers and probes in the range of 0.2–0.9 and 0.1–0.3 μM, respectively.1 showed a mismatch (A and G) among some HTLV-2 Brazilian isolates at position 4,780 of the pol target region. Thus, to overcome this problem, a degenerate base (R) was included in the probe design  website,pol and tax) were employed as positive controls for standard curves construction.Double recombinants plasmids of HTLV-1- and HTLV-2-albumin for both the gene targets  was kindly provided by Jorge Casseb from the Instituto de Medicina Tropical, São Paulo, and it was constructed by cloning a segment of 246 bp of the pol region of HTLV-1  and a 1,183 bp segment  of intron 12 of the human albumin gene, according to pol gene of HTLV-2  and a 170 bp fragment of the human albumin gene .The double recombinant plasmid of the tax target genes were designed at IAL and synthesized by Invitrogen GeneArt Gene Synthesis . The pHTLV1tax-Alb plasmid consisted of a 321 bp synthetic DNA sequence , and the pHTLV2tax-Alb plasmid consisted of a 297 bp fragment , and both the plasmids contained a 170 bp fragment of the human albumin gene . The plasmids containing the HTLV-1 or HTLV-2 tax and albumin target genes were expanded in E. coli cells in Luria Bertani (LB) medium containing 0.2 mg/mL ampicillin, and subsequently extracted and purified using the QIAprep Spin up Miniprep kit . The sequences of the cloned inserts were determined and confirmed by performing a sequencing reaction on ABI 3130xl equipment and subsequently compared with reference sequences in GenBank .The double recombinant plasmids for the The plasmid concentrations (ng/μL) were determined using the NanoDrop 2000 spectrophotometer , and the Avogadro’s constant was used to convert it to the number of copies per microliter.6 to 1 copy/5 μL in Tris-EDTA solution  and pH 8.0, in duplicate. The cycle quantification (Cq) values obtained were plotted against the logarithm of copy number.To determine the performance of the plasmids in mqPCR_HTLV assays, their standard curves and the limit of detection (LOD) were obtained by amplification of 10-fold dilutions of the plasmids containing 10pol) assay with concentrations of plasmids varying from 100 to 10 copies was tested in six replicates among the range of 100–15 copies and in 12 replicates for 10 copies.Since HTLV-2 is known to have fewer copy numbers in relation to HTLV-1 , the LODslope) and R2 ≥ 0.98  −1 ; the par2 ≥ 0.98 .tax probe, which was used at 0.1 μM. Passive reference dye (ROX) was added to the reaction mix when using the ABI platform, according to the manufacturer’s instructions. PCR thermocycler conditions for amplification were as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 50 cycles at 90°C for 50 s, and 1 cycle at 60°C for 1 min  and the 7.500 Applied Biosystems—ABI (Applied Biosystems) platforms. Each mqPCR_HTLV reaction was 25 μL in volume and contained: 5 μL of DNA template, 12.5 μL of master mix reagent, 0.2 μM of each set of oligonucleotides, except for the or 1 min . The rea®Quantitative PCR SuperMix-UDG (Invitrogen), GoTaq®Probe qPCR Master Mix (Promega), PerfeCTa qPCR ToughMix (Quantabio); Sso Advanced™ Universal Probes Supermix (Bio-Rad), Kapa Probe Fast qPCR Kit Master Mix (2X) Universal (Kapa Biosystems) and LuminoCt® qPCR ReadyMix™ (Merck/Sigma).Seven different commercial master mix brands for hydrolysis probes were analyzed in the mqPCR_HTLV assays, using the ABI/Roche platforms: LightCycler Probes Master (Roche), Platinumpol and tax) at six points along the standard curves.To validate the multiplex qPCR format using plasmids, we compared the performance of multiplex and singleplex reactions in detecting HTLV-1 and HTLV-2 . These professionals reproduced the reactions without previous knowledge of the mqPCR_HTLV results, having received only the initial concentration of the plasmids and the LOD protocol.pol and tax), we employed genomic DNA samples obtained from patients who were HTLV seropositive in screening assays, and for calculating the sensitivity and specificity of assays we considered the WB results. In case of divergent results, we observed the results of another confirmatory serological assay  and/or the PCR-RFLP (tax) assay, as previously described  and 152 patients infected with HIV and suspected of HTLV-1/-2 coinfection (Group 2). The specificity of assays was evaluated using genomic DNA samples of 30 HTLV-1/-2-seronegative patients infected with HIV-1 (Group 3), which included 22 HIV-monoinfected, 2 HIV/HBV-coinfected, 3 HIV/HCV-coinfected, and 3 HIV/HBV/HCV-coinfected.pol and tax) assays (reproducibility intra- and inter-assays) was assessed in DNA samples of patients that had different positive Cq values (profiles): Cq ≤ 30 (high); Cq from 31 to 35 (median); Cq from 36 to 37 (low); Cq from 38 to 40 (scarce). Eight clinical DNA samples were processed (4 HTLV-1 and 4 HTLV-2) in duplicate and the experiments were conducted daily across 4 consecutive days.The precision of mqPCR_HTLV (pol), which was conducted in duplicate using the ABI equipment. Using 5 μL of the 100 μL of DNA samples extracted from 1 × 106 PBMCs of 16 HTLV-1/2-infected patients in follow-up (Group 2), we inferred that the Cq value obtained corresponds to 0.5 × 105 cells. Thus, for calculating the number of copies of HTLV-1 and HTLV-2 in 105 cells, we plotted the Cq value obtained in the standard curve and multiplied the number of copies by 2.In addition to the qualitative use of the assays, we evaluated their possible use in PVL quantification. Unfortunately, due to the low volume of the remaining DNA samples, this analysis could not be performed for all the mqPCR_HTLV assays, but only mqPCR_HTLV  and the Kappa index were calculated. The Grubbs test was employed for the comparison of the methods and the creation of the linearity graphs.SD) values, and coefficient of variation (CV) . CV of Cq values of intra- and inter-assay reproducibility of 3 and 10%, respectively, are considered acceptable according to The intra- and inter-assay reproducibility were calculated in percentage using the mean Cq, standard deviation (pol and tax) assays were optimized using 0.2 μM primers and probes, except for the tax probe which performed better at a concentration of 0.1 μM.After testing several concentrations of primers (0.2–0.9 μM), and probes (0.1–0.3 μM), the mqPCR_HTLV  assays analyzed using the ABI and Roche’s equipment are shown in tax target gene.The efficiencies and LODs of mqPCR_HTLV  agree with the acceptance performance parameters for mqPCR assays, but because the LOD for HTLV-2 pol ranged from 10 to 100 copies per reaction, it was decided to conduct additional evaluations in this range of plasmid dilutions to determine the best final LOD using intra-assay reproducibility analyses (pol).All results obtained  assays conducted with respect to the plasmid dilutions prepared by the three analysts, and those prepared by the robotic workstation for high-precision PCR, are presented in pol and tax) assays, 10 copies of HTLV-2 in mqPCR_HTLV (tax) assay, and approximately 10–100 copies of HTLV-2 in mqPCR_HTLV (pol) assay, thereby corroborating previous results.The intra- and inter-assay reproducibility results of the mqPCR_HTLV (pol and tax) assays we compared the WB results. Of the 37 DNA samples from patients infected with HTLV-1 alone analyzed by WB (Group 1), 36 were positive in both mqPCR_HTLV (pol and tax) assays . The single mqPCR_HTLV negative sample showed discordant results on screening (data not shown).To calculate the sensitivity of the mqPCR_HTLV (pol and tax assays showed different and interesting results (pol) assay, 101 out of the 152 samples were positive , and as per the mqPCR_HTLV (tax) assay, 95 out of the 152 samples were positive . The mqPCR_HTLV (pol) assay was able to detect more cases of HTLV-2 than the mqPCR_HTLV (tax) assay and WB (The testing of 152 DNA samples from HIV-infected patients that were HTLV reactive on screening (Group 2) using the mqPCR_HTLV  results . Per WB y and WB .pol) assay identified ten as positive (45.5%): nine as HTLV-2 , and one as HTLV-1 (Patient N. 7). By the mqPCR (tax) assay, seven were positive (31.8%): six were HTLV-2  and one was HTLV-1 (Patient N. 7). Regarding the nine WB HTLV untyped samples, six (66.7%) were identified as positive: three HTLV-1  and one HTLV-2 (Patient N. 84), using both the mqPCR (pol and tax) assays, one HTLV-2 using mqPCR pol (Patient N. 129) and one HTLV-1 by mqPCR tax (Patient N. 108)  .pol), and was repeatedly detected positive for HTLV-1 and HTLV-2 by mqPCR-tax and for HTLV-1 by PCR-RFLP (tax)  assays (Patients N. 11 and 56). In one of these samples the LIA was indeterminate.Furthermore, three discrepant results were observed among the confirmatory assays, one sample confirmed HTLV-2 infection in three tests  assays compared to that of WB in HTLV-1 monoinfected patients was 97.4%; in HIV/HTLV-coinfected was 77.1%, Kappa index—0.405 (95% CI 0.238–0.575) (mqPCR_HTLV pol) and 74.6%, Kappa index—0.435 (95% CI 0.277–0.593) (mqPCR_HTLV tax). No sample from Group 3 (HIV- and/or HBV- and/or HCV-infected) and or samples negative per WB and LIA assays from Group 2 were positive in mqPCR_HTLV assays. Additionally, all samples tested negative for HTLV-1 and HTLV-2 in molecular assays were positive for the reference gene (Albumin).Thus, the relative sensitivity of the mqPCR_HTLV (pol and tax). The DNA samples from eight individuals with different positive Cq profiles were tested in duplicates across four consecutive days. The mean Cq and CV values were determined and the results showed minor variation and good reproducibility of results in samples with low DNA concentrations (Cq > 36). Although the same input volume of DNA was used for both the assays to detect HTLV-2, a difference around 3 Cq was detected, the greater value being for pol target gene detection. Despite the fact that the mqPCR_HTLV tax presented lower Cq values in relation to the mqPCR_HTLV pol, the tax target gene was more difficult to detect in samples with scarce DNA copy number, corroborating previous findings of assays standardization.pol). Four (25%) were negative, seven (43.7%) were HTLV-1 positive, and five (31.3%) were HTLV-2 positive. The results of provirus copy number calculated as described in section “Materials and Methods” are presented as Sixteen DNA samples from patients of Group 2 in clinical and laboratory follow-ups were analyzed for PVL quantification using the mqPCR_HTLV , since this kind of reference material is stable, provides reproducible results, and could be used for absolute PVL quantification . Bandeirpol, and tax, and utilized probes labeled with FAM (HTLV-1), CY5 (HTLV-2) and HEX . We used two assays employing different target genes to avert possible false negative results due to the presence of point mutations in primers and probes binding regions or the presence of defective provirus particles (absence of such genes) in HTLV-1 and -2 infected individuals  of assays were 10 and 10 copies for HTLV-1 and 10 and 70 copies for HTLV-2 using tax specific target primers and human β-globin as reference gene (NED). However, the reference gene to quantify cells number was detected separately and the dye NED is exclusive to the ABI platform.Although previous studies of mqPCRs disclosed greater sensitivities for detect and quantify HTLV-1/2 compared to the present study, they are restricted to equipment and reagents, with trademark exclusivity. For instance, tax gene common to HTLV-1 and HTLV-2, and as reference gene another single qPCR for HLA-DQ alpha; both assays employed the Sybr Green dye. Thus, although these qPCR showed high sensitivity (1 copy per reaction), and could be employed in different PCR platforms, the biplex qPCR does not distinguish HTLV-1 from HTLV-2. tax segment of HTLV-1 and a segment of the 5′ LTR of HTLV-2 as targets and the singleplex assay for human β-globin for PVL quantification. In these assays, the ABI equipment and supplies were utilized and a LOD of ∼60 copies were detected for both HTLV-1 and HTLV-2.pX regions of HTLV with LOD of 1 copy for HTLV-1 and 10 copies for HTLV-2. Nevertheless, the probe used for HTLV-1 pX detection was labeled with ROX. Since this dye is a passive reference that the ABI platform needs to normalize the fluorescence data, this component does not allow the use of such fourplex qPCR for quantitative analyses in this equipment, as well as commercial reagents that have ROX or similar passive reference in their formulation.pol of HTLV-1, tax of HTLV-2, and the human albumin as reference. They used the labeled probes HTLV-1 (JOE), HTLV-2 (FAM) and Albumin (NED) and detected sensitivities varying from 1 to 10 copies for all target genes but solely using the ABI platform. Thus, considering the previous information, the present mqPCR_HTLV assays have the advantage of being used in two different platforms, representative of the PCR platforms available in Public Health Laboratories in Brazil.Furthermore, the present mqPCR_HTLV showed similar sensitivity (LOD) using seven different master mix formulations, solving the problem of using varied brands of master mix depending on availability and cost.pol as target gene, differences in sensitivity (LOD) of assays were detected. In contrast, when we used the tax target gene a significant LOD was detected, corroborating with previous descriptions. The majority of mqPCR assays above employed the tax gene for HTLV-1/2 detection. We do not know the cause of these results, but despite the lower LOD of the mqPCR pol, this assay was able to detect 1 copy of HTLV-1 and 10 copies of HTLV-2, but without consistent reproducibility. In this regard, according to the Poisson’s distribution, LOD < 3 copies, and the detections of 1 copy of DNA are considered only hypothetical to qPCR assays (The concern reported around the LOD (more sensitivity) in the previous qPCR assays compared with the present mqPCR_HTLV assays, is that the discordant results could be partially explained by the target choice. When we used the R assays , and neeR assays . DetectiR assays , corrobopol) assay using plasmids, when this assay was employed in clinical DNA samples, no sample showed Cq values > 40. Thus, we could suggest in the future, after gaining experience from testing many samples, to reduce the number of cycles repetition from 50 to 45, and consider the Cq value ≤ 40 for positive samples.Curiously, although Cq values > 40 were obtained in mqPCR_HTLV (pol) for detecting samples infected with HTLV-2, which seems contradictory with the fact that this assay has a lower LOD compared to mqPCR_HTLV (tax). HTLV-2 is more difficult to be confirmed by serological and molecular confirmatory assays, as HTLV-2 accounts for more indeterminate WB results  and 74.6% (mqPCR_HTLV tax) were detected and the negative results could be due to: (i) provirus mutations and defective HTLV particles circulating in HIV/HTLV-coinfected patients  flpatients , (iii) upatients , and  could be evaluated, the results obtained support its use for monitoring infection and stratifying the risk for HTLV-1-associated disease development , and (v) no inter-labs comparative analysis, the present mqPCR_HTLV assays were validated for routine diagnosis considering the parameters previously described for qualitative assays , without false positive results.Finally, although the present study had some limitations regarding: (i) limited volume of biological samples to conduct all comparative experiments, (ii) absence of samples of patients infected only with HTLV-2, (iii) possible low PVL in HIV-infected patients on HAART treatment, (iv) insufficient volume of samples for validate PVL quantification for both target genes (pol and tax) assays displayed feasibility in diagnosing HTLV-1 and HTLV-2 in different core facilities laboratory, conditions, and supplies.In conclusion, the present mqPCR_HTLV (The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Research Ethics Committee of Instituto Adolfo Lutz. The patients/participants provided their written informed consent to participate in this study.MG designed the work, performed all the experiments, figures and tables, analyzed the results, and wrote the manuscript. LF designed and expanded the plasmids, conducted some experiments, analyzed the results, and wrote the manuscript. FH conducted the experiments of reproducibility. KC conducted experiments that supported to determine the diagnostic performance of the new assays. ACA conceived and administrated the project, acquired funding, supervised the experiments, analyzed the results, and wrote the manuscript. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Homologous DNA recombination is an essential pathway necessary for the repair of double-stranded DNA breaks. Defects in this pathway are associated with hereditary breast cancer, ovarian cancer, and cancer-prone syndromes. Although essential, too much recombination is also bad and can lead to genetic mutations. Thus, cells have evolved “antirecombinase” enzymes that can actively dismantle recombination intermediates to prevent excessive recombination. However, our current understanding of how antirecombinases are themselves regulated remains very limited. Here, we study the antirecombinase Srs2 and its regulation by the recombination accessory factors Rad54 and Rdh54. Our data suggest that Rad54 and Rdh54 act synergistically to function as key regulators of Srs2, thus serving as “licensing factors” that enable timely progression of DNA repair. Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors. Homologous recombination (HR) is a universally conserved DNA repair pathway involved in the repair of DNA double-strand breaks (DSBs) and the rescue of stalled or collapsed replication forks –4. The D5Mutations that impair HR function invariably lead to a loss of genome integrity –7. HowevSaccharomyces cerevisiae, have furnished a general paradigm for understanding antirecombinase mechanisms and the contributions that they make to the maintenance of genome integrity  helicase in the budding yeast ntegrity –30. As antegrity –19. Singntegrity . Multiplntegrity . Specifintegrity , 33.rad55 or rad57 deletions or mutations yield relatively mild phenotypes (The rapid translocation velocity (∼150 nt/s) and high processivity  of Srs2 as it dismantles Rad51 filaments in vitro raises the question of whether Srs2 itself might need to be down-regulated to allow for appropriate HR to take place . In factenotypes , 38. Radenotypes , 40, altenotypes . These oS. cerevisiae Rad54 and Rdh54 are 38% identical and 65% similar, and they are also highly conserved between yeast and humans  to directly visualize the behavior of Srs2 on Rad51–ssDNA filaments bound by Rad54, Rdh54, or both proteins. Surprisingly, we find that either Rad54 or Rdh54 can restrict the translocation of Srs2 on ssDNA bound by Rad51. Moreover, we show that Rad54 and Rdh54 can act synergistically to block Srs2 translocation to result in potent inhibition of the antirecombinase activity of Srs2. Our findings thus support a model in which Rad54 and Rdh54 associated with the Rad51–ssDNA presynaptic complex greatly attenuate the antirecombinase activity of Srs2. We propose that the antirecombinase activity of Srs2 may be operational only within a limited time window during the earliest stages of presynaptic complex assembly. In this context, Rad54 and Rdh54 act as key licensing factors to enable the initiation of HR, and they function by restricting the antirecombinase attribute of Srs2.We have previously established ssDNA curtain assays for visualizing the assembly and disassembly of Rad51–ssDNA filaments A 63–65)–6563–65)6363We have previously established single-molecule DNA curtain assays for visualizing the binding of Rad54 and Rdh54 to both RPA–ssDNA filaments and to Rad51–ssDNA filaments , 66. The−1 , Rdh54 (30 nM), or a combination of both Rad54 and Rdh54 . In reactions with Rad51 alone, upon ATP removal, the Rad51 filaments dissociated with a rate of 0.18 ± 0.1 s n = 33; B and C.  n = 30; B and C.  n = 66; B and C.  n = 31; B and C. 1–898, which retains the same biochemical properties as full-length Srs2 but is much more soluble  , 67, 68. soluble , 31; for no Srs2 A and B,  no Srs2 . An addi no Srs2 A. Analys no Srs2 B. Quanti no Srs2 B. These There could be multiple mechanisms by which Rad54 and Rdh54 might attenuate the ability of Srs2 to prevent D-loop formation. For example, they might act through a mechanism similar to the meiosis-specific recombinase Dmc1, which acts as a physical barrier to Srs2 translocation on ssDNA and in doing so drastically down-regulates the ATP hydrolysis activity of Srs2 . If Rad5We have previously established ssDNA curtain assays to monitor the translocase activity of Srs2 as it disrupts Rad51 filaments bound to ssDNA . Here, wn = 140/333; n = 33/77; n = 60/303; n = 0/100; When no Rad54 was present, ∼45% of the bound GFP–Srs2 molecules underwent 3′ → 5′ translocation, consistent with our previous studies . Importantly, this inhibitory effect of Rad54 became even more pronounced with higher protein concentrations; for example, at 10 nM Rad54, the velocity of Srs2 was reduced to 45 ± 26 nt/s, corresponding to a 70% decrease . As indicted above, we did not observe any Srs2 translocation in reactions with 100 nM Rad54 , consistent with our prior studies . It should be noted that although we do not see measurable Srs2 translocation in assays with 100 nM prebound Rad54, it is possible that Srs2 may in fact be translocating at a very low velocity or over much shorter distances, thus eluding detection within our optical resolution limits. Nevertheless, we can conclude that Rad54 dramatically reduces the translocation velocity and processivity of Srs2 as it acts upon Rad51 filaments.Rad54 also affected the distance over which Srs2 translocated on Rad51-bound ssDNA. Srs2 is highly processive in the absence of Rad54, exhibiting an average translocation distance of 18,548 ± 7,310 nt , and at 100 nM Rdh54, we did not observe any evidence of Srs2 translocation . These values decreased even further to just 79 ± 36 nt/s and 6,831 ± 5,130 nt in reactions with 10 nM Rdh54, corresponding to a 51% reduction in Srs2 velocity (P < 0.001) and 36% reduction in Srs2 processivity (P < 0.001) compared to reactions without Rdh54 . No evidence for any Srs2 translocation was observed in reactions with 100 nM Rdh54  were preincubated with the preassembled Rad51–ssDNA filaments, and excess Rdh54 was flushed from the sample chamber, as described , 66. GFPn = 71/297; SI Appendix, Table S2). Thus, in comparison with either 1 nM of Rad54 alone or Rdh54 alone, the combination of both Rad54 and Rdh54 reveals a strong synergistic effect with respect to the inhibition of Srs2 antirecombinase activity .Rad54 and Rdh54 can simultaneously bind to the Rad51–ssDNA presynaptic complex , and, asSI Appendix, Table S2). Notably, even though the combined presence of either 2 nM or 10 nM total concentration of Rad54 + Rdh54 was sufficient to completely block Srs2 translocation, when assayed individually, we could detect slow Srs2 translocation at either 10 nM Rad54 or 10 nM Rdh54 . These findings provide further evidence that Rad54 and Rdh54 act synergistically to greatly restrict the ability of Srs2 to mediate the disruption of Rad51–ssDNA presynaptic complexes.Higher concentrations of Rad54 and Rdh54 had an even more pronounced effect on Srs2 activity. We detected no evidence for any Srs2 translocation along the Rad51–ssDNA filaments when either 1 nM or 5 nM each of Rad54 and Rdh54 were both incubated with the preassembled Rad51–ssDNA filaments  or continued pushing of Rad54 without evidence of Srs2 stopping  was that Srs2 pushed the Rad54 along the ssDNA for a short distance but then stopped . One possible explanation for this differential effect is that, when assayed at the same protein concentration, there may be more Rad54 bound to the Rad51–ssDNA filaments than Rdh54, or Rad54 may simply associate with the Rad51 filaments more stably. We disfavor both of these hypotheses because our previous studies have shown that Rad54 and Rdh54 exhibit very similar Rad51–ssDNA binding characteristics  were preincubated with the Rad51 filaments, as compared to the finding that 10 nM of either Rad54 or Rdh54 alone slows but does not fully inhibit Srs2 antirecombinase activity. We have previously shown that a Rad54 or Rdh54 concentration of either 5 nM or 10 nM is insufficient for fully saturating the Rad51–ssDNA filaments , 66. Theeristics , 66. An Based on our previously published work and results presented here, we propose that there exist multiple strategies for restricting the antirecombinase attribute of Srs2. We have previously shown that the meiosis-specific recombinase Dmc1 is also able to counteract the antirecombinase activity of Srs2  in concoIn HR, the ends of DSBs are processed to yield long 3′ ssDNA overhangs that serve as a platform for the ordered recruitment of proteins necessary for assembly of the presynaptic complex . The ass1111173151Our results also raise the question of why Srs2 drives general protein turnover from ssDNA during early HR stages. We surmise that this remarkable attribute of Srs2 helps to eliminate potentially toxic nucleoprotein intermediates that would otherwise be fed into the HR pathway. For instance, timely Srs2 action would help dismantle Rad51–ssDNA filaments to prevent Rad54 and Rdh54 accumulation at inappropriate locales such as replication forks or ssDNA gaps , 26–30. 