PIK3CA leads to the hyper-activation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway in more than 20% of ovarian cancer (OC) patients. Therefore, PI3K therapies are under clinical evaluation for this subset of patients. Evidently, in clinical trials testing the efficacy of isoform-specific inhibitors of PI3K (PI3Ki), patients having a stable disease eventually relapse, as tumors become resistant to treatment. Hence, there is an urgent clinical need to develop new therapeutic combinations to improve the efficacy of PI3Ki in PIK3CA-driven OC patients. Here we identified the molecular mechanism that limits the efficacy of the beta-sparing PI3Ki, Taselisib (GDC0032), in PIK3CA-mutated OC cell lines (IGROV1 and OAW42) that acquired resistance to GDC0032. By comparing the molecular profile of GDC0032-sensitve and -resistant OC cell lines, we found that AKT/mTOR inhibition is required for GDC0032 efficacy. In resistant cells, the sustained activation of AKT/mTOR was regulated by the upregulation of the insulin growth factor 1 receptor (IGF1R). Knockdown of IGF1R re-sensitized cells to GDC0032 in vitro, and the combination of AEW541, an IGF1R inhibitor, with GDC0032 exhibited potent anti-tumor activity in vitro and in vivo. We further demonstrated that IGF1R regulates tumor cell proliferation in IGROV1 cells, whereas in OAW42, it determines autophagy as well. Overall, our findings suggest that the dual inhibition of PI3K and IGF1R may be considered as a new therapeutic strategy in PIK3CA-driven OC.Genomic alterations (GA) in  This pathway is often hyperactivated due to genomic alterations (GA) in PI3K pathway-related genes such as deletions in PTEN, and mutations or amplifications in PIK3CA, AKT3, AKT2, and AKT17. In ovarian cancer (OC) patients, point mutations or amplifications of the PIK3CA gene, which encodes the p110α catalytic subunit of the PI3K complex, are present in >20% of all OC types6. Around 240,000 OC patients are diagnosed annually; of these, ~50,000 harbor GA in PIK3CA8. Therefore, identifying potent anti-cancer therapies for this subset of OC patients is essential.The phosphatidylinositol 3-kinase (PI3K) pathway is a key regulator of the survival, growth, and metabolism of normal and malignant cellsPIK3CA have been shown to be more susceptible to isoform-specific inhibitors of the PI3K pathway (PI3Ki) in vitro, in vivo, and in patients than tumor  cells with wild-type PIK3CA19. One of the most promising PI3Ki is GDC0032, a small molecule that blocks p110α, p110δ, and p110γ (p110β sparing). GDC0032 has exhibited clinical activity in tumors harboring PIK3CA alterations in early clinical trials15, but eventually all patients relapsed and developed resistance. Such results suggest that treating PIK3CA-driven OC with PI3Ki is a good therapeutic strategy, but new therapeutic approaches, such as drug combinations are needed to improve and prolong the positive clinical response.Cancer cells with GA in 20. In OC, ongoing clinical trials have been testing combinatorial PI3K therapies only with chemotherapy (NCT02069158 and NCT02476955). Hence, a better understanding of the molecular response and resistance to PI3Ki can lead to investigating more potent drug combinations in this malignant disease.Currently, two-thirds of the ongoing clinical trials testing PI3K-related therapies focus on combining at least two modalitiesPIK3CA-driven OC, to identify new therapeutic targets, and to examine new drug combinations.Our present study sought to identify the molecular mechanism that limits the efficacy of GDC0032, in PIK3CA-mutated cancer cells were found to be more sensitive to PI3Ki than were PIK3CA-wild-type cells18, we explored the Cancer Cell Line Encyclopedia (CCLE) databases21, searching for PI3Ki-sensitive OC cell lines with GA in PIK3CA   Fig. . Lastly,nes Fig. .Fig. 1Ch25, we measured the activity of the AKT/mTOR and MAPK signaling pathways in sensitive and resistant cell lines, in response to GDC0032. We analyzed the phosphorylation (activation) of AKT , mTOR , and MAPK (pERK42/44) over time. The pAKT and pS6 levels revealed that GDC0032 inhibits the AKT/mTOR pathway in sensitive cell lines, whereas sustained activation was observed in both resistant cell lines , compared with their sensitive counterpart cells, were used to create gene set enrichment lists. We then looked for pathways that were upregulated in both cell lines and calculated the average combined KEGG score for each. We found nine shared pathways, including P53 and the insulin signaling pathway  or GDC0032 for 14 days, and analyzed the number of all shRNA under each condition. The analysis revealed that the proliferation of IGROV1Res cells, infected with shRNAs targeting IGF1R, MET, AKT, and others, were strongly inhibited in GDC0032 compared with DMSO-treated cells , PHA-665752 (MET inhibitor), MK2206 (AKT inhibitor), and others , METi (PHA-665752), and GDC0032 compared to their combinations. Importantly, we found that combining GDC0032 with AEW541 has a potent synergistic anti-proliferative effect, whereas combining GDC0032 with PHA-665752 displayed an additive effect  analysis revealed that the basal expression levels of IGF1R are higher in resistant cells compared with their counterpart sensitive cells. Furthermore, treatment of cells with GDC0032 increases the expression of IGF1R in both tumor cell line models Fig. . This diWe further investigated the biological and molecular response to single and combined treatment with GDC0032 and AEW541, with both sensitive and resistant cells. An imaging-based proliferation assay showed that combined treatment with AEW541 and GDC0032 further reduced cancer cell proliferation in sensitive and resistant cell lines compared with the single drug inhibition treatment Fig. . WesternSen cells (OAW42 tumor cells were not tumorigenic) into NOD/SCID mice and treating them with vehicle, GDC0032 (10 mg/kg per day) and AEW541 (30 mg/kg per day), separately and combined. As expected, GDC0032 induced a growth delay of IGROV1Sen tumors, since in the first 2 weeks no tumor progression was detected. Similar to the observation in patients, the tumors eventually started to grow (after 2 weeks of treatment), and tumor volumes were increased by 4-fold, despite daily treatment using GDC0032. Treatment with AEW541 had no effect on tumor growth, compared with the vehicle-treated group. However, combining both drugs (GDC0032 and AEW541) resulted in superior anti-tumor activity, with no appreciable increase in tumor volume, even following 30 days of treatment  or autophagy (vesicles in the cells). In contrast, in OAW42Sen cells, we observed vesicle formation following GDC0032 treatment, together with reduced cell proliferation  and autophagosome accumulation (using a florescence-based dye), following GDC0032 treatment. Analysis of IGROV1Sen and OAW42Sen tumor cells revealed that treatment with GDC0032 induces the accumulation of LC3B and the appearance of autophagosomes only in OAW42Sen tumor cells , in ate Fig. . Blockin20. In our acquired-resistant model, we found that overexpression of IGF1R plays a major role in maintaining AKT/mTOR pathway activation. Inhibition of IGF1R enhances the sensitivity of PIK3CA-driven OC to GDC0032 and BYL719 treatment in vitro and in vivo.Resistance to PI3K inhibitors is a major obstacle in developing these agents in oncology. The molecular mechanisms that induce resistance to PI3K therapies vary from epigenetic regulators, autocrine, or paracrine secretion of pro-survival factors, to overexpression or activation of RTK31. The signaling of IGF1R plays a role in tumor cell motility and adhesion; thus, it contributes to tumor spreading and metastasis32. IGF1R is considered an attractive target in oncology;33 however, years of clinical research yielded only modest efficacy in cancer patients treated with a single agent of anti-IGF1R therapies35. These clinical results highlight the insignificant role of IGF1R as a driver oncogene. Owing to its important role in cancer progression and in limiting the efficacy of anti-cancer therapies, blocking IGF1R combined with other inhibitors is under clinical development in various cancer types, including melanoma36, lung37, breast38, and prostate cancers39. In OC, IGF1R is known to confer resistance to cisplatin, paclitaxel, and cisplatin-taxol40, and to BEZ235 inhibitor as well41. Here we show the first evidence that IGF1R triggers resistance to isoform specific inhibitor of the PI3K in PIK3CA-driven OC.IGF1R is frequently overexpressed in tumors such as thyroid, colorectal, prostate, breast, and ovarian, and regulates tumor cell proliferation and survival via apoptotic signaling25, the integrin alpha2/FAK complex42, or pyruvate dehydrogenase kinase (PDK1)44, in various cancers. In our OC cell line models, p110β seems to play no role in resistance to GDC0032, since AZD6482, a p110β inhibitor, did not re-sensitize the acquired resistant cells to GDC0032  RNA expression , treatment with GDC0032-induced autophagy, whereas in the OAW42-resistant cells, it did not. This phenomenon can be explained by our finding that upregulated IGF1R re-activates the AKT/mTOR pathway. Furthermore, we suspect that IGROV1 cells did not develop GDC0032-induced autophagy due to the interplay between P53 and autophagy47. For example, colon cancer cells with wild-type, but not mutated P53, were re-sensitized to chemotherapy by inhibition of autophagy48, emphasizing the importance of P53 in determining autophagy. OAW42 cells have a wild-type P53 and thus induce autophagy in response to GDC0032 treatment, whereas IGROV1 cells with mutated P53, do not21.Autophagy is a well-known resistance mechanism used in anti-cancer therapiesPIK3CA-mutated and amplified OC patients.In summary, we provide here the first evidence that IGF1R and autophagy play a role in resistance to isoform specific inhibitors of the PI3K in OC. Since GDC0032, BYL719, and AEW541 are under clinical evaluation, our results warrant the clinical testing of the dual inhibition of IGF1R and PI3K for treating 9. Both tumor cell lines were maintained at 37 °C in a humidified atmosphere at 5% CO2, in DMEM or RPMI-1640 medium, respectively, supplemented with 1% l-glutamine 200 mM, 100 units of penicillin, streptomycin 10% fetal bovine serum, and 200 nM antimicrobial cocktail  to avoid mycoplasma infection. Testing for mycoplasma was done every 6 months using Hy-Mycoplasma PCR kit .OAW42 and IGROV1 cells were obtained and grown as previously describedGDC0032- acquired resistant cell lines were derived from their original parental cell line by continuous exposure to GDC0032. Tumor cells were cultured initially with low concentration of GDC0032  for 2 months. The concentration of GDC0032 in the culture was increased gradually by 50–100 nM every 2 weeks. This development period was carried out for 6 to 8 months.The antibodies anti-pAKT Ser473 (#4060), anti-pAKT thr308 (#9275), anti-AKT (#4691), anti-pS6 S235/236 (#4858), anti-pS6 S240/244 (#5364), anti-S6, anti-pERK1/2 Thr202/Tyr204 (#9101), and anti-ERK1/2 (#4695) were from Cell Signaling Technology. Anti-LC3B was obtained from Sigma-Aldrich (L8918). KI67 was purchased from Vector laboratories (#VP K451). Anti-Actin was from MP Biomedicals (691001). Novartis Pharma AG  provided BYL719 and AEW541. LDE225, MK2206, AZD6482,AZD6738, GDC0032, PHA-665752, 17-AAG, LEE011, LY2835219, LBH589, ABT737 and Navitoclax were purchased from Selleckchem . TUNEL- TREVIGEN, cat no-4815-30-K).Cells were washed with ice-cold PBS and scraped into ice-cold lysis buffer with phosphatase inhibitor cocktail . Lysates were cleared by centrifugation at 14 000 rpm for 10 min at 4 °C, and supernatants were collected and assayed for protein quantification using the Bradford protein assay . Quantified samples were then analyzed by western blotting. Twenty micrograms of