R. rita ˃O. niloticus ˃C. marulius ˃S. sarwari ˃C. idella ˃C. catla ˃N. notopterus ˃E. vacha ˃L. rohita ˃C. carpio, respectively. Hg as O. niloticus ˃S. sarwari ˃R. rita ˃C. marulius ˃C. catla ˃N. notopterus ˃E. vacha ˃L. rohita ˃C. carpio ˃C. idella, respectively. As as O. niloticus ˃R. rita ˃S. sarwari ˃C. marulius ˃C. catla ˃C. carpio ˃N. notopterus ˃C. idella ˃E. vacha ˃L. rohita, respectively. Cr accumulation recorded as L. rohita ˃C. idella ˃O. niloticus ˃C. marulius ˃E. vacha ˃R. rita ˃C. catla ˃C. carpio ˃S. sarwari ˃N. notopterus, respectively. Pb accumulation in different fish species was recorded as C. idella ˃C. carpio ˃N. notopterus ˃L. rohita ˃O. niloticus ˃C. marulius ˃R. rita ˃S. sarwari ˃E. vacha ˃C. catla, respectively. Cd accumulation in different organs was recorded as kidney ˃liver ˃gills ˃muscle ˃skin ˃scale. Hg accumulation in different organs was recorded as kidney ˃gills ˃liver ˃skin ˃muscle ˃scale. As accumulation in different organs was recorded as kidney ˃liver ˃gills ˃muscle ˃skin ˃scale. Cr accumulation in different organs was recorded as gills ˃ liver ˃skin ˃muscle ˃kidney ˃scale. Pb accumulation in different organs was recorded as gills˃ kidney˃ skin˃ liver˃ muscle˃ scale.The present study was conducted on Head Punjnad (HP) and Head Taunsa (HT) to evaluate the contamination of Pb, Cr, As, Hg, and Cd in water, soil, sediment, fish as a whole and fish organs. Fish, water, soil and sediment samples were collected from different sites of HT and HP on a monthly basis for 8 months. Heavy metals in water, soil, and sediment were determined by a polarized Zeeman atomic absorption spectrophotometer and in fish and fish organs by an atomic absorption spectrophotometer. Contamination of Cd, Hg, and As was significantly (P<0.05) higher in water of HP as compared to HT, while Cr showed a non-significant (P>0.05) difference at HP and HT. Pb was significantly (P<0.05) higher in water of HT as compared to HP. In the case of soil, Cd, Hg, and Pb were higher at HT as compared to HP, while As and Cr were significantly (P<0.05) higher at HP as compared to HT. In sediment, contamination of Cd, Hg, and As were significantly (P<0.05) higher at HP as compared to HT, while the Cr difference was non-significant (P>0.05) but Pb showed a significantly (P<0.05) higher value at HT than HP. Cd accumulation in different fish species was recorded as  Different terminologies are used for heavy metals like trace metals, hazardous or toxic metals, micronutrients, non-essential and essential metals, and trace elements  de de33] deR. rita (112.02ppb), while C. carpio had the lowest concentration (43.26ppb) , whereas L. calbasu had a lower concentration (0.14ppm or 140ppb). In the lower Indus river, C. mrigala contained a high amount of Cd (0.89ppm or 890ppb), whereas C. chitala had a low amount (0.03ppm or 30ppb). In the Chenab river, C. carpio exhibited a high Cd level (1.48ppm or 1480ppb), while C. garua had a low level (0.22ppm or 220ppb). Similarly, in the Kabul river, C. carpio showed high contamination of Cd (1.13ppm or 1130ppb), while T. macrolips had a lower level (0.08ppm or 80ppb) . Co. Co35]. C. idella accumulated the highest levels of Pb, while L. rohita absorbed Cr the most. Cd, Hg, and As were observed as the highest in O. niloticus. Fish gills and kidneys displayed the highest loads of Pb and Cr followed by Cd. Overall, our findings exhibited that head Taunsa is more polluted than head Punjnad. Simultaneously, almost all the commercially important fish species are at a higher risk of high bioaccumulation of heavy metals that may cause severe human health crises if the fish consumption from the polluted sites remains continued. This study achieved all its targeted investigations. The results may help revise the controlling strategies of the industrial effluents so that we may save our freshwater fish species and allied industries.In conclusion, this study reported the levels of five heavy metals  in water and sediment samples collected from two famous tourist spots i.e., Head Punjnad and Head Taunsa. We also investigated loads of the heavy metals in ten different fish species considered important as commercial species and preferred by the local and visiting people. We also targeted six different fish organs to estimate the heavy metals’ presence and concentrations, and the organs included gills, kidney, liver, muscle, scale, and skin. Our outcomes indicated that Cd was the most prevalent metal in both sites, with head Taunsa as the most polluted site. Cr concentration was found to be the lowest at both study sites. In the case of metals concentration in fish, Pb was the highest, followed by Cr and Cd. The


Interestingly, BET measurements revealed the high surface area of Fe3O4-κ-Carr/MIL-125(Ti) of about 163.27 m2/g, while VSM showed its excellent magnetic property (20.34 emu/g). The comparison study pointed out the synergistic effect between Fe3O4, κ-Carr, and MIL-125(Ti), forming a composite with an excellent adsorption performance toward o-NP. The adsorption data obeyed pseudo-second-order kinetic model, and Freundlich isotherm model was better fitted than Langmuir and Temkin. Furthermore, Langmuir verified the supreme adsorption capacity of o-NP onto Fe3O4-κ-Carr/MIL-125(Ti) since the computed qmax reached 320.26 mg/g at pH 6 and 25 °C. Furthermore, the XPS results postulated that the adsorption mechanism pf o-NP proceeded via H-bonding, π-π interaction, and electron donor–acceptor interactions. Interestingly, Fe3O4-κ-Carr/MIL-125(Ti) composite retained good adsorption characteristics after reusing for five cycles, suggesting its viable applicability as an efficient, renewable, and easy-separable adsorbent for removing nitro aromatic pollutants.Water pollution is a dreadful affair that has incessantly aggravated, exposing our planet to danger. In particular, the persistent nitro aromatic compound like nitrophenols causes anxiety to the researchers due to their hazardous impacts, excessive usage, and removal difficulty. For this purpose, a novel multi-featured composite was constructed based on κ-Carrageenan (κ-Carr), MOF (MIL-125(Ti)), and magnetic FeThe online version contains supplementary material available at 10.1007/s11356-023-25678-2. Water pollution by the fatal aromatic compounds is swiftly exacerbated, threatening humanity’s existence . Assorted characterization tools were utilized to infer the successful fabrication of Fe3O4-κ-Carr/MIL-125(Ti) and study its chemical and physical properties. The adsorbability of Fe3O4-κ-Carr/MIL-125(Ti) composite was examined in the adsorption of o-nitrophenol (o-NP) from an aqueous solution. Besides, the reusability of Fe3O4-κ-Carr/MIL-125(Ti) composite was confirmed by executing the recyclability test for five cycles. More importantly, the mechanism of the o-NP adsorption onto Fe3O4-κ-Carr/MIL-125(Ti) composite was understood thoroughly based on XPS results.Herein, we adopted a developed avenue to foster the adsorption performance of κ-Carr through its combination with MIL-125(Ti). Furthermore, to overcome the separation difficulty, κ-Carr/MIL-125(Ti) composite was decorated by magnetic nanoparticles (Fe2.4H2O), glutaraldehyde, and ethanol were supplied from Aladdin Industrial Corporation, China. Ammonium solution (NH4OH), 1,4-benzene dicarboxylic acid (BDC), and N,N dimethyl formamide (DMF) were purchased from Sinopharm Chemical Reagent, China. κ-Carrageenan, ferric chloride hexahydrate (FeCl3.6H2O), o-nitrophenol, and titanium tetraisoproproxide (Ti(O-iPr)4) were provided from Sigma-Aldrich, USA.Ferrous chloride tetrahydrate  was prepared as follows: 995 mg BDC was dissolved into 25 mL DMF under magnetic stirring for 15 min. Then, 1.35 mL Ti(O-iPr)3O4-κ-Carr/MIL-125(Ti) composite was prepared as follows: a particular amount of κ-Carr was dissolved into 25 mL dist. H2O at 70 °C. Then, 0.02 g Fe3O4 was dipped into κ-Carr solution and kept under stirring for 1 h, allowing the magnetic nanoparticles to diffuse into κ-Carr layers. MIL-125(Ti) was added bit-by-bit to Fe3O4/κ-Carr composite under vigorous stirring, followed by adding 6 mL of glutaraldehyde. After 1 h, Fe3O4-κ-Carr/MIL-125(Ti) composite was collected, washed, and dried at 50 °C for 24 h. Fe3O4-κ-Carr/MIL-125(Ti) composite was prepared with three mass ratios of κ-Carr/MIL-125(Ti) = 1:1, 1:3, and 3:1 (Scheme Fe3O4-κ-Carr/MIL-125(Ti) composite and its components were characterized by Fourier transform infrared spectra , Brunauer–Emmett–Teller method , X-ray diffractometer , zeta potential , X-ray photoelectron spectroscopy , vibrating-sample magnetometer , scanning electron microscope , and transmission electron microscope .Fe3O4-κ-Carr/MIL-125(Ti) composite were defined in a batch mode. A series of the o-NP adsorption process proceeded at a pH range from 2 to 10 to identify the optimum pH. Furthermore, the impact of the Fe3O4-κ-Carr/MIL-125(Ti) dose on the adsorption aptitude of o-NP was determined at a dose range from 0.005 to 0.02 g. Moreover, the influence of the temperature on the removal efficiency of o-NP was examined at a temperature range from 25 to 55 °C. Finally, the impact of the initial concentration of o-NP was studied at a concentration range from 50 to 200 mg/L. The residual concentration of o-NP was measured according to the standard methods to examine water and wastewater  and the volume of o-NP solution, respectively.The optimum conditions to adsorb o-NP onto Fe3O4-κ-Carr/MIL-125(Ti) composite, the used composite was separated