26Our study unveils a mechanism of HR regulation by Rad54 and Rdh54. It will be important to determine whether an analogous regulatory mechanism is also operational in human cells and its relevance to the maintenance of genome integrity.Rad51, RPA, Srs2, Rad54, and Rdh54 were purified as previously described , 32, 61.D-loop experiments were performed in HR buffer  using a 90-nt Atto-647N ssDNA substrate that was homologous for a region on the pUC19 plasmid, as previously described , 67. RadATP hydrolysis assays were performed using a malachite green‐based kit  in accordance with the manufacturer’s instructions. Reactions were initiated by the addition of ATP (500 nM) to a reaction mix containing 100 ng ssDNA , 50 nM RPA, and 50 nM Rad51 with total Rad54 only, Rdh54 only, or Rad54 + Rdh54 concentration of 30 nM. Reactions were terminated at the indicated time points by addition of 100 μL of the malachite green reagent solution to 5 μL of the reaction sample in a 96‐well microplate . After a 20-min incubation at room temperature, the absorbance at 620 nm was measured on a Spectra Max M5e multimode reader (Molecular Devices). Standard solution was used to calculate the amount of free phosphate present in each sample.All experiments were conducted with a custom-built prism-type total TIRF microscope (Nikon) equipped with a 488-nm laser , a 561-nm laser , and two Andor iXon electron-multiplying charge-coupled device (EMCCD) cameras , 83.sn-glycero-3-phosphocholine, 0.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), and 8% 1,2-dioleoyl-sn-glycero-3-phosphoethanoloamine-N- . Lipid bilayers were deposited in preformed flow chambers through sequential deposition of a lipid master mix in lipid buffer .Chrome barriers were deposited on quartz microscope slides via e-beam lithography and thermal evaporation, as described , 84. LipAll data were collected with a 100-ms integration time, and laser shuttering was varied to minimizing photobleaching. Images were collected using Nikon software, and images were exported as individual TIFF images as described , 83. TIF2, 0.2 mg/mL BSA, 1 mM DTT, 0.1 nM RPA). Once ssDNA molecules were aligned, the flow rate was adjusted to 1.0 mL/min, and 0.5 mL of 7 M urea was injected into the flow cell to further extend the ssDNA. HR buffer + RPA was then flushed through the sample chamber at 1.0 mL/min for an additional 10 min. The sample chamber was then flushed with HR buffer + ATP  at 1.0 mL/min for 3 min. Rad51 (1 μM) was injected into the flow cell, flow was stopped, and the reactions were incubated at 30 °C for 15 min. When GFP- or mCherry-tagged RPA was used, the Rad51 filament assembly was monitored with the appropriate laser at an image acquisition rate of 3 frames/min. After 15 min, free Rad51 was removed with HR buffer + ATP. Protein binding experiments with Rad54, Rdh54, and Srs2 were conducted in HR buffer plus ATP.ssDNA was generated by rolling circle replication using phi29 DNA polymerase with a biotinlyated primer annealed to M13 circular ssDNA as a template , 83. TheRad51–ssDNA filaments were preassembled in HR buffer in the absence of RPA as described above. RPA–mCherry (100 pM) was then injected in HR buffer + 5 mM ATP at 0.2 mL/min for 10 min. The experiment was monitored at a frame rate of 1 frame per 20 s. After 10 min, flow was switched to HR buffer plus RPA–mCherry − ATP, and the binding of RPA–mCherry to the ssDNA, which represents the dissociation of Rad51, was monitored for an additional 45 min. For data analysis, kymographs were generated as described above. The increase in RPA–mCherry intensity was measured for each kymograph and normalized by setting the highest value in each kymograph to 1 and normalizing all other signals to that value, generating an association curve. The association rate was then determined by fitting the normalized association data for each molecule using the single-phase association curve equation in GraphPad Prism, as described , 66. TheFor single-molecule Srs2 translocation experiments, Rad51–ssDNA complexes were preformed, and then Rad54, Rdh54, or both were allowed to bind for 10 min in HR buffer − RPA. After 10 min, excess Rad54, Rdh54, or both were removed by flushing the sample chamber with HR buffer + 5 mM ATP at 1 mL/min for 2 min. GFP- or RFP-tagged Srs2 (1 to 898; 500 pM; Refs. Supplementary File


The results showed significant enhancement in the yield, seed quality (protein and oil content), and uptake of nutrients  by various parts viz., seed, husk, and haulm of both varieties with foliar nutrition of silicic acid. However, the short duration variety, KBS-23, responded well under low rainfall conditions (2016) with two sprays of foliar silicic acid @ 4 mL L−1 and MAUS-2 variety in the second season under higher rainfall (2017) with three sprays of foliar silicic acid @ 2 mL L−1, along with the recommended dose of fertilizer. This research revealed that the effectiveness of foliar silicic acid nutrition differs with the duration of the varieties, number of sprays given, and water availability in the soil during the cropping period.The foliar nutrition of silicic acid is considered to be a novel approach in enhancing the performance of many crops worldwide. The present study aimed to assess if the foliar application of silicon (Si) could influence the performance of soybean varieties with distinct crop duration, MAUS-2 (long duration) and KBS-23 (short duration). Field experiments were conducted in two consecutive years (2016 and 2017) of varied rainfall with foliar application of silicic acid @ 2 and 4 mL L Glycine max L. Merril) contributes 25 per cent to the global oil consumption and about two-third of the world’s supply of protein. India constitutes 10 per cent of the world soybean area, but its contribution to total world soybean grain is only 4 per cent, indicating the poor levels of productivity of the crop in the country (1.1 t ha−1), as compared to other countries (world average 2.2 t ha−1) ,,80], K , Ca 81]81], Mg , and Si Soybean seed, haulm, and husk samples were microwave digested  and esti−1.The protein content in the seeds was analyzed by estimating nitrogen content by the micro-Kjeldahl method , then muThe data obtained from field observations and chemical analysis of soil and plant samples in both years were subjected to statistical scrutiny using analysis of variance (ANOVA) to find out the influence of silicic acid treatments on the yield, quality, and nutrient uptake by soybean varieties and the effects were tested at 5 per cent level of significance . Correla−1 three times and 4 mL L−1 two times, along with the recommended dose of fertilizer, was found to be effective in long duration (MAUS-2) and short duration (KBS-23) varieties, respectively. Silicon nutrition thus emerges as a novel approach to exploit the immense potential of soybean crop to meet global needs.Soybean is considered a wonder crop due to high protein (40–43%) and oil content (20%) and hence forecasted to mirror the world’s demand for protein and edible oil. Our investigation aims at improving quality parameters in soybean (protein and oil content) through foliar application of silicic acid, and has proven the same, besides enhancing yield and nutrient uptake. Accordingly, in our study, application of foliar silicic acid @ 2 mL L


Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events. The cellular signaling network consists of highly intertwined pathways that control the functional response of the cell. Thus, receptor stimulation does not only result in the activation of a particular pathway but instead typically involves signaling proteins across the network, mediated by signal integrators and cross-talks between the pathways. Therefore, an input signal is not processed in a linear pathway but rather in a network, in which all events are more or less connected and result in a combined network output. Furthermore, signaling processes are highly dynamic and the duration and subcellular localization of the activation can determine whether it is passed on or halted. Chemical approaches to control intracellular signaling have been successfully applied . HoweverUsing molecular optogenetics, genetically encoded photoreceptors connected with signaling proteins can orthogonally control the activity of the signaling protein by light. This approach has a number of advantages ,3. For eHere, we will provide detailed information on the control of intracellular signaling in mammalian cells by opsin-independent molecular optogenetics . SeveralIn the following, detailed information is provided on how optogenetic switches function and which optogenetic switches are available to control intracellular signaling in mammalian cells.Optogenetic switches consist at least of a photoreceptor that undergoes a conformational change upon illumination with a specific wavelength. Depending on the switch, different modes of action are possible. Optogenetic switches can interact with themselves, thereby forming either homodimers or homo-oligomers. Those are suited as fusion partners for proteins of interest that are active as dimer or oligomer, such as most of the receptor tyrosine kinases. Other switches engage an interaction partner (heterodimerization) which allows artificial split proteins such as in the case of split transcription factors  or to taOptogenetic switches are currently available across the visible spectrum and beyond. Optobase.org is a database including a curated up-to-date list of available opto-switches developed so far . The mosArabidopsis thaliana, which uses the cofactor phycocyanobilin that is not endogenous to mammalian cells . I. IArabidrodimers D 21]. T. TArabidVaucheria frigida (VfAU1-LOV) forms homodimers . I. IAvena Receptor protein kinases (RTK) are transmembrane proteins with intrinsic tyrosine-protein kinase activity . Most RT2+ signaling pathways independent of its ligand brain-derived growth factor (BNDF) in a time-reversible, spatial, and cell type-specific manner. Prolonged patterned illumination caused sustained ERK activation inducing neuronal differentiation. The first light-inducible fibroblast growth factor 1 receptor (FGFR1), optoFGFR1, was based on the same optogenetic switch . C. C56]. CTwo of the main downstream pathways of RTKs are the PI3K/AKT and RAF/MEK/ERK pathways. In the case of the latter one, RTK recruits the cytoplasmic complex composed of the adaptor protein growth factor-regulated binding protein 2 (GRB2) and the guanine nucleotide exchange factor son-of-sevenless (SOS) to the plasma membrane where SOS catalyzes the generation of active GTP-bound RAS. The main effector of GTP-RAS is the protein kinase RAF that headed the three-tiered protein kinase cascade formed by RAF, MEK, and ERK. Meanwhile, several optogenetic switches are available to control SOS, RAF, and MEK .Drosophila, the early embryogenesis was sensitive to the spatial distribution of optoSOS-induced ERK signaling disrupting the normal course of morphogenesis. A follow-up study demonstrated that cell fate decision between endoderm and ectoderm depends on the dynamics of ERK signaling in the early Drosophila embryo . T. T80]. TClustering of stimulated integrins recruits the focal adhesion kinase (FAK) to integrin signaling complexes. Subsequently, a conformational change releases the intramolecular inhibition of FAK monomers facilitating dimerization, transphosphorylation, and activation of its kinase activity. Full stimulation of FAK activity involves an SRC-dependent positive feedback loop. OptoFAK represents a single component, blue light-activatable FAK based on FAK-CRY2 oligomerization B 83]. L. L83]. LPhysical deformation of the plasma membrane generates mechanical forces that modulate cell behaviors like migration. To mimic this process, an optogenetic approach based on the switch iLID was applied to accumulate high levels of a fluorescent protein to the plasma membrane inducing local inward membrane curvature and tension decrease C 82]. L. L82]. LTo directly control the activity of integrins, the interaction between integrins and the extracellular matrix acts as a model. OptoIntegrin comprises β3 carrying an insertion of PIF in its extracellular domain co-expressed with αV wild type and a matrix functionalized with PHYB (optoMatrix) D 81]. U. U81]. UIntegrin-mediated cell adhesion is based on its interaction with specific motifs in proteins of the cellular matrix such as the arginine-glycine-aspartic acid (RGD) motif. For engineering, a photoswitchable synthetic ECM protein, opto-ECM, the RGD cell adhesion peptide was inserted in the Jα-helix of LOV2 resulting in a hidden RGD motif in the dark and its accessibility in blue light . Thus, aSo far, these extracellular optogenetic approaches to control the interaction of endogenous to RGD functionalized surfaces are not applied for studying integrin signaling.The binding of foreign peptide-major histocompatibility complexes (pMHCs) to the T cell receptor (TCR) stimulates T cells . Self- aTwo groups independently developed an optogenetic approach to test the kinetic proofreading model. The optogenetic tool opto-ligand-TCR consists of PIF that was inserted into the extracellular domain of a secretion-optimized TCRβ and a tetramerized PHYB as a ligand A 87]. Y. Y87]. YThe light-controlled chimeric antigen receptor (CAR) is based on the switch LOVTRAP . AsLOV2,Apart from the TCR-ligand binding half-life, temporal stimulation patterns play a major role in T cell activation. In the case of the iLID-based CAR, CAR was split to separate the ligand-recognition and the signaling domain. Blue light illumination induced dimerization of the split CAR through the interaction of AsLOV2-SsrA with SspB and, in combination with antigen stimulation, activated CAR signaling. Using gene expression as readout, optoCAR was exposed to various light oscillation durations. The results showed that T cells could temporally filter minute-scale signals via a band-stop mechanism, besides second-scale TCR-ligand interactions according to the kinetic proofreading model .2+ influx ..7].Since its first applications, optogenetics evolves rapidly and now provides valuable tools for basic research and applied sciences. In this review, we focused on the applications of optogenetics in controlling signaling pathways in cells by light. Future aspects of optogenetics comprise the improvements of the technology. To increase the throughput, there are multiple approaches to improve the reproducibility and the variety of conditions tested in a single experiment. The amount of time that light is applied to the optogenetic system is as essential for the effectiveness of the system as the light intensity. 96-well opto-plates are a great way to decrease the workload and identify the ideal conditions for each experiment in an efficient manner . A challAn interesting perspective is the combinations of optogenetics with nanobodies to enable light-dependent targeting of endogenous proteins to enable reversible alteration of their activity and function . It is aSo far, medical applications of optogenetic tools are rare. There is the problem of the low permeability of the skin by blue light and the resulting question of how to apply directed light to the area of interest. One way to direct the light to the area of interest are fibers that are applied in neurotechnology to guide the light to the desired area. Recently, biodegradable polyethylene glycol diacrylate (PEGDA) linked with DL-dithiothreitol (DTT) could be used to photo induce cell migration and drug release, while being able to have its lifetime adjusted to the desired time frame of the application . To takeWith all the advances that were accomplished in the last decade, optogenetics shows its potential in the precise and unique control of cells and their signaling spatially and temporally. The limit of optogenetics have not been reached and the next years will be able to show even more of the potential of this rather new technology.