total lysate were resolved on NuPAGE 4–12% Bis-Tris gels and electrophoretically transferred to PVDF membranes . Membranes were blocked for 1 h in 5% BSA in Tris-buffered saline (TBS)-Tween and then hybridized using the primary antibodies in 5% BSA TBS-Tween. Mouse and rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies  were diluted in 5% BSA in TBS-Tween. Protein-antibody complexes were detected by chemiluminescence with ECL λernova (Cyanagen) and images were captured with a c300 azure camera system. Protein bands were analyzed by comparing their size to SMOBIO-PM-2600 protein marker.About 3500 cells were seeded in a 96-well plate. At 24 h after seeding, cells were treated with increasing concentrations of the indicated drug, left to right, and for the synergy assay with another drug, top to bottom. At 96 h post treatment, the medium was aspirated, and cells were fixed using Trichloroacetic acid (TCA) 0.6 M for 1 h at 4 °C. TCA was then washed, plates were dried on filter paper, and then incubated with 0.6% Crystal violet staining solution at room temperature on a bench rocker. After 10 min, plates were washed, air dried, and incubated with Acetic Acid (10%) for 10 min. The optical density of each well was measured at 570 nm using an Epoch Microplate Spectrophotometer.TM Bioinformatics Software and the synergistic effects were calculated based on the statistic Loewe Excess model and the equation S = fcov ln fX ln fY ∑ max max, which is a positive-gated, inhibition-weighted volume over Loewe additivity. Complete description of formula and calculation is available in http://chalice.horizondiscovery.com/chalice-portal/documentation/analyzer/analyticMethods.jsp.For the synergy assay, the colorimetric results were analyzed using Chalice6 IGROV-1 cells were suspended in 50 μl PBS and injected subcutaneously (S.C) into six NOD/SCID mice . When tumors reached a volume of 400 mm3, the tumors were dissected into 2 × 2 × 2 mm chunks and implanted S.C in 24 naive female NOD/SCID mice (6–8 weeks old). All 24 mice developed tumors and as tumor volume reached 150–200 mm3, mice were randomized into six groups of four mice (n = 8 tumors per group). Daily treatment of GDC0032 (10 mg/kg), BYL719 (25 mg/kg), AEW541 (30 mg/kg) or the combinations were given to mice by oral gavage.A total of 5 × 10Drugs were dissolved in 5% Carboxymethylcellulose (CMC). Vehicle-treated mice (control group) received 5% CMC. Tumors were measured 3 times a week by caliper and tumor volume was calculated using the formula: Volume = (Length × Width x Width) / 2. Mice were maintained and treated in accordance with the institutional guidelines of Ben-Gurion University of the Negev. Animal experiments were approved by the Institutional Animal Care and Use Committee (IL.80-12-2015). Mice were housed in air-filtered laminar flow cabinets with a 12 h light/dark cycle and food and water ad libitum. Tumor volumes are plotted as means ± SEM, and ANOVA test was applied.Immunohistochemistry (IHC) was performed using the ABC KIT (VECTASTAIN®ABC). After mice were euthanized, the dissected tumors were fixed in 4% formalin buffer overnight at room temperature, transferred to 70% ethanol, and embedded in paraffin, sectioned at 5 μm, loaded onto microscope slides, and deparaffinized at 60 °C for 1 h.2O2) in Methanol buffer for 20 min; the buffer was washed 3 times with PBS for 3 min, and slides were blocked with 5% BSA in PBS-T for 1 h at room temperature. Primary antibodies were diluted in blocking solution and sections were incubated overnight at 4 °C with the antibodies. The next day, slides were washed with 1 × PBS with Tween three times, incubated with a specific biotinylated secondary antibody at room temperature for 1 h; the color developed by adding substrates. Last, slides were dehydrated and covered using mounting medium.After the rehydration, antigen was retrieved. The slides were incubated in 10 mM citric acid buffer, pH 6.0 at 100 °C for 20 min, then cooled in buffer at room temperature for 20 min and rinsed with double distilled water for 3 min, 3 times. Next, endogenous peroxidases were inactivated by 0.3% hydrogen peroxide .For each condition, both cells and growth medium were collected into 15 ml tubes, centrifuged for 10 min at 4 °C, then a pellet was fixed using 70% ice-cold ethanol and stored for at least 24 h at −20 °C. After fixation, the pellet was washed twice with ice-cold 1X PBS, treated for 30 min at 37 °C with 100 μl of RNase solution (100 μg/ul), and stained in the dark for 20 min with 200ul Propidium iodide solution (100 μg/ml). Last, the cell phase was analyzed using BD FACSCANTO II.Cells were treated with 50 nM GDC0032 or DMSO for 24 h. RNA was extracted by using the PureLinkTM RNA mini kit (Invitrogen). After quality control of BioAnalyzer(RIN > 7), IGROV1 samples were sent to the g-INCPM institute at the Weizmann Institute of Science. Briefly, 500 ng of total RNA were fragmented, followed by reverse transcription and second strand cDNA synthesis. The DS-cDNA was subjected to end repair, a base addition, adapter ligation, and PCR amplification to create libraries. Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of 12 samples in one lane. A total of 17–20 million single-end 60-bp reads were sequenced per sample on an Illumina HiSeq 2500 V4 instrument.TM Stage Real-Time Cell History Recorder. Punctate structures were imaged using both the ImageStream and Zeiss Axio Observer 7. IHC slides were scanned using the Panoramic MIDI II scanner, 3DHISTECH.Live cell imaging and time series recording were performed using the Juli49. Briefly, IGROV1 cells were infected with pools of retroviral shRNA. 3 days post infection, cells were selected with puromycin for an additional 3 days (1 µg/ml) to remove the minority of uninfected cells. Thereafter, cells proliferated in culture for 3 days and then an initial population-doubling 0 (PD 0) sample was taken. The remaining population was divided into 9 groups: 3 groups kept as a control, 3 groups treated with GDC0032 (100 nM), and 3 groups treated with BYL719 (1 uM). Cells grew in the presence or absence of drugs for an additional 12 doublings before the final, PD 13 sample was taken. For the PD 0 and PD 13 samples, the shRNA HH barcode was PCR-recovered from genomic samples and samples were sequenced to calculate the abundance of the different shRNA probes. The change in the relative abundance of each shRNA in the library over time was measured using the normalized PD 13/PD 0 ratio of its reads. A log2(PD 13/PD 0) ratio of < 0 indicates that shRNA was depleted in the population over time, and a log2(PD 13/PD 0) ratio of ˃ 0 indicates that shRNA was enriched. To identify shRNAs that are synthetically lethal with GDC0032 or BYL719, the mean log2(PD 13/PD 0) ratios of the treated cell triplicates were compared to the control triplicates to derive the log2 ratio difference. A p-value of the difference between the two triplicates was calculated using the Student t test. Targets were filtered by the presence of at least two different shRNAs for the same gene and when P < 0.05.ShRNA screening was performed as described50. The dispersion estimation, model fitting, and testing were implemented using the functions estimateDisp, glmFit  and glmLRT, respectively, in the edgeR package51To analyze any differential expression in the IGROV1 cell lines, a generalized linear model likelihood ratio test was used. Specifically, a negative binomial generalized linear model was fitted, with shRNA-wise or gene-wise dispersions estimated using the Cox-Reid profile-adjusted likelihood method. Then, a likelihood ratio test was employed to check the differential expression52.To interpret the testing results in a biological context, gene ontology (GO) enrichment analysis and KEGG pathway enrichment analysis were employed as downstream procedures, using the online available tool EnrichrFor pathological analysis, IHC images were analyzed by Histoquant software (3D Histech).P values of 0.05 (*), 0.01 (**),0.001 (***) and 0.0001(****) were considered statistically significant.The remaining experiments were repeated at least three times and representative data/images are shown. Statistical analysis was performed using GraphPad Prism software, presented as mean ± SEM. For comparisons between two groups, P values were calculated. Supplementary Figures and legendsSupplementary Table 1


Triticum aestivum). A total of 179 TaLEA genes were identified in T. aestivum and classified into eight groups. All TaLEA genes harbored the LEA conserved motif and had few introns. TaLEA genes belonging to the same group exhibited similar gene structures and chromosomal locations. Our results revealed that most TaLEA genes contained abscisic acid (ABA)-responsive elements (ABREs) and various cis-acting elements associated with the stress response in the promoter region and were induced under ABA and abiotic stress treatments. In addition, 8 genes representing each group were introduced into E. coli and yeast to investigate the protective function of TaLEAs under heat and salt stress. TaLEAs enhanced the tolerance of E. coli and yeast to salt and heat, indicating that these proteins have protective functions in host cells under stress conditions. These results increase our understanding of LEA genes and provide robust candidate genes for future functional investigations aimed at improving the stress tolerance of wheat.Late embryogenesis abundant (LEA) proteins are involved in the responses and adaptation of plants to various abiotic stresses, including dehydration, salinity, high temperature, and cold. Here, we report the first comprehensive survey of the LEA gene family in “Chinese Spring” wheat ( Therefore, plants have evolved complex regulatory mechanisms in their defense responses to adverse conditions. All mechanisms are based on proteins that directly function in abiotic tolerance and that regulate various signaling pathways to indirectly improve abiotic tolerance. The late embryogenesis abundant (LEA) protein gene family is an important group of functional proteins to reduce cell damage and protect cells under abiotic stress conditions3.Abiotic stress, such as drought, high temperature, cold, and salinity, is a primary factor of reduced crop productivity due to serious disruptions in plant growth and development4. Since then, they have been detected in seedlings, leaves, stems, roots and other organs of many other plants mostly under abiotic stress conditions that result in cellular dehydration6. LEA genes have also been identified in bacteria and invertebrates8. LEA proteins accumulate during late embryonic development and can be induced by various abiotic stresses9. Compared with other proteins involved in abiotic stress tolerance, LEA proteins have no significant enzymatic activity; however, LEA proteins, as intrinsically disordered proteins (IDPs), are highly hydrophilic and intrinsically unstructured in the hydrated state but partially fold into mainly α-helical structures under dehydration conditions7. This feature allows them to function as chaperones via preventing protein aggregation during abiotic stress11. In addition, LEA proteins contribute to the stabilization of membranes, binding of calcium and metal ions, interactions between DNA and RNA, and the protection of functional proteins against aggregation15.LEA genes were first observed in late-stage mature cotton seeds18. Genome-wide characterizations of LEA family proteins have been performed in several plant species, such as Arabidopsis thaliana18, Brassica napus19, Citrus sinensis20, Dendrobium officinale21, Hordeum vulgare22, legumes23, Malus domestica24, Oryza sativa25, Pinus tabuliformis26, Populus trichocarpa27, Prunus mume28, Solanum tuberosum29, and Zea mays30. However, few LEA genes have been identified in wheat.Based on conserved motifs, amino