after the o-NP adsorption and subsequently soaked into 25 mL 1 M NaOH under magnetic stirring to desorb o-NP from its surface. Then, the recycled composite was washed with distilled H2O and utilized in the next cycle, repeating this adsorption/desorption cycle five times.To evaluate the regeneration potential of Fe3O4-κ-Carr/MIL-125(Ti) was added to o-NP/NaCl solution under stirring. After 60 min, a sample was withdrawn and measured to determine the concentration of the un-adsorbed o-NP.The influence of the ionic strength on the o-NP adsorption aptitude was assessed as follows: a specific weight of NaCl (0.2–1.0 mol/L) was soaked in 20 mL of o-NP at pH 6. Then, 10 mg of Fe3O4-κ-Carr/MIL-125(Ti) composite and its pure components. The SEM image of κ-Carr  depicts a quasi-spherical morphology with irregular size  composite suggested the incorporation of Fe3O4 and MIL-125(Ti) particles into κ-Carr  composite  onto the fiber-like particles of κ-Carr, denoting the successful combination between the composite’s components.The TEM image Fig. E exhibit3O4, κ-Carr, MIL-125(Ti), and Fe3O4-κ-Carr/MIL-125(Ti) composite were analyzed by XRD , (311), (400), (511), and (440), respectively  composite illustrates the characteristic peaks to the pure components but with a diminution in the crystallinity which is most likely due to the amorphous character of κ-Carr.The crystallite phases of Fe3O4, MIL-125, and Fe3O4-κ-Carr/MIL-125(Ti) composite. For κ-Carr, the peaks at 847 and 1260 cm−1 are assigned to C-O-S and O = S = O, respectively  composite, the FTIR spectrum reveals the discriminative peaks of κ-Carr, Fe3O4, and MIL-125(Ti) with slight peaks shifting and lower peaks intensity, suggesting the successful combination between the pure components.Figure 3O4 into the Carr matrix. The XPS survey  composite consists from C1s, Ti2p, Fe2p, S2p, and O1s. The C1s-spectrum  backbone since the peaks at 532.80, 531.42, and 529.66 eV are assigned to SO42−, Fe–O/Ti–O, and C-O, respectively.XPS spectra inferred the incorporation of MIL-125(Ti) and Ferum Fig. . Moreove3O4 and Fe3O4-κ-Carr/MIL-125(Ti) composite since the coercivity values were 199.86 and 94.56 G, respectively. Moreover, an expected decline in the saturation magnetization of Fe3O4 (Ms = 52.34 emu/g) occurred which is most likely due to its blinding with the non-magnetic Carr and MIL-125(Ti). Notably, Fe3O4-κ-Carr/MIL-125(Ti) composite possesses a propitious magnetic property (Ms = 20.34 emu/g), endowing it the easy separation advantage by an external magnet instead of the conventional techniques that consume a long time.VSM hysteresis loops Fig. A confirm3O4-κ-Carr/MIL-125(Ti) composite at a pH ranging from 3 to 11. It is apparent that Fe3O4-κ-Carr/MIL-125(Ti) composite displayed a negatively charged surface, while the ZP value was amplified from − 27.9 to − 39.9 mV with a raising of the pH from 2 to 10, respectively. This finding suggests the suitability of Fe3O4-κ-Carr/MIL-125(Ti) to adsorb zwitter ionic, neutral, and cationic pollutants.Figure 2-adsorption/desorption isotherm  composite showed a type II with H4-type hysteresis loop according to the IUPAC classification, suggesting the mesoporous structure of the composite. Moreover, the specific surface area of Fe3O4-κ-Carr/MIL-125(Ti) composite was 163.27 m2/g, and the average pore diameter was 2.861 nm  were 17.35, 28.35, and 49.55% and the adsorption capacity were 22.86, 33.12, and 52.91 mg/g, respectively. Furthermore, the removal % and the adsorption capacity of Fe3O4-κ-Carr/MIL-125(Ti) composites with κ-Carr: MIL-125(Ti) ratios 3:1, 1:1, and 1:3 were 60.99, 65.19, and 77.55% and 63.60, 67.51, and 79.05 mg/g, respectively  composite with a ratio of 1:3 between κ-Carr and MIL-125(Ti) was chosen for the rest of the batch experiments. For assessing the amelioration of the adsorption performance of Carr toward o-NP after blinding with MIL-125(Ti), a comparison test was executed between the pure materials and the three fabricated composites  composite was investigated at a wide scale of pH media  at pH 6. This finding could be explained by the pKa of o-NP = 7.23, meaning that o-NP exists in the molecular form in acidic conditions , and there are other chemical and physical interactions such as π-π interaction, H-bonding, and electron donor–acceptor interaction could take place between o-NP and Fe3O4-κ-Carr/MIL-125(Ti) in the acidic medium  composite  composite onto the adsorption efficiency o-NP. It is apparent that the augmentation in the composite dose from 0.005 to 0.02 g causes an increase in the removal % of o-NP from 58.28 to 92.29%, respectively, which is most likely due to the increase in the number of active sites. On the contrary, a decline in the adsorption capacity of o-NP from 122.12 to 46.40 mg/g was observed with the raising in the Fe3O4-κ-Carr/MIL-125(Ti) dose which may be attributed to the aggregation of the extra amount of the composite, resulting in a diminution in the surface area.Figure 3O4-κ-Carr/MIL-125(Ti) composite. It was recorded an enhancement in the adsorption aptitude of o-NP with the increase in the NaCl concentration from 0.2 to 1.0 mol/L since the adsorption capacity and removal % incremented from 79.31% and 81.33 mg/g to 87.93% and 92.45 mg/g, respectively. This behavior is most likely due to the salting out effect as the presence of NaCl declines the o-NP solubility, agreeing with Mengzhi Yang et al. Figure 3O4-κ-Carr/MIL-125(Ti) since the adsorption capacity and removal % of o-NP dwindled from 79.05 mg/g and 77.55% to 65.82 mg/g and 63.38% with raising the process temperature from 25 to 55 °C, respectively  surface directly diminished.It was deduced the exothermal nature of the o-NP adsorption onto Fe3O4-κ-Carr/MIL-125(Ti) surface. Thence, such potent forces could overcome the mass transfer resistance to the migration of o-NP from the bulk solution to Fe3O4-κ-Carr/MIL-125(Ti) surface  surface  composite not only exhibited efficient adsorption performance toward the detrimental o-NP but also fast adsorption since the equilibrium time was 60 min.Figure ace Fig. D  composite may occur via diverse mechanisms, depending on the heterogeneity of the binding sites onto the composite surface and physicochemical conditions. Therefore, various kinetic models: pseudo-first-order (PFO), pseudo-second-order (PSO), and Elovich, were utilized to analyze the resultant experimental data  composite since R2-PSO > R2-PFO  was greater than the desorption rate.It was deduced from the computed es Table  that PSO3O4-κ-Carr/MIL-125(Ti) composite at equilibrium was scrutinized by bountiful isotherm models, including Langmuir, Freundlich, and Temkin  is more fitted than Langmuir (R2 = 0.984) and Temkin (R2 = 0.983) to model the equilibrium data of the o-NP adsorption onto Fe3O4-κ-Carr/MIL-125(Ti). Additionally, the b value from Temkin model evinced the same result since b < 80 kJ/mol. Moreover, it was found that n > 2 reflects the favorability of the o-NP adsorption process onto Fe3O4-κ-Carr/MIL-125(Ti) composite. Furthermore, the calculated qmax of o-NP onto Fe3O4-κ-Carr/MIL-125(Ti) composite under Langmuir was 320.26 mg/g at pH 6 and 25 °C.The obtained isotherm parameters Table  point ou3O4-κ-Carr/MIL-125(Ti) composite has remarkable recyclability where its adsorption performance toward o-NP was still high after the 5th cycle (q = 58.94 mg/g and R% = 56.01%). This finding confirmed the significance of these magnetic adsorbents that provide a fast, easy, and impeccable separation after the adsorption processes using an external magnet.It is apparent from the recyclability test Fig. D that Fe3O4-κ-Carr/MIL-125(Ti) composite was studied based on XPS analysis, ZP measurements, and the experimental results. XPS survey of Fe3O4-κ-Carr/MIL-125(Ti) after the adsorption of o-NP  as well as the plentiful O-containing groups onto the Fe3O4-κ-Carr/MIL-125(Ti) surface and the H-atoms of o-NP; (ii) π-π interaction between the aromatic ring of BDC in the composite and the benzene ring in o-NP; (iii) electron donor–acceptor interaction between the e-donor groups in Fe3O4-κ-Carr/MIL-125(Ti) composite  and the e-withdrawing group in o-NP (NO2) as well as the e-withdrawing group in the composite (COOH) and the e-donor groups in o-NP (OH and benzene ring). The occurrence of these physicochemical interactions between o-NP and Fe3O4-κ-Carr/MIL-125(Ti) was confirmed by the peak shift of the XPS spectra of O1s and S2p after the o-NP adsorption  composite for removing the organic o-NP pollutant. The characterization stage inferred the successful formulation of the composite adsorbent. Surprisingly, increasing MIL-125(Ti) ratio three times than κ-Carr significantly boosted the removal (%) of o-NP from 60.99 to 77.55%, and the maximal adsorption capacity of o-NP attained 320.26 mg/g at pH 6 and 25 oC. Moreover, data obtained from kinetics and isotherm studies were fitted to Freundlich model and followed the pseudo-second-order model. The reusability test attested the potential capability of Fe3O4-κ-Carr/MIL-125(Ti) composite to adsorb o-NP after five repeated cycles with removal (%) exceeded 60%. Overall, the higher adsorption performance, facile separation, and recyclability features nominate the potential use of the formulated composite as an efficient adsorbent candidate for advanced water treatment.This study reported the construction, characterization, and adsorbability evaluation of a new FeSupplementary file1 (DOCX 17 KB)Below is the link to the electronic supplementary material.