Ursolic acid (UA) is a well-studied natural pentacyclic triterpenoid found in herbs, fruit and a number of traditional Chinese medicinal plants. UA has a broad range of biological activities and numerous potential health benefits. In this review, we summarize the current data on the bioavailability and pharmacokinetics of UA and review the literature on the biological activities of UA and its closest analogues in the context of inflammation, metabolic diseases, including liver and kidney diseases, obesity and diabetes, cardiovascular diseases, cancer, and neurological disorders. We end with a brief overview of UA’s main analogues with a special focus on a newly discovered naturally occurring analogue with intriguing biological properties and potential health benefits, 23-hydroxy ursolic acid. There is substantial evidence supporting the role of phytochemicals, which are categorized as secondary plant metabolites, as major contributors to the health benefits of diets rich in fruits and vegetables . In contTerpenoids are derived from one or more 5-carbon isoprenoid units and can be further characterized by the number of isoprene units and carbon atoms  ,7,8,9.Triterpenoids are composed of six isoprene units formed via the MVA pathway and are in plant resin, cork, waxy coatings and frequently associated with polysaccharide gums . TriterpUrsolic acid (UA) is a pentacyclic triterpenoid primarily synthesized through the MVA pathway, similar to steroid hormones in plants and mammals . UA is f2 resulted in plasma UA concentrations that peaked at 7.5 μM after 4 h [The bioavailability and pharmacokinetics of UA have been characterized in both rodents and humans. In the Biopharmaceutics Classification System (BCS), UA is considered a class IV compound, exhibiting poor oral bioavailability, low solubility, and intestinal permeability, yet UA demonstrates surprisingly strong pharmacodynamic properties and bioactivities . Severalfter 4 h .In addition to the pharmacokinetics of UA, several groups have studied the compounds’ toxicity as well as that of many of its naturally occurring analogues. Our group demonstrated in human THP-1 cells that at concentrations below 30 μM, UA and ten of its naturally occurring analogues did not exhibit any significant toxicity . Using aInflammatory diseases have become major targets for drug development because their effects are wide and debilitating . Major mCOX-2 is one of the many genes regulated by NF-κB, a family of transcription factors that regulates the expression of genes involved in tumorigenesis, adhesion molecules, chemokines, proinflammatory cytokines, and cell cycle genes . NF-κB iChecker et al. showed that UA’s effects on NF-κB, AP-1, and NF-AT are at least partly responsible for its potent anti-inflammatory effects in mouse lymphocytes . They sh50 = 5.5 μM) [Interestingly, UA appears to be an inhibitor of human neutrophil elastase (HNE), an enzyme that regulates local inflammatory processes . Feng et 5.5 μM) .Finally, we would like to mention an important caveat in working with UA initially reported by Ikeda et al. . These aStudies highlighting UA’s anti-inflammatory and antioxidant properties have spurred research focused on utilizing UA to either treat or prevent various metabolic diseases, including obesity, hypertension, diabetes, cardiovascular disease, and liver and kidney diseases, which are discussed below.4)-induced liver injury [UA has hepatoprotective properties that were first discovered in the mid to late 1980s using traditional Chinese medicine preparations to protect against carbon tetrachloride  injections with UA, Dahl salt-sensitive rats showed reduced hypertension, lower blood glucose and total cholesterol levels, and increased expression of two key antioxidant enzymes, glutathione peroxidase (GPx), and superoxide dismutase (SOD) in of UA . In subsAs mentioned above, UA also lowers blood glucose levels. One mechanism through which UA reduces blood glucose levels is via the inhibition of protein tyrosine phosphatase 1B, an important phosphatase inhibitor of insulin-mediated signaling . FurtherRecent studies, including two from our group, have reported cardioprotective properties of UA. UA supplemented in a HFD strongly suppressed atherosclerotic plaque formation and increased survival in a mouse model of diabetes-accelerated atherosclerosis . UA-trea−/− mice [In apparent contradiction to our reports, Messner et al. found that UA supplementation in the drinking water induces endothelial cell apoptosis, inflammation, and increased atherosclerosis in APOE−/− mice . However−/− mice . In fact−/− mice .In addition to protecting against atherosclerosis, UA protects against isoproterenol-induced myocardial infarction (MI) in rats as evidenced by reduced enzyme markers of disease (creatine kinase-MB and lactate dehydrogenase), lipid biomarkers , DNA fragmentation through upregulation of anti-apoptotic protein , downregulation of apoptotic proteins, including Bax, caspase-3, -8, and -9, cytochrome c, TNF-α, and FAS, and reduced oxidative stress in the plasma and heart tissue of these animals ,58. Oral2 (sPLA2) and fatty acid synthase. Thus, in addition to their cardio-and vasculoprotective properties, both UA and OA also appear to exert anti-hypertensive effects.Both UA and its analog, OA, also have anti-hypertensive properties. When OA from Greek olive oil and Cape Town cultivar, or a 1:1 mixture of OA and UA extracted from African wild olive leaves was administered to Dahl salt-sensitive (DSS) rats, an insulin-resistant rat model of hypertension, these rats were protected from the development of severe hypertension and atherosclerosis . In addiEvidence from several studies suggests that UA protects against diabetic-induced kidney disease. UA supplemented in the diet  of STZ-induced diabetic mice preserved kidney function as measured by creatinine clearance, diminished flux through the renal polyol pathway, and decreased advanced glycation end products (AGEs) formation in urine ,84. In aIn summary, oral administration of UA greatly improves health outcomes in a variety of rodent models of human metabolic diseases. A large body of data suggests that UA may be an effective oral therapy for both the preventive and treatment of metabolic disorders in humans as well as the chronic inflammatory diseases associated with these disorders. To date, only a single small clinical trial has been conducted to examine these potential health benefits in humans. In this randomized, double-blind, placebo-controlled clinical trial, 24 patients between 30 and 60 years of age, with a diagnosis of metabolic syndrome