acid sequences and phylogenetic relationships, LEA proteins are essentially classified into eight groups: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, dehydrin (DHN) and seed maturation protein (SMP)31, making the identification and analysis of the LEA gene family in wheat possible. Therefore, in the present study, we performed a genome-wide analysis of LEA genes in wheat to characterize their sequences, evolutionary relationships, putative functions and expression patterns in response to different abiotic stresses.Recently, the chromosome-based draft genome of the bread wheat “Chinese Spring” was publishedA. thaliana and T. aestivum. The TaLEA genes were classified into eight subfamilies based on their conserved domains  have two or three introns  were distributed across an unassembled scaffold , B (61), and D (59) subgenomes. For example, the 1A, 1B and 1D chromosomes had similar numbers of LEA_2, LEA_3, LEA_4, and LEA_5 genes, and the evolutionary relationships in each subfamily were close  and then searched the PlantCARE database to identify and count the cis-acting elements associated with plant growth and development and stress response -responsive elements (ABREs) (92.7%), which respond to ABA and regulate downstream gene expression, were found in most TaLEA gene promoters. In addition, TaLEA_3-8 had the most (11) ABREs in its promoter. CGTCA motifs and TGACG motifs involved in the methyl jasmonate (MeJA) response were also identified, accounting for approximately 67.6%. There were also a few TaLEA gene promoters that included auxin response elements (TGA-elements), ethylene response elements (EREs), gibberellin response elements (GAREs), and salicylic acid response elements (TCA-elements). At the same time, we also identified a large number of cis-acting elements related to plant stress response, MYB (91.6%), MYC (64.2%), W box (27.4%) and TC-rich repeats (10.6%), which can participate in a variety of plant responses to abiotic stress. DRE, LTRE and STRE are capable of responding to drought, low-temperature and heat stress, respectively. WRE is associated with plant damage and infection by pathogens. These results indicate that the TaLEA genes may participate in the growth and development of plants through the ABA pathway and respond to various stresses.The presence of multiple different nse Fig. . For cisLEA genes in wheat under abiotic stress, a microarray analysis of TaLEA genes was carried out .For the salt treatment, ess Fig. . Among tE. coli strains carrying six  of the eight TaLEA genes had mean viability ratios 2-3-fold higher than those of the control strain under salt stress  showed the highest viability ratio after induction at 50 °C for 30 min. After they were induced at 50 °C for 60 min and 120 min, TaLEA_1-5 (~1.98-fold) and TaLEA_3-3 (~2.76-fold) had the highest viability ratios, respectively. In addition, the viability ratio of TaLEA_1-5, TaLEA_3-3, TaLEA_4-1, TaLEA_5-1, and TaDHN43 was higher than that of the control strain after induction at 50 °C for 180 min. There were no differences in the mean viability ratios between other TaLEA genes and the control strain. Consistent with response of the E. coli recombinants under heat stress, the yeast transformed with pPI3.5k-TaLEA_1-5, TaLEA_3-3, TaLEA_4-1, TaLEA_5-1, TaSMP8, and TaDHN43 showed better growth than the control, and the transformed yeast reached the stationary phase faster than the control.For the heat treatment, our results demonstrated that ess Fig. . Among tLEA genes play crucial roles in embryonic development and in response to abiotic stress5. The LEA gene family has been reported in many crops; in contrast, genome-wide identification and annotation of LEA genes have not been reported in T. aestivum. In this study, 179 TaLEA genes were identified in the T. aestivum genome  TaLEA genes have less than one intron. Low intron numbers have also been observed in other stress-response gene families, such as the glutathione transferase family27. Introns can have a deleterious effect on gene expression by delaying transcript production. Introns can delay regulatory responses by extending the length of the nascent transcript, resulting in an additional energetic cost due to increased transcript length32.In recent years, some research has reported that stress-response genes generally contain relatively few intronsArabidopsis18 and tomato33. There is a strong implication that LEA proteins from the principal groups are ubiquitous within cells and their respective tissues, suggesting that their function is required in all cellular compartments during stress34.Subcellular localization analyses showed the presence of TaLEA proteins in all subcellular compartments, including the nucleus, cell membranes, cell walls, mitochondria, chloroplasts, Golgi apparatus, and cytoplasm, as also reported for D. officinale and P. tabuliformis26. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., IDPs, but they may acquire α-helical structures upon dehydration35. This characteristic of LEA proteins allows them to change their conformation according to the changes in their ambient microenvironment, resulting in multiple functions in abiotic stress10.Analysis of the physicochemical properties revealed that the TaLEA proteins had common characteristics, such as small molecular weights and rich hydrophilic amino acids Table , as repo36. Moreover, LEA gene subgroups have evolved different adaptive effects against abiotic stress, as reported in D. officinale and P. tabuliformis26. According to the results of the microarray analysis, the response of most TaLEA genes to salt, cold and drought stress was obvious, and genes were especially significantly expressed in the TaDHN group, followed by the TaLEA_1 and TaLEA_4 groups  and Ensembl (http://plants.ensembl.org/Triticum_aestivum/Info/Index) servers. The open reading frames of TaLEA genes were repredicted and corrected using Fgenesh and Fgenesh + software (http://www.softberry.com) in the wheat database. In addition, we also identified LEA proteins in wheat protein sequence data using the BLAST HMM profile (http://pfam.xfam.org) of LEA_1 (PF03760), LEA_2 (PF03168), LEA_3 (PF03242), LEA_4 (PF02987), LEA_5 (PF00477), LEA_6 (PF10714), SMP (PF04927) and DHN (PF00257). All of the identified TaLEA candidates were analyzed using the Hidden Markov Model of the Pfam database (http://www.ebi.ac.uk/Tools/hmmer/) and the NCBI Conserved Domain database (https://www.ncbi.nlm.nih.gov/cdd) to confirm the conserved domains of the LEA proteins. Finally, we retrieved 179 TaLEA genes classified into 8 LEA protein subfamilies.LEA genes were identified in 42. The exon-intron structures were identified using the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) by comparing CDSs and genomic DNA sequences43. Multiple EM for Motif Elicitation (MEME) (http://meme.sdsc.edu/meme) was used to identify the conserved motifs encoded by the TaLEA family genes44. The chromosomal locations of each TaLEA gene were mapped to each chromosome according to their positions in the wheat genome. The identified positions were then marked on the chromosomes using the MapChart tool45.The phylogenetic tree was constructed by the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0 softwareTaLEA genes, were acquired from the wheat database, and the cis-elements in the promoters were analyzed with the PlantCARE database46.The promoter sequences, which were approximately 2000 bp upstream of the TSS of the TaLEAs were submitted to the Plex database (http://www.plexdb.org/) to search for corresponding probes. These probes were then used as queries to obtain the expression data of the TaLEA genes in GeneVestigator software (https://genevestigator.com/gv/).The coding sequences of 47. In each treatment, the leaf tissues were collected every 12 h for 48 h, frozen in liquid nitrogen, and stored at −80 °C.Wheat seeds of “Chinese Spring” were germinated on moist filter paper at 25/18 °C (day/night) with a photoperiod of 16 h/day. For abiotic stress treatment, seedlings grown in hydroponic culture for two weeks were exposed to 100 μM ABA, high temperature (42 °C), high salinity (800 mM NaCl), cold (4 °C), and 20% PEG 6000 (w/v), as described previouslyActin gene was used as an internal control. The real-time PCR cycling parameters were 94 °C for 30 s, followed by 45 cycles at 94 °C for 5 s and 55 °C for 30 s, with a melting curve analysis from 60 °C to 90 °C at a rate of 0.5 °C/5 s. All reactions were performed in triplicate to ensure the reproducibility of the results.RNApure Plant Kit (CWBIO) was used to isolate total RNA from each frozen sample, and first-strand cDNA was synthesized from total RNA (1 μg) by using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. The sequence was amplified using gene-specific primers Table  with Tra48.The endogenous ABA content of wheat leaf tissues was measured using an enzyme-linked immunosorbent assay (ELISA), as described previouslyTaLEA genes representing each group, inserted the genes into the pET28a vector and then transformed the vectors into the E. coli host strain BL21 (DE3). The primers are shown in Supplementary Table E. coli BL21 (DE3) density reached an OD600 of 0.6 in LB liquid medium containing 50 μg/mL kanamycin at 37 °C. The E. coli cells were harvested by centrifugation, resuspended in phosphate buffered saline (PBS), and then analyzed by SDS-PAGE  cell cultures were diluted to the same OD600 value, transferred to 50 °C, sampled at 60, 120, 180 and 180 min, and plated (20 μL) onto LB plates. For the salt treatment, after IPTG induction, 20 μL of each sample  was spotted onto LB plates containing 300 mM, 400 mM, 500 mM, and 600 mM NaCl. The viability ratio of the transformants under heat and salt conditions was calculated by counting the number of colonies after incubation of the plate overnight at 37 °C. Cell viability ratio = (colony number on stressed plate/colony number on unstressed plate) × 100%. For all experiments , the means of three experiments were determined from three independent transformants. Here, E. coli with the empty vector (pET28a) was the control group.Heat and salt tolerance assays were performed as described previouslyTaLEA genes were inserted into the Ppic3.5 K vector and then transformed into the Pichia yeast strain GS115, as described previously51. The primers are shown in Supplementary Table 3PO4, 4 × 10−5 mM biotin, and 1% glycerin) to induce TaLEA gene expression at 30 °C until the stationary phase of growth was reached. Then, the cells were collected by centrifugation and resuspended in 200 mL induction buffered methanol-complex medium  and then incubated at 30 °C for 4 days. Methanol was added every 24 h to a final concentration of 0.5%. Cell cultures of induced yeast (Ppic3.5K-TaLEA) were diluted to the same OD600 value of 0.8, and 1 mL cells was inoculated into 150 mL of BMGY medium containing 1.2 M NaCl and then induced at 30 °C. For heat stress treatments, 1 mL cells was inoculated into 150 mL of BMGY medium and induced at 40 °C. Growth was monitored with a spectrophotometer by measuring the OD600 every 5 h.The eight selected Figure S1, Figure S2, Figure S3, Figure S4, Table S1, Table S2, Table S3, Table S4Table S5Table S6Table S7