Aspergillus Carneus. Arugosin C, Averufin, Averufanin, Nidurifin and Versicolorin C were analyzed using NCI-SRB assay on 10 different cell lines of breast cancer, ovarian cancer, glioblastoma and non-tumorigenic cell lines. Averufanin showed highest cytotoxicity with lowest IC50 concentrations especially on breast cancer cells. Therefore, Averufanin was further investigated to enlighten cell death and molecular mechanisms of action involved. Cell cycle analysis showed increase in SubG1 phase suggesting apoptosis induction which was further confirmed by Annexin V and Caspase 3/7 Assays. H2A.X staining revealed accumulation of DNA damage in cells treated with Averufanin and finally western blot analysis validated DNA damage response and downstream effects of Averufanin treatment in various signaling pathways. Consequently, this study shows that Averufanin compound induces cell cycle arrest and cell death via apoptosis through causing DNA damage and can be contemplated and further explored as a new therapeutic strategy in breast cancer.Cancer is one of the leading causes of death worldwide, accounting for nearly 10 million deaths in 2020. Current treatment methods include hormone therapy, γ-radiation, immunotherapy, and chemotherapy. Although chemotherapy is the most effective treatment, there are major obstacles posed by resistance mechanisms of cancer cells and side-effects of the drugs, thus the search for novel anti-cancer compounds, especially from natural sources, is crucial for cancer pharmaceutics research. One natural source worthy of investigation is fungal species. In this study, the cytotoxicity of 5 metabolic compounds isolated from filamentous fungus  By 2040, the number of new cancer cases per year is expected to rise to 29.5 million and the number of cancer-related deaths to 16.4 million2 To be able to restrain this probable outcome, investigation of new treatment strategies is vital.Cancer is among the leading causes of death worldwide, accounting for around 10 million deaths in 20203. Although chemotherapy is one of the most effective treatment options, acquired resistance of cancer cells remains as an obstacle to overcome4. Therefore, discovery of new anti-cancer compounds to be used as chemotherapeutic agents is still an attractive field5.Cancer treatment today includes, surgery, radiation therapy, hormone therapy, immunotherapy, targeted therapy, photodynamic therapy, and the most widely used one, chemotherapy6. Cancer cells can also get multidrug resistance (MDR), through increasing efflux mechanisms and decreasing intake rate7. One of the important elements in cancer emergence, progression and resistance mechanisms are tumor suppressor genes. A well-known and widely studied example is TP53 gene which is found mutated in 50% of human cancers8. Protein p53 is most frequently activated in response to DNA damage; and cell-cycle arrest followed by apoptosis are the most prominent outcomes of its activation9. It functions through activating cyclin dependent kinase inhibitors such as p21Cip1 and apoptotic proteins such as Bax, PUMA and Noxa, respectively10. Although these are the prevalent roles of p53, studies show that these are not the only pathways p53 is involved. Some examples are p53 and lncRNA interactions11, miRNA processing12 and CyclinA2/CDK2 complex activating. The latter was recently demonstrated by a study that Cyclin A2/CDK2 complex is not just an S phase cell cycle regulator but also a DNA damage response and takes a role in double-strand repair coupling with Ku proteins13. The mechanisms activating p53 is also widely studied and among several other proteins in regulatory roles, GSK3β phosphorylates p53 so that it is in its active form14. GSK3β is also activated under stress and DNA damage conditions, upon dephosphorylation and is also capable of downregulating Cyclin D1 along with the downstream p53-p21-Cyclin D1 pathway15. In the presence of an active GSK3β, Cyclin D1 levels decrease affected by both pathways. Even though treatment options emerged rapidly and promisingly in the last years in breast cancer, patients acquire resistance quickly in most of the cases. Due to the status of its receptors, the susceptibility to alternative therapy methods, such as hormonal therapy, can be altered. For instance, an Estrogen Receptor (ER) positive cell population can be treated with drugs like Tamoxifen, only until the cells gain resistance and bypass receptor related therapies. In addition, there are different types of breast cancer classifications due to their receptor expressions such as Triple Negative Breast Cancer (TNBC) cells, HER2/ER/PR negative or positive cells. On the other hand, breast cancer possesses p53 mutations lower than other types of cancer according to several statistical data10.Resistance mechanisms may vary between patients and some of the identified mechanisms can be listed as, inactivation of drugs, alterations of drug metabolism, inhibition of cell death, epigenetic altering, amplification or changing of target genes, apoptosis pathway blocking and enhancing DNA damage repairAspergillus Carneus. Aspergillus Carneus is primarily a soil fungus that colonizes mostly in tropical or subtropical terrestrial environments but also found and can grow worldwide16. Recent studies also found the species in marine environments19. Anti-cancer effects of fungi metabolites is a broad area of research and different molecules have been tested in different cancer cell lines including lung fibroblast cells19, pancreatic cancer cells20, liver cancer cells21 and more. Marine fungi secondary metabolites such as the ones in the current study have been previously reported as potential anti-cancer22, anti-microbial or anti-oxidant23 compounds however, there are a large variety of compounds from different species remains to be identified and their effects to be investigated. Aspergillus spp. renders many metabolites, some of which are tested for their cytotoxicity on some tumorigenic cell lines and their respective IC50 values are reported and reviewed in many studies27. There is a study showing anti-tumor growth effects of compound Aspergiolide A isolated from filamentous fungus Aspergillus Glaucus in mice xenograft model27. Bioactive metabolites from the species Aspergillus Carneus has not been extensively studied and reported for their anticancer activity and more importantly, other than its isolation and classification there is no publication to date on the anti-cancer, anti-microbial or anti-oxidant activity of Averufanin which makes this study novel in the area.This study investigated the cytotoxic effects of 5 compounds isolated from filamentous fungus Aspergillus Carneus and tested against human cancer cell lines. We documented that Averufanin was highly cytotoxic towards breast cancer cells and relatively low toxicity on non-tumorigenic cell line. Treatment with Averufanin resulted in DNA damage, cell cycle arrest and apoptotic cell death on breast cancer cell lines by modulating p53 mediated cell signaling pathways. Therefore, Averufanin can be a novel candidate compound for therapeutic strategies in cancer research, especially holding a potential for breast cancer.In this study, 5 chemical compounds were isolated from Aspergillus Carneus species, and their chemical formulas were drawn using ChemDraw is shown in Fig. All chemicals were isolated from Aspergillus Carneus  or DMSO negative controls for 48 h and stained with a DNA dye, propidium iodide (PI). Propidium iodide, which is a DNA-intercalating fluorescent dye, is frequently used for cell cycle analysis. Cells that are in G1 phase of cell cycle or at quiescent state (G0) contain one DNA copy (2N), whereas cells at G2/M phase have double copies of DNA (4N). Moreover, if the cells are in S phase of the cell cycle, they have varying copy number. Healthy cancer cells would have about 60–70% of the cells to be in G1 phase, 20% in S phase. Moreover, G2/m phase cells would comprise 20% of the total cell population. During apoptosis, one of the cellular responses is DNA fragmentation into smaller pieces, which is also visible in a PI Flow Cytometry assay as the “SubG0/G1” phase. Experiment results from this assay showed significant increase in SubG1 phase in cells treated with Averufanin compared to DMSO negative controls on both cell lines  Due to the effect of Averufanin it was captured within 48 h that respective population of cells are detaching from the surface looking spherical. The live, attached portion of cells show no significant morphology changes.To further elucidate the effect of Averufanin on cell cycle, breast cancer cells were treated with Averufanin  is a well-established marker of accumulation of dsDNA breaks. Therefore, human breast cancer cells were treated with Averufanin or DMSO negative control for 48 h and then stained with both DAPI and γ-H2Ax antibody, blue and red respectively. Our results indicated that Averufanin treatment resulted in accumulation of γ-H2Ax positive cells when compared to DMSO negative controls Fig. .Figure 6Based on the previous data on apoptotic induction, initially proteins involved in apoptosis pathway such as PARP, or p53 were analyzed. Western blot analysis on breast cancer cells treated with Averufanin reveled increase in phosphorylation of p53 Fig. A and in Aspergillus Carneus and further focused on the compound Averufanin.In 2020 Breast cancer was the world’s most prevalent cancer and deaths caused by it endures to be at an important rate even with all the treatment options. Therefore, search for novel anti-cancer compounds keeps its importance. One of the natural sources worthy of investigating is fungal species and their metabolites mycotoxins, which are natural protective molecules for fungus itself in their habitats, allowing fungus to toxicant other organisms that may grow in the vicinity and act invasive. This study initially investigated 5 compounds that are metabolites of filamentous fungus 50 concentration than non-tumorigenic cell lines in the panel was the triple negative cell line MDA-MB-231. TNBC is the most lethal subtype of breast cancer because it has high heterogeneity and aggressive nature28, and lack of treatment options still remains an obstacle to overcome. Although TNBC is considered a histological subtype it also has subtypes repeatedly being described by researchers, most recent classification having five members29. This level of heterogeneity makes it harder to emphasize any one single differential pathway or trait and target TNBCs accordingly30. Same reason complicates our discussion for Averufanin’s mechanism of effecting cells. Although the lowest IC50 concentrations come from cell lines positive for one or more receptors among estrogen receptor (ER), progesterone receptor (PR), human epidermal growth receptor-2 (HER-2), and one is tempted to make the deduction of correlating Averufanin with receptor status, this hypothesis needs further investigation and is worth looking into in the future.Averufanin was not only highly cytotoxic for almost all cancer cell lines in our study but also less toxic for non-tumorigenic cell lines MCF12A, HGRC1. Only cell line that had higher ICIn addition to this compliant feature as an anti-cancer agent, it is also novel because it had not been studied in any field, including antioxidant, agriculture, cancer, and toxicity research. Our study shows that it is indeed a candidate for novel anti-cancer compounds however extensive studies