without treatment, were randomly assigned to two groups of 12 patients each, which either received orally 150 mg of UA or homologated placebo once a day for 12 weeks . The autUA’s anti-cancer properties have initially been described for the prevention of skin tumors , but morApoptosis, i.e., programmed cell death, is triggered by intrinsic and extrinsic cellular pathways. At high doses, UA has been shown to activate the intrinsic pathway by inhibiting anti-apoptotic pathways such as NF-ĸB ,91,92 anWAF1-dependent manner [Carcinogenesis is characterized by excessive cell proliferation. UA has been shown to inhibit cell proliferation by inhibiting MAPKs ,119,120 t manner .Metastasis is dependent on tumor angiogenesis and the regulation of proteases, peptidases, and adhesion molecules. UA and other triterpenoids reduce the angiogenic potential through down-regulation of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and IL-8 ,118,122.While many of UA’s anti-cancer effects have been reported in cell lines, UA shows similar potency in rodent tumor models. Topical application of UA extracted from rosemary reduced the number of tumors formed in a CD-I mouse model of skin tumor . DMBA anw/w) demonstrated reduced tumor formation and tumor size. This effect was attributed to the induction of apoptosis and disruption of cell cycle by UA [Dietary studies show that even low doses of UA are effective against cancer. In a mouse breast cancer model, mice on a 3-week diet of UA  or 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal cell damage in a concentration-dependent manner [Neurons are susceptible to oxidative damage which is thought to be the underlying cause of many neurodegenerative diseases. UA’s role has been investigated in preventing neurodegeneration through the reduction in ROS production and inflammation through the upregulation of antioxidant enzymes and the downregulation of inflammatory pathways . In varit manner . InteresOf note, UA has been proposed as a potential therapeutic for Alzheimer’s disease due to UA’s ability to reduce amyloid β binding to CD36, an important step in microglial activation and the onset of neuroinflammation . FurtherIn addition to UA, many of its naturally occurring analogues have also been investigated for their biological effects and potential health benefits. Interestingly, many of the analogues are found in olive oil, which is thought to be responsible for many of the beneficial health effects of the Mediterranean diet, suggesting a possible additive or even synergistic effect of these compounds in human health. Based on the reported health benefits and potential relevance for human disease prevention, we will limit our discussion to the following three natural UA analogues: asiatic acid (AA), corosolic acid (CA), and 23-hydroxy UA  has also been investigated as an anti-cancer compound. Studies in breast cancer cells , human mCorosolic acid (CA) has also been demonstrated to have protective effects against metabolic syndrome in rats  Table 3Table 3. −/− mice, an established mouse model of human atherosclerosis, both dietary UA and 23-OH UA supplemented at 0.05% to the HFD prevented dyslipidemia-induced loss of MKP-1 activity and hyper-chemotactic activity, hallmarks of blood monocyte dysfunction, without affecting plasma lipids or blood glucose levels or white blood cell and monocyte counts. Despite their similar mechanism of action, dietary 23-OH UA was significantly more effective in preventing atherosclerotic lesion formation and weight gain than UA. In a follow-up study, the same group confirmed the potent anti-obesogenic properties of 23-OH UA in a mouse model of diet-induced obesity and reported that 23-OH UA also improves glucose tolerance, prevents hyperleptinemia, preserves blood monocyte function, and reduces the recruitment of monocyte-derived macrophages into adipose tissues during nutrient stress. The authors provide evidence that the mechanism of action of 23-OH UA appears to involve the conversion of macrophages into anti-inflammatory, potentially inflammation-resolving phenotypes, which appears to contribute to the reduced adipose tissue inflammation seen in 23-OH UA-supplemented mice. Together these data suggest that 23-OH UA may serve as an oral therapy for patients at risk for obesity, impaired glucose tolerance, and cardiovascular diseases.Based on structure-function studies using ursolic acid and nine of its naturally occurring UA analogues, Nguyen et al. identified 23-hydroxy ursolic acid (23-OH UA) as a novel, naturally occurring triterpenoid with potential health benefits  Table 3Table 3. Other less studied naturally occurring analogues of UA include erythrodiol (ED), hederagenin (HG), and madecassic acid (MA). They have all been investigated as anti-cancer agents. ED, another analogue found in olive oil, was also found to have anti-cancer properties. ED inhibited proliferation and promoted apoptosis in HT-29 adenocarcinoma cells . Adding UA’s structural isomer OA, an oleane-type triterpenoid , was tra50 = 1.7 μg/mL or 3.7 μM). OA has also been reported to have anti-cancer effects. In a cell culture study with four different liver cancer cell lines, Yan et al. found OA, along with UA, to decrease cell viability, increase DNA fragmentation, and increase caspase-3 and caspase-8 levels, indicating their potential as anti-cancer agents [Like UA, OA has been reported to have anti-inflammatory properties such as ameliorating formaldehyde-induced arthritis in rats . These ar agents . In addir agents , leukemir agents , and is r agents .Betulin is a plant-derived pentacyclic triterpene metabolite of the lupine type  and founSeveral synthetic triterpenoids have also been shown to protect cancer cells, macrophages, and neutrophils from oxidative stress, inflammatory stimuli, and cell death ,161,162.The data reviewed here reveal that UA has a wide range of biological activities and is able to both prevent and treat a variety of pathologies in animal models of human diseases, ranging from metabolic disorders and chronic inflammatory diseases to cancer and neurological diseases. Whether these promising results will translate into real health benefits in humans remains to be explored as do UA’s bioavailability, pharmacokinetics, efficacy, and safety for humans. Furthermore, pharmaceutical efforts should focus on identifying the molecular target(s) of UA and on developing UA derivatives with better bioavailability. We hope this review will stimulate more research into UA and its analogues and derivatives and will ultimately lead to more human trials with this exciting and promising compound.