Escherichia coli system, as a capture antigen.Chikungunya (CHIKV) virus is an important mosquito-borne virus causing outbreaks of acute febrile illness with arthropathy. The detection of specific antibodies against CHIKV is used for diagnosis after the acute viremic phase of the disease. However, a major challenge for serologic diagnosis of CHIKV and other alphaviruses is the cross-reactivity of antibodies to common antigens among these viruses. In the present study, we have developed an enzyme-linked immunosorbend assay using a recombinant envelope protein 2 of CHIKV produced in High titers  of anti-CHIKV antibodies were detected in human sera analyzed by the CHIKV assay, suggesting it may detect low levels of the antibodies presence. On the other side, cross-reactivity was not observed in mouse hyperimmune sera to Mayaro virus and other alphaviruses analyzed by the CHIKV immunosorbend assay, suggesting it is a CHIKV-specific test. Fifty-nine human serum samples of CHIKV infection suspected cases were tested for immunoglobulin G (IgG) and M (IgM) antibodies detection using the CHIKV immunosorbend assay. A total of 44% (26/59) of samples were positive for IgG to CHIKV, determining 89.66% sensitivity and 100% specificity when the assay is compared to a CHIKV-specific neutralization assay. In addition, 40.6% (24/59) of samples were positive for IgM, determining 92.48% sensitivity and 79.04% specificity by a Bayesian method in the absence of a gold standard. Moreover, CHIKV immunosorbend assay showed similar sensibilities to a commercial immunochromatography assay  for CHIKV IgG and IgM detection.In short, we have developed a rapid, simple, specific and sensitive CHIKV immunosorbend assay for IgG and IgM detection and our results showed potential applicability on the diagnosis of infections by this virus.The online version of this article (10.1186/s12985-018-1028-1) contains supplementary material, which is available to authorized users. Alphavirus genus (Togaviridae family). CHIKV genome consists of a linear, positive-sense, single-stranded RNA of ~ 11.8 kb in length including two open reading frames (ORFs) that encode four non-structural proteins (nsP1–4) and five structural proteins  -diammonium salt (ABTS) peroxidase substrate  per well and incubated for ~ 15 min at 37 °C for test revealing. The plates were read in Titertek Multiscan MMC/340 Microplate Reader at an optical density (O.D.) of 405 nm. ELISA cutoff values were calculated as the mean O.D. of negative controls plus three standard deviations (SDs). All samples were tested in duplicate. Sample with an average O.D. above the cutoff value was considered as positive [The rE2 of CHIKV was used as an antigen in an indirect ELISA for diagnosis of CHIKV infection. Concentrations of rE2, from 0.5 μg/ml to 8 μg/ml, diluted in 0.05 M Carbonate-Bicarbonate buffer pH 9.6  were added to wells of microplates  and as negative control, In order to evaluate cross-reactivity of antibodies to alphaviruses in the rE2-CHIKV ELISA, mouse hyperimmune sera to AURV strain BeAr10315, EEEV strain BEAN-1999, MAYV strain BeAr20290, MUCV strain BeAn-8 and WEEV strain SpAn14723 were tested at different dilutions (1:100 to 1:800). These hyperimmune sera had their reactivity previously confirmed by indirect immunofluorescence tests  against A total of 59 serum samples from patients clinically suspected of CHIKV infection were tested by rE2-CHIKV ELISA. Human assays used the HRP-conjugated goat anti-human IgG ‘Fab’ specific  diluted at 1:2000 or the HRP-conjugated goat anti-human IgM ‘Fc5μ’  diluted at 1:25000. Positive samples were diluted from 1:100 to 1:25,600.Results obtained for the 59 human sera in IgG and IgM rE2-CHIKV ELISAs were compared to those obtained by an in-house EIA-ICC using C6/36 cells  and also50 was performed in Vero cells. Briefly, human sera previously inactivated were serially diluted  in Dulbecco’s Modified Eagle’s Medium (DMEM) . Subsequently, 1.75 × 102 PFU of CHIKV, strain BzH1, cordially provided by professor Benedito Antônio Lopes da Fonseca, were mixed with each serum and these mixtures were incubated for 1 h at 37 °C. Then, 200 μl of each mixture was inoculated into Vero cell monolayers in 6 well plates. Cells were incubated 1 h at 37 °C under gently rocking. In sequence, 2 ml of pre-warmed DMEM containing 1% agar  and 3% fetal bovine serum (FBS)  were added to each cell monolayer well, and plates were incubated for 2 days at 37 °C at 5% CO2 atmosphere. Finally, the cells were fixed in the wells with 4% formaldehyde  solution for 2 h and stained with 1% crystal violet  during 5 min, for plaque visualization. Plaque reduction was calculated for each serum by comparing their respective number of plaques to the positive control, which was inoculated with 1.75 × 102 PFU of CHIKV. The cut off titer for positivity was set as 80.To evaluate the specificity of anti-CHIKV antibodies in human serum samples, a PRNTHuman IgG and IgM positive samples in the rE2-CHIKV ELISA were submitted to an avidity assay . Brieflywww.mrc-bsu.cam.ac.uk/software/bugs). Posterior inferences were based on summaries of 1,000,000 iterations with a sampling lag of 10, after a burn-in of 10,000 iterations. The final results were presented as a median and a 95% probability interval  of each parameter estimate.The sensitivity and specificity of rE2-CHIKV ELISA, EIA-ICC and immunochromatographic test for IgM detection were estimated by the Bayesian method introduced by Joseph et al. . The BayAdditional file 1:Table S1. Detection results of human samples obtained by rE2-CHIKV ELISA, EIA-ICC, CHIKV commercial assay and PRNT50. Bold or POS.: positive sample, ND: Not detected. (DOCX 18 kb)Additional file 2:Table S2. The relative avidity index for IgG and IgM of each sample. Samples with RAI > = 60% were considered of High Avidity, between 40 and 60% of Medium Avidity and = < 40% of Low Avidity. ND: Not detected. (DOCX 15 kb)Additional file 3:Figure S1. Immunofluorescence assay of infected cells. Specific homotypic antibodies detection by infected Vero cells to the respectively hyperimmune sera of alphaviruses. Green: hyperimmune sera labeling viruses; Blue: Nuclei, Red: Cytoplasm; Pos.: Positive infected cells and Neg.: Negative non-infected cells. (PDF 599 kb)


Grafting was then performed according two distinct routes: (i) using a blend of both homopolymers (PCL and PTCL) or (ii) using pre-synthesized copolymers with controlled CL:TCL ratios. Hexamethylene diisocyanate was used as a grafting/coupling agent through urethane bonds with high yield. Grafting preferentially occurred at sulfur sites. The results indicated that PTCL is more reactive and favorable than PCL for grafting onto chitosan. With the homopolymers blend grafting route, the corresponding materials mostly had a higher PTCL portion than expected. To obtain polyester grafted chitosan with a determined CL:TCL ratio, the copolymer grafting route would yield better results.Polyester and/or polythioester grafted chitosan copolymers were synthesized. For that, poly(ε-caprolactone) (PCL), poly(ε-thiocaprolactone) (PTCL), and their copolymers were first synthesized by ring opening polymerization. Copolymers with caprolactone:thiocaprolactone (CL:TCL) molar ratios of 2:1, 1:1, 1:2 were synthesized. All of the synthesized macromolecular architectures were characterized using different spectral (Fourier transform infrared (FTIR), proton nuclear magnetic resonance ( Increasing environmental concerns, the depletion of some fossil resources fractions, and the necessity to develop new macromolecular architectures have motivated the strong increase of environmentally friendly (green) alternatives for fossil-based materials during the last decade ,2,3. UsiN-acetylglucosamine units, is the second most abundant polysaccharide on earth; it constitutes e.g., the exoskeletons of crustaceans and cell walls of some fungi dec-5-ene (TBD) were purchased from Sigma Aldrich  hexamethylenediisocyanate (HDI) from Fluka , they were used as received without further purification. Anhydrous toluene was freshly distilled over sodium under argon atmosphere. Other solvents (GC grade) were purchased from Acros Organics  and were used without further purification.Chitosan  was used for data reduction in order to find out the dominant factors. A typical FTIR spectrum was composed of several hundreds of points with a correlation to each other. Using PCA, this correlation was removed in a way creating uncorrelated variates known as principal component (PC) scores. The PCs could be defined as new, orthogonal axes where variances were maximized. Thus, the projection of PCs against one another could reveal the clustering or structural information regarding the major components that are responsible for the change in certain region of the spectrum. PCA was performed on first derivative FTIR data (including the homo- and copolymers) in order to show the evidence of the grafting onto chitosan using R software (version 3.4.3) running on a 64-bit Linux platform (kernel version 4.14.15). First-order derivatives of the spectra in range between 1800 and 700 cm1H-NMR spectra were measured on a Bruker instrument at 400 MHz (1H) at 25 °C using deuterated solvents (32 scans).n), Mw, and the dispersity (Ð) of the samples. Measurements were performed in chloroform (HPLC grade) in a Shimadzu liquid chromatograph that was equipped with a LC-10AD isocratic pump, a DGU-14A degasser, a SIL-10AD automated injector, a CTO-10A thermostated oven with a 5 μ PL-gel Guard column, two PL-gel 5 μ MIXED-C and a 5 μ 100 Å 300 mm columns, and two online detectors, a Shimadzu RID-10A refractive index detector, and a Shimadzu SPD-M10A diode array (UV) detector, respectively. Molar masses and dispersity were calculated from a calibration with polystyrene standards .Size exclusion chromatography (SEC) was performed to determine the number-average molar mass , universal gas constant (8.314 J mol−1 K−1), absolute temperature (K); y was the fraction, which was defined as Equation (2) where Wt, ∞W, and W0 were the sample weight at temperature t, the end and beginning of decomposition, respectively [Thermogravimetric analysis (TGA) measurements were conducted under argon (at a flow rate of 25 mL/min) using a Hi-Res TGA Q5000 apparatus from TA Instruments. The samples (2–5 mg placed in a platinum pan) were heated up to 500 °C at 10 °C/min. Activation energy was obtained from the plot of Equation (1), where ectively .(1)ln(lDSCX) was calculated according to the Equation (3), where ΔmH and Differential scanning calorimetry (DSC) measurements were performed using TA instruments DSC Q200 apparatus. The measurement method had three cycles; first, the sample was heated up to 120 °C at a heating rate of 20 °C/min and was kept at this temperature for 1 min to remove adsorbed water and to erase the thermal history. Subsequently, it was cooled down to −70 °C with a cooling rate of 5 °C/min and was kept at this temperature for 1 min. Finally, the sample was heated up to 150 °C with a ramp of 10 °C/min. All cycles were conducted under nitrogen flow (50 mL/min). Crystallinity (α radiation (λ = 0.1542 nm) operating at 40 kV and 40 mA. The scattering range was 2θ = 10–50°, with a step size of 0.0158° and a scan speed of 0.5 s/step. The mean distance among the closest neighboring chains (R) was determined with Equation (4) [X-ray diffraction (XRD) patterns were recorded with a Bruker AXS D8 ADVANCE using Cu-Ktion (4) .