in other fields as well can be adjuvant and elucidative. To improve the understanding of the underlying mechanisms of how Averufanin functions there can be additional experiments using different cell lines and methods in the future, allowing the compound to gain recognition and widely obtained, studied and used in the appropriate areas.31. This DNA damage resulted in the dephosphorylation of p-GSKβ in Ser9 residue32, allowing it to be active. Active GSK3β and possibly other DNA damage response elements33 increased p53 levels and phosphorylation of the upregulated tumor suppressor in Ser15 residue34 occurred. Both p-p5335 and GSK3β15 downregulated cell cycle regulatory protein Cyclin D136, delaying passing to the S phase of cells and resulted in a cell cycle arrest at SubG1 and G1 phase, supported by the PI staining experiments. Meanwhile p-p53 and possibly other DNA damage response elements also effected DNA damage repair elements13 (as well as being S phase cell cycle regulatory proteins) Cyclin A2 and CDK2 and their levels elevated. Apoptosis regulator p53 also activated the caspase3/7 cascade38, as shown by the MUSE analysis, resulting in PARP cleavage and programmed cell death, apoptosis. Consequently, our data suggested that Averufanin can be contemplated and further explored as a new therapeutic strategy as an anti-cancer compound, especially for breast cancer.This study covers some of the pathways Averufanin affects in breast cancer cell line T47D. Obtained by the extensive western blot results, Fig. Aspergillus Carneus which was isolated from marine sponge Agelas Oroides previously, was cultivated in rice medium (100 g rice and 110 ml distilled water) that was autoclaved for 20 min at 121 °C before cultivation through 4 weeks. Then, medium was extracted with 500 ml ethyl acetate three times and organic solvent was dried under vacuum at 40 °C. The concentrated crude extract was fractioned between n-hexane and 90% aqueous methanol. Methanolic phase dried under vacuum at 40 °C and the metabolite isolation was continued with concentrated methanolic fraction39.2O to yield compounds Versicolorin C (5 mg), Averufanin (7 mg), Aruogosin C (3 mg), Nidurufin (3 mg) and Averufin (10 mg).The MeOH extract from solid rice medium (1.5 g) was loaded to vacuum liquid chromatography over silica gel, eluted with a gradient of n-hexane–EtOAc (100:0 to 0:100) followed by DCM-MeOH (100:0 to 0:100) to yield 5 fractions (Fr1–Fr9). Fr1 (500 mg) was fractioned using semi-preparative RP-HPLC eluted with a gradient of MeOH-H1H, 13C) were recorded on a Bruker ARX 600. HPLC analyses were performed a Dionex ultimate 300 LC system coupled with the photodiode array detector (UVD340S). LCMSMS Semipreparative HLPC separations were carried out via he LaChrom-Merck Hitachi system  at a flow rate of 5 mL/min. LC–MS spectrums were obtained by using a Thermo Finnigan LCQ Deca mass spectrometer, and HRESIMS spectrums were carried out with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer.The NMR spectrums . 1H NMR : 7.14 , 7.26 , 6.69 , 6.53 , 3.62 , 2.24 , 1.84 , 4.14 , 4.21 . 13C NMR ; 160.1 (C-1), 126.5 (C-2), 166.0 (C-3), 109.0 (C-4), 108.0 (C-5), 164.6 (C-6), 107.5 (C-7), 164.4 (C-8), 108.9 (C-8a), 136.4 (C-5a), 189.1 (C-9), 182.4 (C-10), 107.5 (C-9a), 133.0 (C-10a), 128.1 (C-1′), 39.7 (C-2′), 27.1 (C-3′), 66.7 (C-4′)40.Versicolorin C, red powder; UV spectrum λ max (MeOH): 201.3, 221.1 and 289.0 nm: HRESIMS [M + H] + m/z 371.0776 . 1H NMR : 7.13 , 7.26 , 6.68 , 5.17, , 3.70 , 1.91 , 2.31 , 1.59 13C NMR  159.0 (C-1), 115.2 (C-2), 160.0 (C-3), 109.4 (C-4), 108.0 (C-5), 163.2 (C-6), 105.5 (C-7), 165.0 (C-8), 189.0 (C-9), 183.5 (C-10), 133.0 (C-10a), 135.5 (C-4a), 108.9 (C-8a), 108.5 (C-1a), 80.5 (C-1′) 67.5 (C-2′), 40.2 (C-3′), 25.7 (C-4′), 101.2 (C-5′), 27.0 (C-6′)41.Nidurufin, red powder; UV spectrum λ max (MeOH): 222.6, 267.3 and 292.8 nm: HRESIMS [M + H] + m/z 369.0967 . 1H NMR : 7.14 , 7.26 , 6.69 , 6.53 , 3.62 , 2.24 , 1.84 , 4.14 , 4.21 . 13C NMR ; 160.1 (C-1), 126.5 (C-2), 166.0 (C-3), 109.0 (C-4), 108.0 (C-5), 164.6 (C-6), 107.5 (C-7), 164.4 (C-8), 108.9 (C-8a), 136.4 (C-5a), 189.1 (C-9), 182.4 (C-10), 107.5 (C-9a), 133.0 (C-10a), 128.1 (C-1′), 39.7 (C-2′), 27.1 (C-3′), 66.7 (C-4′)42.Averufin, orange powder; UV spectrum λ max (MeOH): 201.3, 221.1 and 289.0 nm: HRESIMS [M + H] + m/z 371.1125 . 1H NMR : 7.20 , 6.73 , 7.28 , 5.16 , 2.02 , 1.64 , 1.78 , 1.50 , 3.82 , 1.32 . 3C NMR  111.6 (C-4), 109.0 (C-5), 109.6 (C-7), 105.8 (C-1′), 31 (C-2′), 33 (C-3′), 23.2 (C-4′), 76.5 (C-5′), 31 (C-6′)43.Averufanin, orange powder; UV spectrum λ max (MeOH): 222.6, 267.3 and 292.8 nm: HRESIMS [M + H] + m/z 425.1954 . 1H NMR : 1.72 , 1.72 , 1.70 , 7.40 , 6.48 , 6.87 , 4.20 , 4.22 , 2.41 , 5.33 , 1.29 , 1.29 . 13C NMR  18.2 (C-1), 25.0 (C-2), 132.0 (C-3), 122.5 (C-4), 23.0 (C-5), 121.0 (C-6), 133.8 (C-7), 109.1 (C-8), 157.1 (C-9), 112.0 (C-10), 162.5 (C-11), 197.2 (C-12), 151.0 (C-13), 154.2 (C-14), 116.0 (C-15), 133.0 (C-16), 148.5 (C-17), 123.8 (C-18), 60.0 (C-19), 58.5 (C-20), 72.0 (C-21), 65.0 (C-22), 28.0 (C-23), 28.0 (C-24), 17.0 (C-25)44.Arugosin C, red powder; UV spectrum λ max (MeOH): 202.7, 293.3 and 306.4 nm: HRESIMS [M + H] + 2 in the following mediums: OVCAR-3, OVSAHO, Kuramochi in RPMI-1640 with 10% Fetal Bovine Serum and 1% penicillin–streptomycin, MCF7, T47D, SK-BR-3, MDA-MB-231 with DMEM High Glucose with 10% Fetal Bovine Serum and 1% penicillin–streptomycin, HGRC1 with DMEM F12 Ham’s with 10% Fetal Bovine Serum and 1% penicillin–streptomycin, MCF12A with DMEM F12 Ham’s with 10% Fetal Bovine Serum, 1% penicillin–streptomycin, 10 ng/ml EGF, 500 ng/ml Hydrocortisone, 10 µg/ml Insulin.Cells were cultured routinely at 37 °C under 5% CO45. DMSO was used as a negative control. The experiment was performed in triplicates.Cells were inoculated in 96-well plates (1000–5000 cell/well) and grown for 24 h before being treated with increasing concentrations of the compounds (0.2–100 µM or 0.1–50 µM). After 72 h and 48 h of incubation, cells were fixed with 10% (v/v) trichloroacetic acid for 1 h at 4 °C. Then plates were washed five times with deionized water and left to dry. Next day, cells were stained with 0.4% (m/v) of Sulforhodamine B in 1% acetic acid solution in the dark at room temperature for 30 min. The plates washed five times with 1% acetic acid before air-drying. Bound SRB was dissolved in 10 mM Tris-base solution. The absorbance was read in a plate reader at 515 nm5 cells) into flat bottom 6-well plates and treated with both IC50 and IC75 concentrations. After 48 h treatment cells were collected, washed with ice-cold DPBS and fixed with ethanol (70%) with constant gentle vortexing. Then cells were centrifuged and resuspended in PI solution (50 µg/ml propidium iodide (Sigma Aldrich), 0.1 mg/ml RNAseA and 0.05% TritonX-100 in ice-cold DPBS) and incubated at 37 °C for 40 min. Samples were washed with DPBS and re-suspended. Flow Cytometry (CytoFLEX) was used for analysis.In order to investigate the effect of the compound on cell cycle, cells were plated (1 × 105 cell/well), and then cells were grown for 24 h and treated with IC50 concentrations of the compound and DMSO control. After 72 h treatment, cells were collected and diluted with 1% FBS/PBS solution to 100/500 cell/µl. 75 µl cell + 75 µl Annexin V dye  were incubated at room temperature in the dark for 20 min before analyzing with MUSE Cell Analyzer46.6-well plates were used for cell inoculation (1 × 105 cell/well) and grown for 24 h and treated with IC75 concentrations of the corresponding compound and DMSO controls. After 48 h treatment, cells were collected and diluted with 1% FBS/PBS solution to 300–500 cell/µl. Results were analyzed using MUSE Caspase 3/7 kit according to the manufacturer’s protocol47.Human breast cancer cells were plated in 6-well plates (1 × 105 cells/well) 24 h later they were treated with IC50, IC75 concentrations of the compound or DMSO control for 48 h. The medium was removed and coverslip was washed with DPBS three times. 4% Paraformaldehyde in 1× DPBS solution was given to each well to enough to cover the surface. It was incubated for 20 min in RT in the dark before DBPS wash for three times again. Later 0.1% Triton-X 100 DPBS solution was given to cells for 15 min in RT in the dark. The permeabilization step was followed by a wash step with 0.1% Tween 20 DPBS solution. Blocking was performed with 5% Blotting Grade Blocker in DPBS for 30 min in RT. Coverslips were incubated with the primary antibody γH2Ax  overnight at 4 °C, in 1:100 dilution with DPBS. Next day, secondary antibody incubation with Alexa Flour 594 was applied for 2 h in RT. (1:100 dilution). Mounting medium containing DAPI (Vectashield) was applied to slides and coverslips were left to dry overnight at RT in the dark. Next day, coverslips were fixed on the slides with varnish and Confocal Microscopy  was used to visualize the samples. Subvolume sizes of x, y dimensions for control (580.68 µm × 1160.8 µm), IC50 (870.74 µm × 580.68 µm) and IC75 (870.74 µm × 870.74 µm) were gathered and different crop regions were cut to analyze with ImageJ and GraphPad PRISM.In order to investigate the effect of Averufanin on DNA damage, cells were inoculated in 6-well plates with a coverslip in the bottom. (1.5 × 106 cells/dish) for 24 h and treated with IC50, IC75 concentrations of the compounds or DMSO control for 48 h. Pellets were collected and lysed with 1× RIPA Lysis Buffer, 10% Proteinase Inhibitor and 1% Phosphatase Inhibitor. Lysates were incubated on ice for 45 min, vortexing every 15 min. Samples were centrifuged in 13000×G for 15 min. The protein concentration of the lysates was determined with the BCA assay . Cell lysates containing 20 µg protein were mixed with 4X Laemmli Sample Buffer, 1/6 β-Mercaptoethanol and denatured for 6 min in 95 °C. Samples were loaded to BIORAD Precast gels of 15 wells (4–15%). After electrophoresis, the proteins were transferred to PVDF membranes (BIORAD) with Semidry Transfer System (BIORAD), followed by overnight incubation in blocking solution (5% Blotting Grade Blocker in 1× TBS-T (0.2% tween)). PARP , p53 , Phospho-p53 (Ser15) , GSK3β , phosphor-GSK3β (Ser9) , CyclinD1 , Cyclin A2 , CDK2  primary antibodies were used in a ratio of 1:500 to 1:1000 in 5% BSA-TBS-T, incubated for 1 h in room temperature. Secondary antibodies, anti-rabbit (Sigma Aldrich), anti-mouse (abcam), were used in 1:2500 dilutions in 5%Blotting Grade Blocker-TBS-T for 1 h at room temperature. β-Actin (abcam), Vinculin  or GAPDH (abcam) primary antibody for equal loading analysis was used in 1:1000 dilution in 5% Blotting Grade Blocker in TBS-T for 1 h at room temperature. For visualization of the results, chemiluminescence was performed with ECL. The membranes were visualized using LI-COR Fc. Sample bands were visualized with Chemi light exposed for 10 min and 700 light exposed for 1 min for the protein standards (ladder). All proteins were run in the same gel with their loading controls and only physical application was vertical cropping. All blots were evenly treated with brightness and contrast modifications (mostly + %20 Contrast and + %20 brightness of Office Powerpoint was applied.) and no bands were exaggerated or lost during the processing. To quantitatively analyze the band intensities, ImageJ was uses, statistical analysis were done with GraphPad PRISM.Cells were cultured in 15-cm culture dishes  p < 0.05, (**) p < 0.01, (***) p < 0.001 and (****) p < 0.0001.Supplementary Information.