Oryza sativa L.) is usually considered two-line male sterile rice because of its dual-purpose in two-line hybrid rice system: under short days and low temperatures, it is fertile and used for self-propagation, but under long days and high temperatures, it is sterile and used for hybrid seed production. Therefore, photoperiod and temperature conditions are extremely important for the fertility transition of two-line male sterile rice. In recent years, there have been frequent occurrences of abnormally low-temperature (ALT) resulting in failure of two-line hybrid rice seed production. The daily average temperature (DAT) during ALT events is sometimes higher than the critical sterility-inducing temperature (CSIT) of two-line male sterile rice, of which the night temperature is lower than the CSIT. DAT has been traditionally used as the single indicator of pollen fertility transition, but it is unknown why the fertility of two-line male sterile rice in seed production restored fertility under ALT conditions.Photo-thermo-sensitive genic male sterile (PTGMS) rice (For Hang93S (H93S), a newly released PTGMS line, we hypothesized fertility transition is determined mainly by the cumulative effective low temperature (ELT) and only a certain duration of low temperature is required every day during the fertility-sensitive period. This study simulated ALTs where the DAT was higher than the CSIT while some segments of night temperature were lower than the CSIT. The results showed H93S exhibited a fertility transition to varying degrees. Moreover, fertility was restored under simulated ALT conditions and pollen fertility increased with increasing cumulative ELT, indicating that the fertility transition was affected primarily by the cumulative ELT. Results also indicated that pollen fertility increased as the number of treatment days increased.The fertility transition is caused mainly by the cumulative ELT. In two-line male sterile rice breeding, the effects of day length, ALT at night, and continuous response days should be considered together. The present study provides new insight into fertility transition so breeders can more effectively utilize the two-line male sterile rice, H93S, in breeding programs.The online version contains supplementary material available at 10.1186/s12284-021-00514-8. Oryza sativa L.) cultivars developed via the three line system involving a cytoplasmic male sterile A-line, maintainer B-line and restorer R-line . The objectives of this study were to: 1) validate our hypothesis that the fertility transition is determined mainly by the cumulative ELT and a certain duration of low temperature exposure is needed every day during the fertility-sensitive period, and 2) characterize and explore the regulation of ELT during the fertility transition in two-line male sterile rice.These variations in temperature represent the primary bottleneck limiting reliable hybrid seed production via a two-line male sterile rice system  and M-93 , followed by backcrossing and selfing  spaced 12.5 cm × 25 cm were transplanted into fields at the South China Agricultural University Experimental Station, Guangzhou, China . Plants whose distance between the ligule of the flag leaf and that of the next leaf on the main-culm was about 2.0 cm, were transplanted into the plastic buckets  for use in the treatments. At the end of the treatment, the tillers whose distance between the ligule of flag leaf and that of the next leaf on the culm was about 1.0 cm   for t1 and below Tc for t2 to simulate ALT  during the sensitive period, and TΔ is the value of the ELT, i.e., the difference between actual treatment temperature (T) and the Tc. TΔ was set to two different values: TΔ1 = 2.5 °C , and together with the different selected TΔ, t1, and t2 values, 12 treatments were established in which t1 = 24 h-t2–6 h. For example, assuming that the CSIT of H93S is 23.0 °C  and that the TΔ1 = 2.5 °C, t1 = 11 h, and t2 = 7 h, the DAT , and at each temperature level, the plants were treated for 4 d, 5 d, 6 d, and 7 d, resulting in 24 treatments (Supplementary Table The T2-KI), observed under a microscope, and the numbers of fertile and sterile pollen grains were counted in three fields of view for the same slide. Round and deeply stained pollens were considered fertile and pollen fertility percentage (PFP) was calculated from the number of fertile grains divided by the total number of grains. After sampling was performed, the flowered spikelets from the panicle were removed, after which the panicle was bagged. Two weeks later, the SSP was determined.Anthers of five mature florets from a panicle that had initiated a heading were collected for three consecutive days, and they were subjected to microscopic examination. Pollen grains were stained with 1% potassium iodide solution  were subjected to various combinations of light and temperature. Afterward, the fertility rates after the treatment were calculated, the results of which are shown in Table Clearly, at the same temperature in the fertility-restoration temperature range, the fertility of H93S increased as the photoperiod decreased, suggesting that H93S is a photo-thermo-sensitive interaction type. The control, PA64S, also exhibited similar photo-thermo-response characteristics associated with fertility transition, and the CSIT was 24.0 °C under a 13.5 h photoperiod.c = 23.0 °C under a 13.5 h photoperiod), and five treatments presented DATs lower than the CSIT. For example, when t1 = 11 h and t2 = 7 h, two values of ELT (TΔ) were used during the night , and at TΔ1, the plants were treated with 20.5 °C (Tc-TΔ1), which is 2.5 °C lower than the CSIT during the nighttime, corresponding to an ΣTΔ1 = (TΔ1 (2.5 °C)*t2 (7 h)*7 d)/24 h = 5.10 °C·d and a DAT of {(6 h*25 °C) + (t1 (11 h)*26 °C) + (t2 (7 h)*(Tc (23 °C)-TΔ1 (2.5 °C))}/24 h = 24.15 °C. Similarly, at TΔ2, the treatment corresponded to ΣTΔ2 = 4.08 °C·d and a DAT of 24.29 °C. These results indicated that the DATs of the two temperature treatments were higher than the CSIT of H93S (23.0 °C), however, owing to the ELT treatment at night, the fertility of H93S restored, as reflected by PFPs of 3.10% and 1.53% and corresponding SSPs of 1.31% and 1.01%, respectively. In the TΔ1 and TΔ2 treatments under t1 = 9 h and t2 = 9 h, at t1 = 7 h and t2 = 11 h, and with respect to TΔ2 treatment at t1 = 5 h and t2 = 13 h, all the DATs were all higher than the CSIT, and H93S showed varying degrees of PFP and SSP. In the remaining five treatments in which the DAT was lower than the CSIT, the PFP and SSP of H93S were higher than those in the aforementioned seven treatments, with the greatest PFP of 42.36% and the greatest SSP of 10.06% in the TΔ1 treatment at t1 = 1 h and t2 = 17 h, respectively.Both H93S and PA64S were subjected to various ELT treatments , the corresponding TΔ were plotted , after 4–7 d of treatment, H93S and the control were sterile, with a PFP of 0%. At TΔ = 1.0 °C, after 7 d of treatment, the PFPs of H93S and PA64S were 8.79% and 5.63%, respectively. At TΔ = 1.5 °C, 2.0 °C, or 2.5 °C, the fertility transition initiated on day 6 after treatment for H93S and on day 5 for PA64S. Similarly, at TΔ = 3.0 °C, for H93S, fertility transition occurred on day 5, with a PFP of 1.25%, which peaked on day 7 at 44.30%, while that of PA64S was 8.68% on day 5 and peaked at 51.22% on day 7. Figure Δ and the number of treatment days increased, the pollen fertility of H93S and PA64S increased accordingly, even under the same number of treatment days, the fertility also increased along with the increasing TΔ.The results confirmed that there is an effect of cumulative ELT on the fertility transition of H93S as well as PA64S, which requires a certain number of ELT-response days or a certain ΣTc = 23.3 °C when it was released , drifted upward and once reached to 24.2 °C  * 5 d or TΔ (2.5 °C) * 6 d. However, the results of the 12 ELT treatment combinations , the fertility of H93S was restored, which was also the case for PA64S. This phenomenon supports our hypothesis of the effect of cumulative ELT. Based on this hypothesis, we modified the general fertility transition scheme for the photo-thermo-sensitive male sterile line proposed by Yao et al. , resulting self-pollination. The results showed that fertility transition is not only related to the relationship between the DAT and CSIT but also to the effect of the cumulative ELT. Additionally, a long duration of low-temperature exposure plays an important role in the fertility transition. The effect of cumulative ELT serves as another scientific and rigorous factor for breeding and hybrid seed production and effectively changes in response to fluctuating global climatic conditions, reducing the risk and expanding the purity and yield of rice. The present study provides new insight into the fertility transition and breeding of two-line male sterile rice.Additional file 1 Fig. S1. Images of the facility used to apply the treatments described in the text. a and b Plants growing in precise constant temperature cold water baths for the constant temperature treatments listed in Table c At the end of the treatment, tillers whose distance between the ligule of the flag leaf and that of the next leaf was approximately 1.0 cm were marked, and the plants were moved back to the field. d and e Plants were treated in artificial climate chambers for determining the CSIT. Table f and g Plants were treated in a large scale smart greenhouse for the study the effect of cumulative ELT by simulating ALT conditions. h The plants were ready to be transplanted back to the field after the treatments were applied. All the plants under treatment were during their fertility-sensitive period (at the young panicle differentiation IV to VI stage). Fig. S2. Pollen and anther morphology of fertility restoration and abortion of H93S and the control PA64S. The images show the a) Normal pollen and b) anther morphology of the fertility restoration of H93S . c) Aborted pollen and d) anther morphology of sterile H93S . e) Normal pollen and f) anther morphology of fertile PA64S . g) Aborted pollen and h) anther morphology of sterile PA64S . Scale bars are 100 μm  and 1 mm Table S1. Light-temperature conditions during the day at T∆1 = 2.5 °C. Table S2. Light-temperature conditions during the day at T∆2 = 2.0 °C. Table S3. Temperature settings for the study of effective treatment days (photoperiod of 13.5 h).