(4)R=58XRDX) was calculated using Equation (5), where cryI and totalI were the area under the curve of crystalline peaks and total integral of all peaks. Crystalline and amorphous peaks were deconvoluted using Fityk software (version 1.3.1).Crystallinity . Polymers samples (150 mg) were placed between two steel plates that were covered with Teflon layers and pressed at 90 bar and 110 °C for 2 min. After cooling (5 min), the resulting films were removed and photographed.6-mercaptohexanoic acid synthesis protocol was adapted from Shimokawa et al. . The rea−1, acidic O-H stretching as a broad signal between 3500–2400 cm−1, S-H stretching at 2666 cm−1 as a weak band.FTIR of the product displayed C=O stretching at 1704 cm1H NMR : δ = 1.35 ppm , δ = 1.40–1.50 ppm , δ = 1.57–1.70 ppm , δ = 2.36 ppm , δ = 2.52 ppm .The final product was characterized by NMR spectroscopy: 2O5 was added. The flask was quickly fitted with a short distillation head. Subsequently, the flask was immersed in an oil bath at 190 °C and the pressure was lowered to 80 mbar. The distillation rate of thiolactone was maintained by lowering pressure gradually until 1 mbar. After 2 h, thiolactone was solubilized in ether and was washed with aqueous NaHCO3 until it reached neutral pH. Mixture was then dried over magnesium sulfate and ether was stripped off by rotary evaporation, typically giving a yield of 40% : δ = 1.70–1.90 ppm , δ = 2.05–2.15 ppm , δ = 2.85 ppm , δ = 3.05 ppm .The final product was characterized by 1H NMR  was added to the mixture. After 24 h the reaction was stopped by addition of one drop of 1 M HCl solution to the reaction mixture. Polymer was precipitated by addition of heptane, then filtrated and dried first in air then in vacuum for 24 h (yield 97% by weight) (ε-caprolactone (CL)  was dissolved in dry toluene (10 mL) under argon atmosphere and was heated to 110 °C. Afterwards, 1.25 mL of Sn(Oct) weight) .The same procedure was applied with TCL  to obtain poly(ε-thiocaprolactone) (PTCL) (yield 75% by weight).Poly(ε-caprolactone-co-ε-thiocaprolactone) copolymers were synthesized using the above procedure with different ε-caprolactone/ε-thiocaprolactone feed ratios (mol/mol). The use of 2:1 ratio resulted in a yield of 64% by weight and the obtained product was labeled as COP-2:1. Same protocol was repeated using comonomers molar proportions of 1:1 and 1:2. Final product yields were 40 and 32% by weight for COP-1:1 and COP-1:2, respectively.2 equivalent) were stirred in DMF (3.2 mL) at 100 °C, under argon. After 20 min, HDI  was added to the mixture and was left under stirring for 90 min. The mixture was then poured into ice water to precipitate copolymer. The obtained chitosan-graft-polyester copolymer was washed with acetone to remove unreacted polyester. Finally, it was extracted with toluene using Soxhlet apparatus for 24 h and dried first in air then in vacuum. The overall procedure was given in In a typical procedure polyester (300 mg), TBD , and chitosan .Homopolymers and copolymers of CL and TCL were successfully synthesized. Copolymerization that was performed with different monomers molar ratios yielded copolymers in favor of PCL resulting from the easier polymerizability of CL. Although the molar masses of the PTCL and COP were smaller, they showed better thermal stability when compared to PCL. The crystallinity degree of COP samples was found lower when compared to PCL and PTCL homopolymers. The obtained homo- and copolymers were then coupled to chitosan backbone using isocyanate to obtain chitosan grafted copolymers (CG-), according two distinct routes. In the blending route, homopolymers mixtures were used, while in the second route, presynthesized COP with varying ratios were grafted. The latter route was found to be superior as the PCL:PTCL ratio could be adjusted as desired. Obtained CG- samples had even better thermal stability, but lower crystallinity when compared to corresponding polyesters. Grafting onto chitosan via isocyanate, groups preferentially occurred at sulfur sites. Thus, the homopolymer blends grafting route yielded grafted polyesters in favor of PTCL. The hot-melt pressing of grafted copolymers yielded flexible and durable films.To answer the initial hypothesis and objective, grafting polyester chains onto chitosan enhanced the properties of the starting materials, as expected. In comparison to pristine starting materials, grafted copolymers had better thermal stability and film forming capabilities. The PCL/PTCL proportion could be tuned via copolymerization prior to grafting. Then, using presynthesized copolymers for grafting would yield well defined and controlled PCL/PTCL ratios in the final material.Complementary studies should be performed to analyze in details some functional properties, such as the mechanical, drug release, and antimicrobial behaviors of CG- copolymer films in connection with specific biomedical applications, such as e.g., active wound dressing.


Nrf2 expression in H9c2 cardiomyocytes and diabetic (db/db) mice, respectively. Using an oxidative stress RT2 Profiler PCR array, ASP at a dose of 1 µM was demonstrated to protect H9c2 cardiomyocytes against HG-induced oxidative stress, but silencing of Nrf2 abolished this protective response of ASP and exacerbated cardiomyocyte apoptosis. Db/db mice and their non-diabetic (db/+) littermate controls were subsequently treated daily for six weeks with either a low (13 mg/kg) or high (130 mg/kg) ASP dose. Compared to nondiabetic mice the db/db mice presented increased cardiac remodeling and enlarged left ventricular wall that occurred concomitant to enhanced oxidative stress. Daily treatment of mice with ASP at a dose of 130 mg/kg for six weeks was more effective at reversing complications than both a low dose ASP or metformin, eliciting enhanced expression of Nrf2 and its downstream antioxidant genes. These results indicate that ASP maintains cellular homeostasis and protects the myocardium against hyperglycemia-induced oxidative stress through activation of Nrf2 and its downstream target genes.Aspalathin (ASP) can protect H9c2 cardiomyocytes against high glucose (HG)-induced shifts in myocardial substrate preference, oxidative stress, and apoptosis. The protective mechanism of ASP remains unknown. However, as one of possible, it is well known that phytochemical flavonoids reduce oxidative stress via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation resulting in up-regulation of antioxidant genes and enzymes. Therefore, we hypothesized that ASP protects the myocardium against HG- and hyperglycemia-induced oxidative damage by up-regulating  The International Diabetes Federation recently reported a rapid rise in cases of diabetes mellitus (DM) worldwide . The curNrf2) is an emerging regulator of cellular resistance to oxidants. Nrf2 plays a crucial role in controlling basal and induced expression of an array of cytoprotective and antioxidant defense genes important for the regulation of physiological and pathophysiological outcomes in response to oxidative exposure  1 (Nqo1) [Nrf2 is activated by disassociating from its negative regulator Kelch-like ECH-associated protein 1 and translocating to the nucleus, where it binds to the antioxidant response element, activating detoxifying enzymes and genes to inactivate the stressors and restore homeostasis [Humans are equipped with a defense system that controls free radical species and reduces oxidative stress. The nuclear factor (erythroid-derived 2)-like 2  . Under ceostasis .Aspalathus linearis), a popular South African herbal tea and some of its major polyphenolic compounds in ameliorating metabolic complications associated with DM [C-glucosyl dihydrochalcone unique to rooibos, can improve glucose and lipid metabolism by modulating 5′ adenosine monophosphate-activated protein kinase (AMPK) expression in 3T3-L1 adipocytes [Nrf2 [Nrf2 by using an in vitro H9c2 cardiomyocyte model and a leptin receptor-deficient db/db mouse model, respectively.Various phytochemicals from food substances—such as sulforaphane, derived from a major glucoslinolate of broccoli and quercetin, an aglycone, derived from flavonol glycosides, common to plant foods, can prevent oxidative injury by up-regulating Nrf2 expression ,18,19,20 with DM ,23,24,25ipocytes . In addiipocytes ,28,29 anipocytes ,31, in aipocytes . While Aes [Nrf2 . Hence, 2-PCR analysis revealed that ASP post-treatment at a dose of 1 µM was able to enhance mRNA expression of a number of antioxidant genes and phase II detoxification enzymes. Results obtained showed that ASP increased the expression of catalase , Gpx2 (15.8-fold), peroxiredoxin 1, 3, 4, and 6 , and Sod1 and Sod2 (2.1- and 1.2-fold) (Gsh)-associated and reducing genes, including glutamate-cysteine ligase catalytic subunit , glutamate-cysteine ligase, modifier subunit  and glutathione reductase  , thioredoxin 1 , thioredoxin reductase 1 , heme oxygenase 1 , Nqo1 (11.4-fold), and uncoupling protein 2   and down-regulate that of Caspase 8  (High glucose exposure decreased endogenous antioxidant capacity of the cells. RT.2-fold) . The inc.2-fold) . Increas.8-fold) . Interes8; -1.3) .Nrf2, small interfering RNA (siRNA) was employed. H9c2 cardiomyocytes exposed to siNrf2 for 24 h resulted in a significantly reduced Nrf2 mRNA expression  when compared to the scrambled RNA (scrRNA) control (2.0 ± 0.2)  (p ≤ 0.01) (Nrf2 mRNA expression when compared to the scrRNA control (To investigate if ASP activates 0 ± 0.2) A. Furthe≤ 0.001) B. ASP tr ≤ 0.01) B. As ant control B.Gpx2 , Park7 , Sod2 , and Ucp2  when compared to the NG controls  , Park7 , Sod2 , and Ucp2  when compared to the HG controls  A–D. Treacontrols A–D. Intecontrols A–D.Casp3 and Nox4) was up-regulated in HG exposed cells  when compared to the NG controls   when compared to the HG controls  E,F. ASP controls E,F. Howecontrols E,F. The db/db) mice and their heterozygous leptin-receptor-deficient nondiabetic lean littermate controls (db/+) were treated daily with either a low (13 mg/kg) or a high (130 mg/kg) dose of ASP, as well as metformin (MET) at 150 mg/kg for six weeks. The untreated diabetic control (db/db_UC) group displayed elevated FPG levels from week 9 to week 14  when compared to the untreated db/+_UC control group  , though not significantly  A. Althouficantly A.db/db_UC  was compared to the db/+_UC control (9.6 ± 0.50)  B. High d ± 0.50) B. Treatmdb/db_UC group presented with increased HW/BW ratio , LV wall and interventricular septum thickness measurements  occurring concurrent to enhanced cardiac remodeling in comparison to the db/+_UC control group  (p ≤ 0.001), LV wall , and interventricular septum thickness  /body weight (BW) ratio, LV wall and interventricular septum thickness, and occurrence of cardiac muscle remodeling were assessed after mice were treated with ASP for six weeks. Results showed that the 20 ± 29) A–D. Inte≤ 0.001) A–D. NeitNrf2 and its associated downstream cytoprotective genes in vivo, and results showed a significant enhancement of Nrf2 mRNA expression in the db/db_UC group  in comparison to the db/+_UC control group (0.99 ± 1.49) (p ≤ 0.01) significantly increased mRNA expression of Nrf2 when compared to both db/db_UC and db/+_UC groups  A. AlthouC groups A. Nrf2, it was noted that the expression of Gpx2  and Gss  was reduced in the db/db_UC group when compared to the db/+_UC group  (Park7 was not significantly affected (0.94 ± 0.06). Treatment with MET and the low dose ASP did not have any effect, while the high dose ASP significantly up-regulated expression of Gpx2 , Gss , and Park7  when compared to db/db_UC group  B,C. The UC group B–D.Casp3 and Nox4) was significantly enhanced in the db/db_UC group  when compared to the db/+_UC group   and Nox4  when compared to db/db_UC group (Expression of oxidative damage-associated genes (ctively) E,F. no eUC group E,F.Nrf2 and thereby increase its cytoprotective response [Nrf2 activation as demonstrated for shogaol derivatives [Hmox1 and Nqo1 through activation of Nrf2 in HepG2 cells [Nrf2 prevented oxidative stress by increasing the expression of an array of antioxidant genes and enzymes. We showed that a 6 h treatment with 1 µM ASP protected cardiomyocytes exposed to HG against oxidative damage by up-regulating the expression of antioxidant genes and cytoprotective enzymes such as Nqo1 and Hmox1. This was consistent with a dramatic increase in the expression levels of Cat, Txnrd1, Gpx2, and Gsh-synthesizing subunits (Gclc and Gclm). This is an essential result since reduced expression of these antioxidant genes is consistently reported in a diabetic heart tissue [Gpx2, which is mainly expressed in the gastrointestinal tract and liver was not anticipated due to its limited expression in the heart [Accumulative evidence has shown that certain dietary phytochemicals are able