We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells  largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case–control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCR Psoriasis vulgaris (PV) is a debilitating autoimmune dermatosis with a complex etiology and lifelong duration. Psoriatic arthritis, diabetes, and cardiovascular disorders often accompany the skin manifestations, making PV a systemic and highly polymorphic condition ,2,3,4,5.+EOMES+IFNG+) , and 14 sex- and age-matched, unrelated healthy controls  surface markers. Dead cells were excluded using LIVE /DEAD Fixable Near IR Dead fluorescent viability dye  and unspecific antibody binding was prevented by a 10 min pre-staining incubation with the 5% FcR blocking reagent . The γδT cell-count acquisition was performed with the use of the BD FACS Canto II cytometer  and the collected data were analyzed with FlowLogic v7.2.1. software . The gating strategy for peripheral γδT cell populations  was set Before being processed into cDNA, cryopreserved total RNA samples were thawed and the available quantities were measured using the DeNovix QFX Fluorometer . Reverse transcription (RT) of four candidate  and three control miRNAs  was carried out in four sequential steps using the TaqMan Advanced miRNA cDNA Synthesis kit . In short, for initial 3′ poly-A tailing, 5 ng of total RNA was incubated in 5 µL of Poly(A) reaction mixture (45 min at 37 °C) and the 5′ ligation of an adaptor sequence in a 15 µL ligation blend (60 min at 16 °C) was performed to extend all mature miRNAs present, prior to cDNA synthesis. Next, the extended miRNAs were reverse transcribed (15 min at 42 °C) in a 15 µL RT reaction mix composed of 6 µL of 5X RT buffer, 1.2 µL of dNTP mix, 1.5 µL of 20Xuniversal RT primers, 3 µL of 10X RT enzyme mix, and 3.3 µL of RNase-free water. In order to improve the detection of low-expressing miRNA targets while maintaining their relative differential expression levels, 5 µL of each cDNA sample was pre-amplified with 2.5 µL of Universal miR-Amp Primers and 25 µL of miR-Amp Master Mix. The pre-amplified cDNA products were diluted fivefold and the transcript levels of target miRNAs were measured using the QuantStudio 5 Real-Time instrument  in triplicate 15 µL quantitative real-time PCR (qRT-PCR) reactions containing 6.75 µL of the cDNA template, 7.5 µL of TaqMan Fast Advanced Master Mix, and 0.75 µL of TaqMan Advanced miRNA Assay . The cycling conditions were set according to the guidelines in the manufacturer’s leaflet and the list of assays is given in 2), were measured by five-point, fourfold serial dilutions of the arbitrary standards that were run next to the samples in each experiment, providing an insight into the final achieved ranges of efficiency (80–100%) and R2 (0.980–0.998). Intra-assay variability was less than 1.96%, and a variation of less than 2.34% was achieved between different PCR experiments. Among the three tested control miRNAs, only the hsa-miR-423-3p was successfully amplified in our sample set and thus used for the NormFinder stability evaluation (M = 2.098) and normalization of target miRNAs expression levels. Finally, the fold difference in the relative miRNA quantity was determined with respect to the control group levels, using the efficiency corrected model of the 2−ΔΔCt method as described by Pfaffl [EOMES, RUNX3, TBX21, RORC, CCR6, ZBTB16, SELPLG, and IL18R was performed as reported earlier [The threshold cycle (Ct) values were collected using QuantStudio Design&Analysis software, v1.5.2. Amplification efficiency and pipetting precision, as assessed by the linear regression coefficient . For this analysis, we restricted ourselves to gene–gene cross-correlations by adopting the harmonized TPM (transcript-per-million) data from UCSC Xena [The processed single-cell (sc)RNASeq data from Tan et al.  were useCSC Xena .Gaussianity was assessed by the Shapiro–Wilk test, and the homogeneity of variances by Levene’s test. Generally, a nonparametric approach was adopted. Where possible, an equal allocation design was used to maximize statistical power. Continuous data is presented as median with the interquartile range (IQR), except for stacked barplots, where arithmetic means were utilized, because the sum of group-level medians does not readily converge on the grand median. This choice did not significantly affect our results. For downstream analysis, serum CMV IgG levels were winsorized at the upper limit of quantification (250 IU/mL). As most subjects were CMV-experienced, we also explored the effect of past CMV exposure by dividing the sample into two equal subgroups using median CMV IgG quantity. The Mann–Whitney U-test was used for independent group comparisons and the Fisher’s exact test was applied to contingency tables. Pairwise correlations were assessed by the Spearman’s rank test. Shapley’s additive explanations , represep < 0.05 was considered significant. No adjustment for multiple testing was applied. All statistical analyses were performed in R v4.0.3 . The boxplots, barplots, scatterplots, and ternary maps were generated using R-package cowplot v1.1.0, ggplot v3.3.5, ggpubr v0.4.0, ggtern v3.3.5, patchwork v1.1.1, RColorBrewer v1.1.2, reshape2 v1.4.4, rstatix v0.7.0, scales v1.1.1, Ternary v2.1.0, tidyverse v1.3.0, viridis v0.5.1, viridisLite v0.3.0, and xgboost v1.3.2.1.If not otherwise stated, a two-tailed


H-pyrrole-2,5-dicarboxylic acid (1) and one known pyrrole acid (2) were isolated from the fruits of Phyllanthus emblica. The structures of these compounds were elucidated via the comprehensive analyses of IR, HRESIMS, 1D and 2D spectroscopic data. A series of biological assays revealed that compounds 1 and 2 could inhibit LPS-induced over-production of nitric oxide (NO), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor-α (TNF-α) by reducing the phosphorylation of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK) in RAW 264.7 cells. Additionally, compounds 1 and 2 were found to reduce lipid deposition and increase the mRNA expression of ATP-binding cassette transporter A1 in oxidized low-density lipoprotein-treated RAW264.7 macrophages.An undescribed pyrrole acid, 1-(4′-methoxy-4′-oxobutyl)-1The online version contains supplementary material available at 10.1007/s13659-023-00393-0. Atherosclerosis (AS), a chronic inflammatory disease triggered by the accumulation of cholesterol-containing low-density lipoprotein (LDL) particles on the arterial wall, is the principal contributing factor to many cardiovascular diseases . OxidizePhyllanthus emblica L., mainly distributed in subtropical and tropical parts of southeast Asia, has been used for thousands of years in traditional Chinese and Indian medicine for treating diseases such as hemorrhage, diarrhea, jaundice, and dyspepsia etc. , three methylene groups , and a methoxy group [δH 3.56 ]. The 13 C NMR and the DEPT spectra showed 8 carbon signals assigned to one methyl (δC 52.2), three methylene , one methine (δC 118.1) and three quaternary carbons . These data suggested that 1 possessed a 1H-pyrrole-2,5-dicarboxylic acid (2) moiety by comparison of these data with those reported in the literature  + .1-(4′-methoxy-4′-oxobutyl)-1oC in a humidified atmosphere of 5% CO2. According to experiments, cells were randomly divided into three groups, including control (vehicle DMSO); LPS/Ox-LDL treated; LPS/Ox-LDL and compounds 1/2/PE treated. After 24 h of incubation, cells were subjected to compounds 1, 2 and PE extract respectively for 2 h followed by treatment with LPS (500 ng/mL) or Ox-LDL (50 µg/mL) for 24 h. Subsequently, cells were harvested and extracted for further analysis.The RAW 264.7 cell line was purchased from the School of Basic Medicine of Peking Union Medical College . Cells were incubated in Dulbeco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/mL penicillin and streptomycin at 37 5 cells/mL) for 24 h, RAW 264.7 cells were treated with compounds 1 and 2 at different concentrations (50 µM and 100 µM)) respectively for 24 h. Then, RAW 264.7 cells were incubated in 0.5 mg/mL MTT at 37 °C for 4 h. The MTT-containing supernatant was removed after centrifugation. Each well was treated with DMSO (100 µL) at 37 ℃ for 10 min. Subsequently, absorbance values were measured at 450 nm using a microplate reader .After incubated in 96-well culture plates  or PE (500 µg/mL) for 2 h, and then treated with LPS (500 ng/mL) for 24 h. The culture medium was separated and collected the supernatant. NO concentration in the supernatant was measured by Griess assay. The absorbance of NO concentration was determined by a microplate reader at 540 nm. The content of IL-6, TNF-α and MCP-1 levels in the supernatant was quantified by enzyme linked immunosorbent assay (ELISA) kits  according to the manufacturer’s protocols.Cells were administrated with compounds 1, 2 (100 µM) or PE (500 µg/mL) for 2 h, the cells were stimulated by LPS (500 ng/mL) for 24 h. Then, cells were washed twice by using ice-cold phosphate buffer saline  and lysed in whole-cell lysis buffer on ice to yield the cellular proteins. The protein concentration was quantified according to the bicinchoninic acid (BCA) method. Aliquots containing 20 µg of protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride membranes . The membranes were blocked with 5% skim milk in Tri-buffered saline tween (TBST) for 1 h at room temperature. To detect the target proteins, the PVDF membranes were incubated with primary antibodies that were specific to the phospho-extracellular regulated protein kinases, extracellular regulated protein kinases, phospho-c-Jun N-terminal kinase, c-Jun N-terminal kinase and β-actin  overnight at 4 °C. Sequentially, the membranes were rinsed three times with TBST incubated for 1 h with horseradish peroxidase-conjugated secondary antibody . After subsequent washes with TBST and deionized water, membrane bound antibodies were detected using a chemiluminescence (ECL) system  and band intensities were quantified using imaging systems analysis software. .After cells were treated with compounds 1, 2 (100 µM) and PE (500 µg/mL) for 2 h previously, were stimulated by Ox-LDL (50 µg/mL) for 24 h. After being rinsed once with PBS, the cells were fixed with 10% (v/v) paraformaldehyde for 15 min. Then, cells were washed with double-distilled water (ddH2O) and stained with filter 0.5% Oil Red O solution at room temperature for another 15 min. Subsequently, Mayer’s hematoxylin was added to restrain the nucleus for 2 min. Finally, after further washing the cells with ddH2O to remove the background staining, the cells were photographed randomly under the microscope.RAW264.7 cells, which had been pre-treated with compounds 1, 2 (100 µM) or PE (500 µg/mL) for 2 h, were stimulated by Ox-LDL (50µg/mL) for 24 h. Then, cells were extracted with chloroform: isopropanol: IGEPAL CA-630 (7:11:0.1) in a micro homogenizer. Cells were centrifuged to remove insoluble material. The organic phase was transferred and dried at 50 °C to remove chloroform, before being dissolved dried lipids with the cholesterol assay buffer. The content of cellular total cholesterol (TC) and free cholesterol (FC) was determined using a cholesteryl assay kit . The cholesteryl ester (CE) was calculated as follow: CE = TC–FC.Cells, which had been previously treated with compounds 1, 2 (100 µM) or PE (500 µg/mL) for 2 h, were stimulated by Ox-LDL (50µg/mL) for 24 h. According to the manufacturer’s instructions, total RNA was isolated using TRIzol reagent . The concentration of RNA was measured by a spectrophotometer, and the cells with an OD value (A260/A280) between 1.8-2.0 were reverse transcribed into cDNA using the synthesis kits . Quantitative RT-PCR was performed using a QuantiFast SYBR® Green PCR Kit (Roche) and ABI PRISM 7300 PCR Machine (Applied Biosystems). The cDNA was amplified at 90°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 60 s. Final extension was performed for 7 min at 72°C. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. PCR primers were as follows: ABCA1, 5’-GCAGATCA AGCATCCCAAC T-3’ (forward) and 5’-CCAGAGAATGT TTCATTGTCCA-3’ (reverse).Cells, which had been previously treated with compounds t-test were used by SPSS20 , respectively. The results were considered as significantly different at p < 0.05 and that of p < 0.01 was taken to be extremely significant.The results are expressed as a mean ± standard deviation (SD) from at least three independent experiments. Statistical elaborations were performed on GraphPad Prism 7 . For the statistical comparison of means difference between the samples, the Student’s 1 and 2) were isolated and characterized from the fruits of P. emblica. These compounds were identified as 1-(4-methoxy-4-oxobutyl)-1 H-pyrrole-2,5-dicarboxylic acid and 1 H-pyrrole-2,5-dicarboxylic acid through various spectroscopic data. Pharmacological experiments showed that compounds 1 and 2 inhibited the production of LPS-induced NO, IL-6, TNF-α and MCP-1 in RAW264.7 cells, and that these effects were mediated by the ERK and JNK pathway. Moreover, compounds 1 and 2 effectively reduced lipid deposition induced by Ox-LDL in macrophages. These findings indicate that compounds 1 and 2 may be effective in inhibiting the progression of AS and have potential for the prevention and treatment of AS.In conclusion, two pyrrole acids (Additional file 1. Additional information.


The seven-membered 1,3-thiazepan-4-one ring system, like its six- and five-membered-ring analogues, is known to be biologically active and is of potential medicinal use. The crystal structure reported is of the sulfone of a 1,3-thiazepan-4-one in which the seven-membered ring adopts a pucker-chair conformation. 19H19NO3S, crystallizes in space group Pb-axis direction. One C—H grouping of the cyclopropyl ring is in close contact with the phenyl ring of the neighboring independent molecule in C—H⋯π type interactions with carbon atom–ring-centroid distances of 3.544 (5) and 3.596 (4) Å. Other interactions are of the parallel–reciprocal type, with the chiral carbon atom of one molecule donating a proton to an oxygen atom of the sulfone group of a symmetry-related molecule and vice-versa. Symmetry-related molecular pairs also exhibit T-type interactions between aromatic rings with interplanar angles of 74.2 (2) and 69.2 (2)° and intercentroid distances of 4.965 (4) and 5.114 (4) Å.The racemic mixture of the title compound, C We reported similar puckering of the 1,3-thiazepan-4-one ring previously  Å, C—H⋯O = 161°] and C24—H24A⋯O3 [3.413 (5) Å and 165°], wherein the carbonyl oxygen atom of one molecule accepts a C—H grouping of the heterocycle of another, form a chain of alternating crystallographically independent molecules along the b axis direction. Independent neighbors along the . Crystals were grown by slow evaporation of an ethanol solution.6,7-Diphenyl-5-thia-7-azaspiro[2.6]nonan-8-one Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623009379/hb4455sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623009379/hb4455Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623009379/hb4455Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2414314623009379/hb4455Isup4.cmlSupporting information file. DOI: 2303670CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Leukemia 10.1038/s41375-023-01973-2, published online 20 July 2023Correction to: In the original published online version, “<1.08 × 10E+7” in row #5 of Table


In-depth structural characterization of lipids provides a new means to investigate lipid metabolism. In this study, we have conducted deep profiling of total fatty acids (FAs) from RAW 264.7 macrophages by utilizing charge-tagging Paternò-Büchi derivatization of carbon-carbon double bond (C=C) and reversed-phase liquid chromatography-tandem mass spectrometry. A series of FAs exhibiting unusual site(s) of unsaturation was unearthed, with their identities being confirmed by observing anticipated compositional alterations upon desaturase inhibition. The data reveal that FADS2  NotablyFA 16:0) , which iFA 16:0) ; howevern lipids . The stun lipids , 15. In n lipids , 18. Iden lipids , 18, whin lipids . Alternan lipids , ultravin lipids , 22, epon lipids , 24, azin lipids , 26, andn lipids , 30, 31,In this work, we employ charge-tagging PB derivatization in combination with desaturate inhibition to uncover a diverse set of novel fatty acids with an unusual site(s) of unsaturation in RAW 264.7 macrophages. The latter method allows us to achieve high confidence in the identification even in the absence of authentic standards. Furthermore, desaturate inhibition provides evidence for studying FAs derived from SCD1 Δ9-desaturation and FADS2 Δ6-desaturation activities. Our data suggest that both enzymes display broad activity toward both even and odd-chain saturated FAs and give the cells access to an expanded repertoire of even-chain MUFAs with n-5, n-7, n-9, n-10, and n-12 C=Cs. Furthermore, even-chain MUFAs derived from these de novo synthesized families also act as substrates for further desaturation and elongation, yielding a wide array of PUFAs, consisting of the n-7, n-9, n-10, and n-12 families.n-x nomenclature, counting from the methyl terminus. The Z/E stereo-configurations of C=C could not be assigned from PB-MS/MS and thus were not indicated for FAs from biological samples.The shorthand notation recommended by LIPID MAPS consortium was used . In brieHigh-performance LC (HPLC) grade iso-propanol (IPA), acetonitrile (ACN), methanol (MeOH), and water were purchased from Fisher Scientific. Methyl tert-butyl ether (MTBE), formic acid (HCOOH), ammonium formate, and 2-acetylpyridine (2-acpy) were purchased from Sigma Aldrich. Lipid standards were purchased from Avanti Polar Lipids. AMP+ MaxSpec Kit was purchased from Cayman Chemical.RAW 264.7 macrophages (American Type Culture Collection (ATCC); Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and collected by centrifugation. The cell pellets were washed, frozen in liquid nitrogen, and stored at −80°C. For direct inhibition of enzyme activity, RAW 264.7 macrophages were treated for 72 h with 60 μM FADS2 inhibitor  and 2.5 μM SCD1 inhibitor , while the control group was treated with DMSO.g for 10 min. The upper phase was collected. The lower phase was re-extracted with 2 ml of the upper phase of the system MTBE/MeOH/H2O . Finally, the combined organic phases were collected and dried under N2 flow for further use. The cell medium was also subjected to lipid extraction and total FA profiling. The extracted lipids were saponified in 500 μl ACN:15% KOH  at 60°C for 60 min. The solution was acidified with 1M HCl (1 ml). The hydrolyzed total FAs were extracted twice with 1.5 ml isooctane each time. The organic layer was collected, dried under nitrogen, and redissolved in an aliquot of 500 μl MeOH for further derivatization.The cell samples were extracted using a modified MTBE protocol . In brieAMPP derivatization followed the procedure provided by the vendor (AMP+ MaxSpec Kit). AMPP derivatized sample was extracted twice by 600 μl MTBE and dried under a nitrogen stream. The derivatized sample was resuspended in 100 μl MeOH for RPLC-MS/MS analysis.The 2-acpy PB derivatization was performed using a home-made flow microreactor . Total F2O:ACN  and mobile phase B contained IPA:ACN . The flow rate was set at 0.5 ml/min. Oven temperature was set at 60°C. The chromatographic gradient was as follows: 30% B at 0–0.4 min, 30%–45% B at 0.4–0.9 min, 45%–52% B at 0.9–1.1 min, 52%–58% B at 1.1–1.8 min, 58%–66% B at 1.8–2.5 min, 66%–70% B at 2.5–3.1 min, 70%–75% B at 3.1–4 min, 75%–97% B at 4–4.5 min, 97% B at 4.5–6 min, 30% B at 6.1–7 min. The MS parameters were optimized as follows: capillary voltage, 4500 V; end plate