to activate response ,16,18. Pivatives . FurtherG2 cells . In agreG2 cells ,35, our t tissue ,36. Txnrhe heart ,38; howeNrf2 by ASP is crucial for the antioxidant response against HG-induced oxidative injury in H9c2 cardiomyocytes. While it was expected that Nrf2-knockdown may exacerbate susceptibility of such cells to oxidative damage, ASP treatment showed an even higher capacity to enhance Nrf2 expression and its downstream target genes and thus protect against HG-induced stress. However, an isolated in vitro cell-based system is not influenced by any variables, thus the observed decrease in Nrf2 after HG exposure of H9c2 cells. Whereas, in an in vivo system the expression and regulation of genes is influenced by various factors. In this study, we speculate that in the db/db mice, the observed increase of Nrf2-expression in the db/db_UC group was a compensatory mechanism used by the cells to decrease increased oxidative stress.Moreover, by using RNA interference we showed that activation of db/db mice, which represent a type 2 diabetic mouse model. These mice display a whole-body phenotype of DM and are frequently used in the pharmacological study of compounds including ASP [Therefore, following in vitro experiments on H9c2 cardiomyocytes, it was important to confirm such findings in an in vivo system. For this we made use of ding ASP ,39. Moreding ASP . At an ading ASP ,41.db/db mice presented elevated FPG levels, an increased HW/BW ratio that occurred concomitant to a thickened LV wall and cardiac remodelling when compared to the db/+ control group. An enlarged heart LV wall is a conspicuous sign of DCM and is identified as an early defect associated with chronic hyperglycemia in diabetic patients [db/db mouse hearts occurred concurrently to accelerated oxidative stress and a modest increase in Nrf2 expression. It has been previously reported that the expression of Nrf2 is initially elevated in response to stress, and then diminishes at a later stage leading to adverse complications such as apoptosis [Accelerated ROS production induces deleterious modifications to DNA, proteins and lipids concomitant to the pathophysiological state observed within the diabetic heart ,43. To spatients . The latpatients ,8,18,32.poptosis .Nrf2 activation, resulting in reduced LV wall thickness and cardiac remodelling. This was an important result since most polyphenols have been shown to induce pro-oxidant effect at higher concentrations [db/db mice for five weeks as well as improve impaired glucose tolerance at 30, 60, 90, and 120 min. However, based on our model to induce cardiomyopathy at 9–14 weeks, the disease had to be at an advance stage with chronically elevated blood glucose levels and thus we did not expect ASP or MET to have an effect on hyperglycemia. Similarly, another factor that could influence the analysis of results, especially the confidence intervals and p-values, could be the small sample size as this has been reported previously [Despite the limited protective effect displayed by ASP treatment in reducing elevated FPG concentrations, this polyphenol at a dose of 130 mg/kg was able to ameliorate oxidative damage via trations ,45. Howetrations ,30,31. Atrations . The inatrations . In a steviously ,47.db/db mice are treated with a combination of ASP and MET before measuring relevant markers associated with hyperglycemia-induced myocardial injury are of foremost importance.In contrast, while current antidiabetic agents including MET effectively reduce FPG concentrations, there is still an increasing number of cardiovascular-related deaths in diabetic patients . CorrespNrf2 may be a key factor to protect against DCM. However, further studies to assess the impact of ASP on cardiac functional parameters in diabetic rodents are required to confirm this exciting proposal. In addition, an in-depth study is being planned looking at Nrf2, mitochondrial function, and ASP protection, with the use of more robust molecular techniques.In summary, consistent with previous findings, ASP displays strong properties to improve myocardial ultrastructure by preventing HG-associated complications. However, dose selection remains important to induce desired efficacy, since the low dose of 13 mg/kg did not show any effect in protecting the heart when compared to the high dose of 130 mg/kg in our study. In addition to its modulatory effects of AMPK, the ability of ASP to regulate 2 Profiler PCR Arrays , RT2 SYBR Green qPCR Master Mix, RT2 Array First Strand Kit and RNeasy Mini Kit were obtained from Qiagen . All other consumables and reagents were purchased from Sigma-Aldrich Corp. , unless otherwise specified.H9c2 rat derived cardiomyoblasts (ECACC No. 8809294) were purchased from the European Collection of Cell Cultures , while ASP was synthesized according to Han et al.  and obta2). Cells were seeded in a 6-well plate at a seeding density of 2 × 104 cells/well. Confluent H9c2 cardiomyoblasts were differentiated into adult cardiomyocytes by substituting growth media with differentiation media consisting of Dulbecco’s Modified Eagle’s Medium supplemented with 10 nM all-trans-retinoic acid and 1% horse serum for six days [H9c2 cardiomyoblasts were cultured in supplemented Dulbecco’s Modified Eagle’s Medium  overnight under standard tissue culture conditions (37 °C in humidified air and 5% COsix days . On day six days . Cells esix days .A stock solution of ASP (22.1 mM), prepared in dimethyl sulfoxide, was diluted using Dulbecco’s Modified Eagle’s Medium to give a final solution of 1 µM. Concentrations of ASP have been previously optimized . The toxTotal RNA was extracted using Trizol reagent, according to a previously described protocol . RNA was2 First Strand Kit, according to manufacturer’s instructions. Rat Oxidative Stress and Atherosclerosis RT2 Profiler PCR Arrays were used (PARN-065ZA and PARN-065ZA) to analyse the differential expression of multiple genes. Briefly, cDNA was added to the RT2 SYBR Green qPCR Master Mix and aliquoted onto each well of the 96-well RT2 Profiler PCR Array plate. An ABI 7500 Instrument  was used for mRNA quantification at the following cycling conditions: 50 °C for 1 min, 1 cycle of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Analysis of PCR array data was done using a Microsoft Excel sheet with macros made available by the manufacturer . Each array contained five housekeeping genes  against which the sample data were normalized. The transcript level of each candidate gene was quantified according to the ΔΔCt method. Ct values > 35 were not included in the analysis and considered as negative.cDNA was synthesized from 2 µg RNA using the RTCasp3, Gpx2, Nox4, Nrf2, Park 7, Sod2, and Ucp2) and in vivo study ), 1 µL of cDNA, and 3.5 µL RNAse free water to a final volume of 10 µL. The quantitative RT-PCR protocol was conducted as follows: 50 °C for 1 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Gene expression data were normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt). Of note, not all genes quantified in the RT2 Array plate were repeated for the siRNA study, but only those that have been previously shown to be affected by ASP [Quantitative RT-PCR was performed on an ABI 7500 Instrument. cDNA was synthesized from 1 µg of RNA using the High Capacity Reverse Transcription Kit, as per manufacturer’s instructions. Thereafter, the PCR reaction mix was prepared by adding 5 µL Taqman universal PCR master mix, 0.5 µL Taqman gene expression assay  and housed at the Primate Unit and Delft Animal Centre (PUDAC) of the South African Medical Research Council (SAMRC) in a controlled environment with a 12 h light/dark cycle in a temperature range of 23–25 °C (relative humidity: ~50%). The mice received standard laboratory chow pellets  ad libitum and had free access to water. Ethical clearance for the use of animals in this study was granted by the SAMRC Ethics Committee for Research on Animals (ECRA No. 07/13), and the Stellenbosch University Ethics Committee (SU-ACUM13-00021).db/db) mice and their non-diabetic (db/+) littermate controls were treated daily for six weeks through oral gavage with either a low (13 mg/kg) or high (130 mg/kg) ASP dose and compared to MET at a dose of 150 mg/kg. Dose selection for ASP and MET was based on previous studies [n = 6/group) included; (i) db/+ untreated controls (db/+_UC); (ii) db/db untreated controls (db/db_UC); (iii) db/db_MET (db/db_MET); (iv) db/db ASP low dose (db/db_ASP_LD); (v) and db/db ASP high dose (db/db_ASP_HD). ASP and MET were dissolved in distilled water before orally administration at the same time (08:00–09:00) every day for six weeks, while untreated animals were given water in place of treatment.Nine-week old diabetic .After the six-week treatment period, mice were fasted for 4 h before being weighed and anesthetized with halothane. Animals received the anesthetic until no reaction could be recorded by pedal reflex before removal of the heart. The heart tissue was weighed and fixed in 10% formalin for a minimum of 16 h before it was processed using a Leica TP 1020 automated processor  and embedded in paraffin wax. Paraffin embedded tissue was cut into sections and attached to aminopropyltriethoxysilane coated glass slides. Tissues were then stained with H&E as previously described . StainedIn mice fasted overnight for 16 h, FPG concentrations were measured by tail prick using a OneTouch Select handheld glucometer , according to the manufacturer’s instructions.After the six weeks of treatment, OGTTs were performed. Briefly, after a 16 h fast, a glucose bolus of 2 g/kg was orally administered through gastric gavage. Plasma glucose levels were determined by tail prick at time intervals of 0, 30, 60, and 120 min.t-test, and a p-value of ≤ 0.05 was deemed as statistically significant.Data were expressed as the mean ± SEM. Results for in vitro experiments were expressed as the mean of three independent biological experiments with each experiment containing at least three technical replicates, unless otherwise stated. For in vivo experiments, each treatment group contained six mice. Statistical analysis was performed using GraphPad Prism software version 5.00 . Comparisons between groups were performed using one-way multivariate ANOVA, followed by unpaired Student


Camellia sinensis var. assamica in northern Thailand. By the local wisdom, Assam tea leaves are used as the raw material in tea fermentation to produce “Fermented Miang” consumed by people in northern Thailand and the countries nearby. In this study, twenty-eight bacterial isolates were obtained from Assam tea leaf samples collected from Nan province, Thailand. Bacterial isolates were identified within 6 genera including Bacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus and Staphylococcus. Among these, the strain ML061-4 shared 100.0 and 99.4% similarity of 16S rRNA and rpoB gene sequence with F. penangensis JCM 31735T, respectively. This is the first discovery of F. penangensis in Thailand. F. penangensis ML061-4 exhibited probiotic characteristics including lactic acid production (9.19 ± 0.10 mg/ml), antibacterial activities (Escherichia coli ATCC 25922 and E. coli O157:H7 DMST 12743), acid and bile salt tolerance , autoaggregation (97.0%), coaggregation (66.0% with E. coli O157:H7), cell surface hydrophobicity (90.0%), bacterial adhesion (82.9% with Lactobacillus plantarum FM03-1), competitive inhibition (17.8% with E. coli O157:H7) and competitive exclusion (34.9% with E. coli O157:H7). Overall, the data suggested that F. penangensis ML061-4 had a great potential to be a probiotic.Assam tea or Miang is a local name of  Floricoccus penangensis is a bacterium in the family Streptococcaceae and has been classified as lactic acid bacteria (LAB). The genus was first described by Chuah et al.1 as it was clearly distinguished from the genera Streptococcus and Lactococcus. The sequence similarities of the 16S rRNA gene found closely related to lactococcal and streptococcal relatives, 92–94%. Currently, there is no report about discovery of Floricoccus penangensis in Thailand as well as their property reports.2. Nevertheless, many scientists have indicated that using of 16S rRNA gene are not applicable to multiple genera in taxonomic analysis. The 16S rRNA gene may not be effectively multiplied due to differential primer affinity and GC content3. Besides, it also demonstrates low resolution among closely related species4. The rpoB gene encodes the beta subunit of bacterial RNA polymerase. Its sequence is more discriminative than the 16S rRNA gene to distinguish various species of bacteria owing to the divergence levels of rpoB gene sequence that is explicitly higher than 16S rRNA gene5. Moreover, the partial rpoB gene sequence exhibits the precise reading frame leading to the easy verification of sequence accuracy. Therefore, the rpoB gene is a strong tool for bacterial identification6.More than two decades, the 16S rRNA gene sequence has been used to identify microorganisms as well as to decide a novel microbe Camellia sinensis var. assamica or Assam tea or “Miang” is an indigenous perennial plant found in highlands of northern Thailand for a long time7. By local wisdom, fresh Assam tea leaves are fermented to produce a chewing refreshment product called “Fermented Miang”8. Assam tea can generally be found in upper northern Thailand including Chiang Mai, Lampang, Chiang Rai, Phayao, Nan and Phrae provinces9.10. Probiotics can improve gut microbiota composition especially LAB, which have been suggested for health benefits including defense function as well as the nourishment of gastrointestinal tract in the body11. LAB are non-pathogenic bacteria found in fermentation for preservation, food and vitamins production12, help to improve microbial imbalance in the body caused by many factors including antibiotic treatment13 and are considered as generally recognized as safe (GRAS). Some LAB can produce bacteriocins, proteinaceous compounds with antibacterial activity against closely related strains14. LAB was classified within the phylum Firmicutes, which were divided into 14 genera: Carnobacterium, Enterococcus, Floricoccus, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc, Melissococcus, Streptococcus, Oenococcus, Pediococcus, Tetragonococcus, Vagococcus and Weissella15. This study aims to investigate the existence of microbes on Assam tea leaves, to demonstrate the use of partial rpoB gene sequence as a molecular tool for lactic acid bacteria identification and to evaluate probiotic property of the selected lactic acid bacteria found on Assam tea leaves. The results obtained will be useful for elucidating the involvement in tea fermentation process of microorganisms found on Assam tea leaves and will be further used in the food industry in many aspects such as functional ingredient and synbiotic production in the future.Probiotics are defined as live microorganisms that provide health benefits on the host when consumed in adequate amountsIn this study, fresh Assam tea leaves were collected between 12 March 2016 and 24 March 2018 from three sampling sites in Pua (Code ML05 and ML06) and Phu Phiang (Code ML09) districts, Nan province, Thailand at an altitude ranging from 243 to 1,278 m above sea level. Meanwhile, fresh Assam tea leaves in Sakat sub district, Pua district (Code ML06) were collected twice on 12 March 2016 and 24 March 2018  showed a maximum of four morphologically different bacterial colonies and had the highest number of bacterial isolates , 4 species per leaf, including B. altitudinis, F. penangensis, M. aloeverae and S. haemolyticus  had 97.9% similarity of 16S rRNA gene sequence when compared with the type strain Floricoccus penangensis ML061-4 was a Gram positive coccus, non-motile, non-spore forming, and negative for catalase, oxidase, indole and lysine decarboxylase tests. Their cell arrangement was found mostly in chains or occur as single cell and pairs. Some branching was occasionally observed. The SEM micrographs demonstrated that cell division occurred in one or two planes from a second division, producing diplococci and streptococci arrangement  genes partial nucleotide sequences of strain ML061-4 were performed. Phylogenetic analysis based on the 16S rRNA indicated that the strain ML061-4 clustered with F. penangensis, supported by a bootstrap value of 93%. It had 100.0% sequence similarity (99% of query cover) with F. penangensis JCM 31735T and 99.8% similarity (100% of query cover) with F. tropicus JCM 31733T. The rpoB gene sequence of strain ML061-4 had 99.6% sequence similarity with F. tropicus JCM 31733T, supported by a bootstrap value of 85%, and 99.4% similarity with F. penangensis JCM 31735T. The partial rpoB gene sequence of F. penangensis ML061-4 shared 49.5, 74.4 and 76.7% identity with Streptococcus mitis CIP 103335T, Lactococcus plantarum DSM 20686T and Lactococcus chungangensis DSM 22330TrpoB sequence, respectively. The phylogenetic tree of relationships based on the sequences of rpoB gene were analyzed using the 18 aligned sequences, the topology of the phylogenetic tree was supported by bootstrap values 1,000 replications which the main branch explicitly led to three branches of Floricoccus and Lactococcus, and Streptococcus genera , contained hypervariable region and appropriated to use for group separation.The 16S rRNA  and F. penangensis ML061-4 was analyzed using HPLC. The total lactic acid was 9.19 ± 0.10 mg/ml consisting of 0.54 ± 0.07 mg/ml of D-lactic acid and 8.65 ± 0.03 mg/ml of L-lactic acid. The ratio of D:L was 1:16. The enzymes involved will be further elucidated.Lactic acid produced by F. penangensis ML061-4 could only inhibit growth of E. coli ATCC 25922 and E. coli O157:H7 DMST 12743 with the inhibitory clear zone of 7.0 ± 0.0 mm.The culture supernatant of F. penangensis ML061-4 was susceptible to the testing antibiotics with the inhibitory clear zone ranging between 10.3 and 28.5 mm while L. acidophilus TISTR 2365 was susceptible to amoxyclav, ampicillin, ampicillin/sulbactum, cefixime, ceftriaxone, cefuroxime, cefuroxime axetil, cefotaxime, cefoxitin, gentamicin, meropenem, tetracycline and ticarcillin/clavulanic acid with the inhibitory clear zone of 9.9–49.0 mm but not for amikacin, co-trimoxazole and ofloxacin.For the susceptibility evaluation, F. penangensis ML061-4 was higher than 70% after 4 hr of incubation in acidic pH, 1.0, 2.0 and 3.0. At pH 1.0, the survival rate was decreased gradually to 90.4, 79.4, 74.6 and 71.1% at 1, 2, 3 and 4 hr of incubation, respectively  of bile salt was higher than 50% subsequent to 24 hr of incubation. Once F. penangensis ML061-4 exposed to the mentioned media, the survival rate was decreased and maintained at between 54.9 and 66.9% after 24 hr . Meanwhile, at 24 hr of incubation showed 97.0% and 93.2%, respectively, non-significant (p > 0.05)  and E. coli O157:H7 DMST 12743 (66.0%) at 48 hr of incubation (P > 0.05)  lower than that of F. penangensis ML061-4, 27.5 and 90.0%, respectively.05) Fig. . Moreove05) Fig. . Cell suF. penangensis ML061-4 was 21.4 and 82.9% when using L. acidophilus TISTR 2365 and L. plantarum FM03-1 as positive controls (100% adhesion), respectively, while E. coli O157:H7 DMST 12743 demonstrated as 5.4 and 20.9%, respectively  (Table E. coli O157:H7 DMST 12743 between 34.9 and 38.4% (p > 0.05). Nevertheless, none of LAB strains tested was able to displace adhered E. coli ATCC 25922 (Table Adhesion ability of ly Table . In bact5) Table . For com22 Table .Table 3A16 at altitude of 450–1,500 m above sea level8. Interestingly, in this study Assam tea trees can grow at the elevation of 243 m above sea level (Phu Phiang district) which is lower than the previous report and also found cultivated within the community in the areas around villages indicating the close relationship between local people and Assam tea. Beyond the altitude above sea level that affects growth of tea, the climate, alkaline and acidic balance, soil nutrient as well as soil ecology also play a vital role in tea plant growth17. The sampling sites were both in the villages and on the mountains  where located far away from the community. Consequently, the existence of microorganisms on Assam tea leaves will bring about the role of microbes on Assam tea leaves in the tea fermentation process, first report in Thailand, especially lactic acid bacteria and their probiotic properties.Generally, Assam tea or Miang are found in highland forests, hillsides and high resourceful water areasBacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus and Staphylococcus. These bacteria have been widely found in nature which can be contaminated onto surface of leaves and stems20. B. altitudinis. B. subtilis, K. halotolerans and M. aloeverae were reported as endophytic bacteria involving in plant growth promotion and protection24. B. clausii, B. megaterium, B. siamensis and B. subtilis have been reported as probiotic. Bacillus spp. can form endospore and produce a bacteriocin-like substance, which help resisting stomach acidic condition and inhibiting pathogens27. B. aryabhattai produces vanillin from ferulic acid28, hence, it may involve in aroma and flavor occurred in fermented Miang. However, S. haemolyticus, S. cohnii and S. hominis are normal flora commonly live on human and animal skins31. Their existence on Assam tea leaves may be due to human activities as Assam tea growing area is located in the community.All bacterial isolates were member of Actinobacteria and Firmicutes phyla and were classified within 6 genera including 7. Recently, Chaikaew et al.32 reported the diversity of LAB in fermented Miang. The prevalent species found were L. plantarum, L. pentosus, L. panthesis and L. fermentum. Furthermore, Leuconostoc, Enterococcus, Pediococcus, Weisella and Lactococcus were found as minor populations in fermented Miang. Interestingly, Floricoccus that has a close relationship with Lactococcus and Streptococcus was never reported. In this study, Floricoccus was found on fresh Assam tea leaves possibly due to some nutrients occurring on Assam tea leaves33 which enhances its survival. The existence of Floricoccus on an Assam tea leaf was not always as it was not found on all samples collected. The finding of LAB on Assam tea leaves could explain their key role in the tea fermentation process. Some reports proposed that the significant differences of bacterial communities in the environment may be related to soil compositions such as nutrients, acid and base conditions at the moment34. Hence, how Floricoccus exists on Assam tea leaves is to be further elucidated.LAB were known to involve in Assam tea or Miang fermentationFloricoccus is not found in fermented Miang may be due to its lower ability to produce lactic acid than lactobacilli35 which is dominated in fermented Miang. Besides, this study also found that Floricoccus penangensis could not grow in MRS medium. These may be the reasons for the absence of Floricoccus species in fermented Miang. Some reports described that the non-growing LAB may associate with the high concentrations of polyunsaturated fatty acids in media which might inhibit growth36.Reasonably, rpoB gene has been usually used for identification when the 16S rRNA gene is unclear5. Identification of Afipia, Bosea and Bradyrhizobium spp. using rpoB gene showed the percentage cut-off between different genus ranging between 82 and 83%37. Thus far, there is no report about microorganisms on leaves of Assam tea. This is the first discovery report of F. penangensis in Thailand using the primer pair which is rpoB gene specific to identify bacteria in the genus Floricoccus that clearly separated from genera Lactococcus and Streptococcus.The F. penangensis ML061-4 may lie within the amount of acid produced. Undissociated lactic acid could go through cell membrane and discharge hydrogen ion inside the cell leading to disturbance of important cell functions39. Furthermore, some species of LAB can produce bacteriocin or bacteriocin-like inhibitory substance (BLIS) that play a role in formation of bacterial cell membrane pore, inhibition of ATP synthesis and ultimately death of cells40. Notwithstanding, the inhibitory substance of F. penangensis ML061-4 will be further evaluated.The antibacterial activity of 27. Moreover, antibiotic resistance gene transfer also may be related to the change of microbial community in the environment including Assam tea growing area. In this study, F. penangensis ML061-4 was inhibited by all test antibiotics indicating its safety as a potential probiotics while L. acidophilus TISTR 2365 was sensitive to 13 of 16 antibiotics tested.Antibiotic susceptibility is proposed as a vital criterion for potential probiotics to ensure that the antibiotic resistance gene will not be transferred to recipient bacteria in the gut which leads to the development of antibiotic-resistant bacteriaF. penangensis ML061-4 may be based on the histidine decarboxylation pathway, which generates proton motive force resulting in ATP synthesis42. Furthermore, the acid tolerance in LAB also related to glutamate decarboxylase system and arginine deiminase pathway43. Additionally, some reports suggested that bile resistance is associated with enzyme activity of bile salt hydrolase (BSH), which leads to the toxicity reduction42. However, the immediately reduction of F. penangensis ML061-4 in base condition at the beginning can be explained as a result of antibacterial activity of bile salt and its bacterial membrane ability44.The acid tolerance of 47. In this study, F. penangensis ML061-4 exhibited a higher autoaggregation and hydrophobicity than L. acidophilus TISTR 2365.The autoaggregation and hydrophobicity assays are used for determination of adhesion ability of probiotics to intestinal mucosa, which is necessary for host gut bacterial colonization, and bacterial coaggregation can prevent pathogenic bacterial infection and colonization48. Bacterial cell wall components such as proteins, carbohydrates and fatty acids were responsible for the adherence to the intestinal mucosa49. High potential probiotics should demonstrate bacterial competition and competitive exclusion to disturb and interrupt pathogenic infection, colonization and biofilm formation as well as produce polymeric substances such as exopolysaccharides to help probiotic colonization within intestine51. In this study, F. penangensis ML061-4 represented the ability of adhesion, competition and displacement similar to L. acidophilus TISTR 2365 and L. plantarum FM03-1 that are considered the great probiotics. These characteristics are harbored by F. penangensis ML061-4 which has high potential to be a good probiotic.Bacterial adhesion onto epithelial cells is a considerable property of probiotic to reduce enteropathogenic adhesion and infectionFresh Assam tea leaves were obtained from Pua and Phu Phiang district, Nan province of Thailand. The source of Assam tea is presented in Table Each sampling swab was serially diluted with 0.85% (w/v) of NaCl and spread on tryptic soy agar (TSA)  prior to incubation at 37 °C for 24–48 hr. All pure bacterial isolates were stored at −20 °C in tryptic soy broth (TSB) (Merck™) containing 20% (v/v) of glycerol.et al.52. The 16S rRNA gene was amplified using universal bacterial primers, 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′)53. The primers used for amplification of the housekeeping gene rpoB were RpoBLac1F (5′-TAC GGK AAA CAC CGT A-3′) and RpoBLac1R (5′-TCA ARC CAW GCT CCA CGG-3′), which were previously designed and reported by Meucci et al.54. The amplification cycles were initially denatured at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 56 °C (16S rRNA) or 47.5 °C (rpoB) for 30 sec, extension at 72 °C for 1 min, final extension at 72 °C for 5 min, and kept at 4 °C. The PCR products were electrophoresed on 0.8% (w/v) agarose gel containing 1X nucleic acid staining solution  for 50 min at 95 V 300 mA in 1X Tris-acetate-EDTA (TAE) buffer  using electrophoretic gel system  at room temperature. The 1 kb DNA Ladder  was used as a marker. The gels were visualized under ultraviolet light by gel documentation system , and gel images were captured using monochrome CCD camera . The amplicons were purified and sequenced by a DNA sequencing services . The 16S rRNA and rpoB gene sequence were performed by comparing with GenBank and EzBioCloud databases. The sequence data was aligned, and phylogenetic tree was analyzed by a neighbor-joining method55 with a MEGA 7 program56.The chromosomal DNA of each strain was extracted by modification of the protocol described by Pitcher et al.57 with slight modifications using the scanning electron microscope (SEM) . For biochemical characterization, various tests were conducted including carbohydrate fermentation , esculin and starch hydrolysis, indole, lysine decarboxylase, catalase and oxidase production, and Voges-Proskauer and methyl red tests. Various growth conditions were determined including growth in the presence of 3, 4 and 6% (w/v) NaCl, growth at various temperatures and pHs. The media used were de Man, Rogosa and Sharpe (MRS) and brain heart infusion (BHI) (Merck™).The strain ML061-4 was investigated its morphological characteristics. Scanning electron microscopy was performed as described by Arroyo F. penangensis ML061-4 was cultivated in TSB and incubated at 37 °C for 24 hr. The culture broth was centrifuged at 6,000 rpm 4 °C for 5 min and filtered by nylon membrane filter, 0.22 µm. The filtrate was kept at −20 °C before evaluation of lactic acid content. Lactic acid was determined using a high-performance liquid chromatography (HPLC) system , equipped with a UV detector at 254 nm using an Astec CLC-L (150 × 4.60 mm) column  with 0.005 M of CuSO4 as the mobile phase at a flow rate of 1.0 ml/min. The D- and L-lactic acids (Sigma-Aldrich™) were used as standards.The et al.58. The culture broth was tested against Bacillus cereus TISTR 687, Escherichia coli ATCC 25922, E. coli O157:H7 DMST 12743, methicillin resistant Staphylococcus aureus DMST 20625, Pseudomonas aeruginosa ATCC 27853, Salmonella Typhi DMST 22842, Shigella dysenteriae DMST 1511, S. aureus ATCC 25923 and Vibrio cholerae DMST 2873.Agar well diffusion method was carried out according to the method of Bonev ®, India) including amikacin (30 µg), amoxyclav (30 µg), ampicillin (10 µg), ampicillin/sulbactum (10 µg/10 µg), cefixime (5 µg), ceftriaxone (30 µg), cefuroxime (30 µg), cefuroxime axetil (30 µg), cefotaxime (30 µg), cefoxitin (30 µg), co-trimoxazole (25 µg), gentamicin (10 µg), meropenem (10 µg), ofloxacin (5 µg), tetracycline (30 µg) and ticarcillin/clavulanic acid (75/10 µg) according to the method described by Clinical Laboratory Standards Institute (CLSI)59. L. acidophilus TISTR 2365 was used as a control.The antibiotic susceptibility test of the strain ML061-4 was examined using antibiotic discs  prior to evaluation of acid and base tolerant. The acid tolerance was performed in media pH 1.0, 2.0 and 3.0 while the alkaline resistance was done in media containing 0.1, 0.2 and 0.3% (w/v) of bile salt (Himedia®)60. Survival rate was evaluated by a viable plate count method on TSA following formula:The et al.61. Briefly, the F. penangensis ML061-4 was cultured overnight and harvested by centrifugation at 6,000 rpm 4 °C for 5 min. The cell pellets were resuspended in 1X PBS and adjusted equivalent to 0.1 at OD600 (ODi) and undisturbed at room temperature for 48 hr. The upper suspension fluid was measured the OD600 (ODt) at 1, 2, 4, 6, 12, 24, 36 and 48 hr. L. acidophilus TISTR 2365, a well-known probiotic strain, was used as a control. The autoaggregation percentage was calculated according to the equation:Autoaggregation was investigated by modification of the protocol described by Valeriano E. coli ATCC 25922 and E. coli O157:H7 DMST 12743 was carried out62. Cell suspensions of F. penangensis ML061-4 and test pathogenic bacteria were prepared to 1 × 109 CFU/ml each. After that, the strain ML064-1 was mixed with each test pathogenic bacteria . The upper suspension fluid was measured the absorbance at 600 nm. The percentage of coaggregation at 0, 1, 2, 4, 6, 12, 24, 36 and 48 hr was calculated according to the equation:x and Ay represented absorbance of each isolate and A(x + y) demonstrated absorbance of mixture.The coaggregation against et al.27. In brief, the bacterial cell culture of F. penangensis ML061-4 was incubated overnight, harvested by centrifugation and washed twice by 1X PBS. The cell pellet was resuspended in 1X PBS and adjusted equivalent to 0.1 at OD600 . The F. penangensis ML061-4 suspension was mixed with xylene. The mixture was undisturbed for separation of organic and aqueous phases at room temperature for 30 min. Afterwards, the final absorbance of aqueous phase  was measured. L. acidophilus TISTR 2365 was used as a control. The percentage of surface hydrophobicity was calculated according to the equation:Cellular hydrophobicity was determined by measuring the bacterial cell adhesion to hydrocarbon according to the protocol described by Meidong et al.63 with modifications. Briefly, the African green monkey kidney cell line (Vero cell) was grown in Dulbecco’s modified eagle medium (DMEM)  supplemented with penicillin/streptomycin and fetal bovine serum (FBS) (Sigma-Aldrich™) and incubated at 37 °C in a CO2 incubator. The Vero cells were cultivated in 6-well plate and adjusted equivalent approximately 1 × 105 cell/ml in DMEM with FBS, incubated at 37 °C in a CO2 incubator for 18-24 hr and washed three times with 1X PBS afterwards. Subsequently, the suspended ML061-4 cells (1 × 108 CFU/ml) and DMEM were added into each well prior to incubation for 1 hr and washed three times with 1X PBS. The Vero cells were methanol fixed for 3–5 min and stained with Giemsa stain for 10–15 min. L. acidophilus TISTR 2365 and L. plantarum FM03-1 (GenBank accession no. MF599378), known adhesive strains, were used as controls. Morphological alterations were observed under light microscope and percentage of adhesion to cell line was calculated.The bacterial adherence assay was performed following the method described by Llanco E. coli ATCC 25922 and E. coli O157:H7 DMST 12743 by strain ML061-4 was investigated by the method described by Boudeau et al.64 with modification. In competition experiments, the strain ML064-1 and E. coli strain were added together to the cell monolayer  and incubated for 1 hr. For competitive exclusion, the Vero cell monolayer was first incubated with E. coli for 1 hr, washed the non-adherent bacteria three times with 1X PBS, added the strain ML064-1 and incubated for 3 hr.Bacterial competition and competitive exclusion of Student’s t-test was performed to calculate statistical significance of cellular autoaggregation, coaggregation and cell surface hydrophobicity. The bacterial adhesion, competition and competitive exclusion were statistically evaluated using analysis of variance (ANOVA) with SPSS 22.0 and Duncan’s multiple range tests. Significance levels were defined using p < 0.05.Camellia sinensis var. assamica) contained diverse microorganisms including lactic acid bacteria that might participate in Miang fermentation. F. penangensis ML061-4 isolated from Assam tea leaves, first report in Thailand, exhibited some biological properties indicating the high potential for using as a probiotic. The rpoB gene was first used as a tool for effective identification of bacteria in the genus Floricoccus.Leaves of Assam tea or Miang (Supplementary Information