voltage, 500 V; nebulizer, 3 Bar; dry gas, 10 L/min; dry temperature, 210°C; CID energy for MS/MS, 22 eV.RPLC-MS/MS analyses were conducted on an Acquity UPLC I-Class system (Waters) hyphenated with a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer . A CORTECS UPLC C18 column  was used for separation. The mobile phase A contained H1 spectra of AMPP-derivatized fatty acid and corresponding internal standards were imported into the LipidNovelist Extension and subjected to Type-I isotope correction. The most up-to-date version of LipidNovelist and LipidNovelist Extension, along with instructional videos and example data to facilitate its utilization, can be accessed at https://doi.org/10.6084/m9.figshare.22297771.The analysis of RPLC-PB-MS/MS data was carried out using a home-made software, LipidNovelist 1 data which provided relative quantification (I/IIS) at the sum composition level 1 signal to that of all FAs detected in the same RPLC run. Type-I isotope correction was applied to the data prior to relative quanaitation. The associate data are provided in RAW 264.7 cells have been extensively characterized and are well-suited for lipid metabolism studies due to their expression of a wide range of fatty acid desaturation enzymes involved in lipid metabolism , 37, 38.Then, we used 2-acpy derivatization (the PB reaction) followed by RPLC-MS/MS to achieve relative quantitation of unsaturated total FAs at the C=C location level . The PB-A total of 29 groups of unsaturated FA and 9 groups of saturated FA were identified at the sum composition level, covering long-chain (carbon number: 14–21) and very-long-chain FAs (carbon number >21) with up to 6 C=Cs. Our data A showed The retention time (RT) values of AMPP-derivatized FAs were plotted as a function of carbon chain length (14−24) and the number of C=C (0−6) . We clean-xfo and n-xFo, generic structures shown in Since the odd-chain MUFAs and their C=C location isomers have been rarely characterized, we focused our attention to analyze the odd-chain MUFAs. P = 1.6×10−4). These data corroborate that SCD1 is involved in de novo synthesis of these n-8 isomers. However, the %relative composition of FA 21:1 (n-8) showed an increase after SCD1 inhibition . Because FA 21:1(n-8) is the single component found in the culture medium for FA 21:1 , after the inhibition of SCD1 B. It is :1(n-10) . The smaAfter FADS2 inhibition, the relative compositions of the n-11 isomers of the odd-chain MUFAs decreased significantly B, such aAs discussed earlier, FA 17:1 only accounted for 0.43 ± 0.03% of the total FAs in RAW 264.7 cells at the sum composition level, while enzymatic inhibition showed small disturbances to its relative abundance, eg, 0.33 ± 0.03% in the SCD1 inhibition group and 0.47 ± 0.04% in the FADS2 inhibition group A. The anP <0.001, N = 6)  B. The pr2 CID spectrum of FA 18:2. Based on detecting the C=C diagnostic ions (n-xfo and n-xFo), FA 18:2 was identified as a mixture of FA 18:2  (44.2 ± 0.8%), FA 18:2  (29.8 ± 0.5%), FA 18:2 (7.2 ± 0.6%), and FA 18:2 (18.9 ± 0.3%). The presence of FA 18:2  indicates that FADS2 catalyzes Δ8-desaturation of FA 18:1(n-7) to form FA 18:2. Further chain elongation and Δ5-desaturation of FA 18:2 contribute to the n-7 PUFAs identified in FA 20:2, FA 22:2, FA 18:3, and FA 20:3 (P = 3.7×10−11) (P = 9.7×10−12)  B. Simila7×10−12) B. A simi7×10−12) CFig. 6A:P =1.5×10−4) and 3.3 ± 1.1% after FADS2 inhibition (P =1.9×10−5)  indicates that FADS2 catalyzes Δ6-desaturation of FA 18:1(n-9) to form FA 18:2. Furthermore, the n-9 isomer was found to be the most dominant form in FA 20:3 (73.9 ± 0.5%), which is an indicator of essential fatty acid deficiency . Previou2, n-15) . An inhi.9×10−5) B. A simi.9×10−5) .Likewise, the presence of FA 18:2 indicates the possibility for Δ5-desaturation of FA 18:1(n-10) by FADS1, which highlights a point of divergence in enzymatic activity surrounding the FA 18:1(n-10). The subsequent elongation of FA 18:2 results in the n-10 PUFAs, including FA 20:2 and FA 22:2. A similar enzyme branching point can be observed for FA 20:1(n-12), whereby FADS1 mediates Δ5-desaturation towards FA 20:1(n-12) 2, n-15) C.P <0.001, N = 6), except for FA 18:3 and FA 24:5 , FA 20:5 (100 ± 0%), FA 22:5 (100 ± 0%) and FA 22:6 (100 ± 0%), and the n-6 isomers dominated in FA 18:2 (87.5 ± 0.8%), FA 20:3 (75.3 ± 0.8%), FA 20:4 (92.9 ± 0.1%), and 22:4 (64 ± 4%)  and S4. 5, n-18) C. Intere5, n-18) . We thus5, n-18) . This ph5, n-18) .Fig. 7BiP = 2.3×10−12). In the control group, FA 24:4 only contained the n-6 isomer (100%), likely originating from sequential elongation of the n-6 isomer of FA 20:4 (97.2 ± 1.4%) to FA 22:4 (85 ± 3%). The n-6 isomer of FA 24:5 was observed as the only component (100%) in the control and SCD1-inhibition groups but absent in the FADS2-inhibition group and culture medium  to FA 18:3 and convert FA 24:4  to FA 24:5  by catalyzing the desaturation at the Δ6 position . Thus, ae medium . These dNote that the Δ6-desaturase activity of FADS2 was greatly increased by SCD1 inhibition in the conversion of FA 24:3  to FA 24:4 . %Relative composition of FA 24:4  increased from 0% in control to 100% in after SCD1 inhibition. These results also confirm that SCD1 inhibition consolidates FADS2 activity toward PUFA. The biosynthetic pathways of PUFA observed in RAW cells are proposed in 2 CID  (72.2 ± 0.4%) and FA 24:2  (100%) . The str2 CID B. Similaer cells . Non-meter cells . De novoer cells .Fig. 8MSThe exploration of FA C=C isomers within RAW 264.7 cell lipidome in combination with enzyme inhibition reveals that cellular fatty acid desaturation is far more complex and dynamic than previously considered. The biosynthesis pathways of the odd-chain MUFAs, even-chain MUFAs, and PUFAs are proposed based on the experimental observations. This expanded network of FA desaturation provides plasticity to generate certain FA families arising from extreme metabolic stress. For instance, the FADS2 plasticity allows for multiple substrate acceptances (e.g., FADS2 via OzID . In concAll data concerned with this study are presented within this manuscript.This article contains The authors declare no conflicts of interest.


T was isolated from birch (Betula pendula) pollen at Giessen area, Germany. The analysis of 16S rRNA gene-based phylogenies indicated the next-relative genera were Robbsia, Chitinasiproducens, Pararobbsia and Paraburkholderia (96–95.6%). Further comparative genome analysis and phylogenetic tree-based methods revealed its phylogenetic position under the genus Robbsia. The genome of strain Bb-Pol-6 T was 5.04 Mbp with 4401 predicted coding sequences and a G + C content of 65.31 mol%. Average amino acid identity, average nucleotide identity, digital DNA–DNA hybridization and percentage of conserved proteins values to Robbsia andropogonis DSM 9511 T were 68.0, 72.5, 22.7 and 65.85%, respectively. Strain Bb-Pol-6 T was rod-shaped, non-motile, facultative anaerobic and grew optimally at 28 °C and pH 6–7. Ubiquinone 8 was the major respiratory quinone and the major cellular fatty acids were C16:0, C19:0 cycloω7c, C17:0 cycloω7c and C17:1ω6c. The dominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. Based on the genomic physiological and phenotypic characteristics, strain Bb-Pol-6 T was considered a novel species under the genus Robbsia, for which the name Robbsia betulipollinis sp. nov. was proposed. The type strain is Bb-Pol-6 T (= LMG 32774 T = DSM 114812 T).One gram-negative strain designated Bb-Pol-6The online version contains supplementary material available at 10.1007/s00284-023-03344-7. Robbsia belongs to the class Betaproteobacteria [Burkholderiaceae [Robbsia andropogonis, was isolated from sorghum with strip disease and first described by Smith as Bacterium andropogoni [T [R. andropogonis has been reclassified several times into the genus Pseudomonas [Burkholderia [Paraburkholderia [Robbsia proposed by Lopes-Santos et al. [R. andropogonis strains possess a single polar sheathed flagellum [Betula pendula) pollen designated as Bb-Pol-6 T. Based on genomic and phenotypic characterization, the strain is proposed to represent a novel species within the genus Robbsia.The monotypic genus bacteria  and the eriaceae , which heriaceae . The phyropogoni . The DNAropogoni , and is ogoni [T . After iudomonas , Burkholholderia , Paraburholderia , and eves et al. . It is as et al. –12. Infes et al. . Distinclagellum  and are lagellum . In this4P2O7, and serially diluted with 0.02% (v/v) Tween 80 and 0.085% (w/v) NaCl to 10–5. Dilution was plated on 1:10 AC agar medium (Sigma Aldrich), and aerobically incubated at 25 °C for one week. Strain Bb-Pol-6 T was isolated by subculturing in the same medium and the purity was confirmed under light microscope. The pure culture was stored in 20% glycerol at −80 °C.The pollen sample from a birch was collected from the Giessen area, Hesse state, Germany (50° 34′ 10.755″ N 8° 40′ 17.859″ E) in February 2016. The details of sample collection and strain isolation procedure were carried out according to Ambika Manirajan et al. . In brie T was extracted from a late-log phase axenic culture according to Pitcher et al. [ T were identified by searching against the quality-controlled 16S rRNA sequence database in EzBioCloud [Genomic DNA of strain Bb-Pol-6r et al. . The 16Sr et al.  and 1512r et al. , and seqr et al. . The retr et al. . Next reBioCloud  and Genb T was aligned with related taxa and merged with the pre-aligned 16S rRNA gene database LPT_12_2021 (February 2021) [Escherichia coli numbering) [The analysis of 16S rRNA-based phylogenies was performed in ARB (version 7.0)  and the ry 2021) . Phylogembering) . Maximum T genome and the genome of the reference strain (GCA_902833845.1) was performed using formula 2 of the Genome-to-Genome Distance Calculator 3.0 with the recommended BLAST + alignment tool (https://ggdc.dsmz.de/ggdc.php) [Extracted genomic DNA was further sequenced with the Illumina Miseq V3 2 × 300 bp) service from LGC Genomics. The draft genome assembly was performed using SPAdes 3.15.4  and open0 bp servgdc.php) . Comparagdc.php)  with def GCA_9028845.1 wasgdc.php) . Plant igdc.php) . T were determined together with the reference strain R. andropogonis DSM 9511 T. The Gram-staining was conducted as described in Gerhardt et al. [2O2 and oxidase test strips (Roth), respectively. Bacterial growth was tested on different media including R2A agar (Roth), Luria–Bertani agar , trypticase soy agar , nutrient agar , MacConkey agar (Merck) and AC 1:10 agar (Sigma). The optimal growth conditions were assessed by incubation in AC 1:10 broth at a range of temperatures , pH  and salinity . The broth pH-value was adjusted by 0.2 M Na2HPO4/0.4 M citrate for pH 2.0–8.0, and 0.2 M glycine/0.4 M NaOH for pH 8.5–11.0 with slight modification as described by Lin et al. [ T and R. andropogonis DSM 9511 T were grown for five and three days respectively, except for inoculants at 4 °C, which were grown for two weeks. Strain Bb-Pol-6 T was incubated on AC 1:10 agar medium anaerobically using Anaerocult A system (Merck) to examine its oxygen requirement. Hypersensitive response was examined on tobacco (Nicotiana tabacum cv. Xanthi) and birch (Betula pendula) according to Klement and Goodman [ T is a slow-grower, the incubation time of assimilation tests on API 20 NE and API 50 CH strips for strain Bb-Pol-6 T was extended to five days for a clear result. The assimilation of acetate, succinate, malonate and propionate that are not involved in API system was also tested on API AUX medium.Physiological characteristics of strain Bb-Pol-6t et al. . Morpholt et al.  while cen et al. . Before  Goodman . Rhizobi Goodman . Biochem T and the reference strain were analyzed using corresponding DSMZ services. Late-log phase bacterial culture in AC 1:10 medium was harvested and freeze dried. Cellular fatty acids were converted into fatty acid methyl esters (FAMEs) using a modified method of Miller [Cellular fatty acid, respiratory quinones and polar lipid composition of strain Bb-Pol-6f Miller  and Kuykf Miller , separatf Miller , 44, purf Miller , separatf Miller . T is KX450422. The whole-genome shotgun BioProject number is PRJNA875284 with the GenBank assembly accession number JAPMXC000000000. The strain has been deposited in the DSMZ under the number DSM 114812, and the BCCM/LMG under the number LMG 32774.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Bb-Pol-6 T was isolated, one gram of sample was found to contain 4.1 ± 3.1 × 105 colony forming units (CFU) [Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. Culture-independent method revealed 441 operational taxonomic units (OTU), and the most dominant phyla were Proteobacteria, Actinobacteria and Acidobacteria [. Compared to insect-pollinated pollen species (autumn crocus and rape), birch pollen possesses significantly higher species richness and different communities [ T has one of the highest measured LPS concentrations in this study with an LPS concentration of 76.6 ng l−1 [From the same birch pollen sample where Bb-Pol-6ts (CFU) . Culturebacteria . Comparemunities . Furthermunities . This po6 ng l−1  and five6 ng l−1  and on h6 ng l−1  are alsoR. andropogonis DSM 9511 T , Chitinasiproducens palmae JS23T , Pararobbsia silviterrae DHC34T  Paraburkholderia elongata 5NT  were identified as the next-relative species with valid names to strain Bb-Pol-6 T. As the similarity is less than 98.7%, strain Bb-Pol-6 T can be considered a potential novel species [ T and its conspecific species were clustered with R. andropogonis DSM 9511 T, forming a stable monophyletic group, which was consistently revealed in the NJ and MP trees . This indicates the potential phylogenetic position of strain Bb-Pol-6 T under the genus Robbsia.According to the highest pairwise gene similarity results in EzBioCloud (Ez) and ARB (Table S1),  species . Similar T consisted of 46 contigs with a total size of 5.04 Mbp and the longest contig was 2.68 Mbp which was the N50 length. This genome contained 4401 predicted coding sequences among which 509 sequences were predicted to encode signal peptides, 57 tRNA genes and three rRNA genes, and had a G + C content of 65.31 mol%. Completeness and contamination of the genome was 98.44 and 0.05%, respectively. Although four copies of 16S rRNA gene were predicted only one 16S rRNA gene sequence from the draft genome was partially recovered with a length of 470 bp. R. andropogonis DSM 9511 T has the closest AAI and ANI values to strain Bb-Pol-6 T (72.42% and 72.53%) compared to the other next-relatives . The dDDH and POCP values of strain Bb-Pol-6 T to R. andropogonis DSM 9511 T were 22.7 and 65.85%, respectively. These values were below the generally accepted species boundary values  [ T a promising novel species. The ML tree calculated from the genome of strain Bb-Pol-6 T and 19 closest-related species using 1307 core genes again revealed the monophyletic group formed by strain Bb-Pol-6 T and R. andropogonis DSM 9511 T, confirming its phylogenetic position within the genus Robbsia , redox-cofactor, phosphonate and terpene . Two antibiotic resistance ontology terms with antibiotic efflux mechanism, qacG and adeF, were predicted using protein homolog model in CARD. The most abundant genes annotated by PIFAR-Pred were toxin-encoding genes (35%) . Among these toxin-encoding genes, the majority encode syringomycin, a necrosis-inducing phytotoxin [Burkholderia glumae in crop diseases [ T.The draft genome assembly of strain Bb-Pol-6DH: 70%) , and aboytotoxin , and toxdiseases , 56, ind T were rod-shaped (0.4–0.7 µm in width and 0.9–1.6 µm in length) with no flagellum, Gram-stain-negative, facultative anaerobic, non-spore-forming and non-motile. Colonies of strain Bb-Pol-6 T were round, convex, viscous, creamy in color and has entire margin after grown on AC 1:10 agar for five days. The strain grew optimally on AC 1:10 agar and growth was also observed on R2A agar, but not on LB, TSA, NA and MacConkey agar. Strain Bb-Pol-6 T was catalase-positive and oxidase-negative. The temperature, salinity and pH value ranges for growth were 4–28 °C (optimum at 28 °C), 0–1% NaCl (optimum without NaCl) and 5–7.5 pH (optimum at 6–7). Unlike R. andropogonis, strain Bb-Pol-6 T cannot induce hypersensitive response on Nicotiana tabacum cv. Xanthi and Betula pendula  and does not produce rhizobitoxine. The differential physiological and biochemical characteristics of strain Bb-Pol-6 T and its reference strain were summarized in Table Cells of strain Bb-Pol-6 T (96.5%) and R. andropogonis DSM 9511 T (97.7%). The major cellular fatty acids of both strains were C16:0, C19:0 cycloω7c, C17:0 cycloω7c and C17:1ω6c (Table S2). Compared to R. andropogonis DSM 9511 T, strain Bb-Pol-6 T contained a higher amount of C12:0 (2.8%), C18:1ω5c (2.9%) and C16:1ω7c (8.8%), and a lower amount of C14:0 (0.4%), C17:0 (0.7%) and C18:1ω7c (0.3%). Strain Bb-Pol-6 T contained trace amount of C10:0, C12:0 3-OH and C18:1ω7c 11-methyl, which were not detected in R. andropogonis DSM 9511 T, while C15:0 3-OH detected in R. andropogonis DSM 9511 T was not present in strain Bb-Pol-6 T. The major polar lipids in strain Bb-Pol-6 T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, one unidentified aminophospholipid, two unidentified phospholipids and six unidentified lipids . Glycolipid was identified in R. andropogonis DSM 9511 T but not in strain Bb-Pol-6 T.On AC 1:10 medium, ubiquinone 8 was the major respiratory quinone of both strain Bb-Pol-6 T belongs to the genus Robbsia. Further phenotypic and biochemical characterization distinguished strain Bb-Pol-6 T from its next-relative reference strain R. andropogonis DSM 9511 T suggesting it represents a novel species, for which the name Robbsia betulipollinis sp. nov. is proposed.In summary, the phylogenetic analysis revealed that strain Bb-Pol-6Robbsia betulipollinis .16:0, C19:0 cycloω7c, C17:0 cycloω7c and C17:1ω6c. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid, one unidentified aminophospholipid, two unidentified phospholipids and six unidentified lipids.Cells are Gram-negative, facultative anaerobic, non-spore-forming, non-motile, rod-shaped (0.4–0.7 µm wide × 0.9–1.6 µm long) and do not have flagellum. Colonies were round, convex, viscous, creamy in color and has entire margin after five days of incubation on AC 1:10 agar. Grows between 4 and 28 °C (optimum at 28 °C), 0–1% NaCl (optimum without NaCl) and 5–7.5 pH (optimum at 6–7). Positive for catalase, esterase (C4), esterase (C8), acid phosphatase, β-galactosidase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, α-Glucosidase, D-galactose, D-mannitol, D-mannose, D-ribose, L-arabinose, D-arabitol, D-glucose, D-fructose and succinate. Negative for oxidase, nitrate reduction, glucose fermentation, arginine dihydrolase, gelatin hydrolysis, esculin hydrolysis, indole production, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, N-acetyl-β-glucosaminidase, lipase (C 14), β-glucuronidase, β-glucosidase, α-mannosidase, α-fucosidase, urease, alkaline phosphatase, adipic acid, amygdalin, arbutin, capric acid, cellobiose, D-fucose, D-gentiobiose, lactose, maltose, melezitose, melibiose, raffinose, sucrose, D-tagatose, turanose, dulcitol, D-xylose, glycerol, inulin, L-arabitol, L-fucose, L-rhamnose, L-Xylose, malic acid, methyl α-D-glucopyranoside, methyl α-D-mannopyranoside, N-acetyl-D-glucosamine, phenylacetic acid, potassium 2-ketogluconate, potassium 5-ketogluconate, potassium gluconate, salicin, starch, xylitol, esculin ferric citrate, trehalose, glycogen, erythritol, methyl-ß-D-xylopyranoside, L-sorbose, trisodium citrate, propionate, acetate, D-adonitol, D-lyxose, D-sorbitol, inositol, D-arabinose, malonate, hypersensitive response and rhizobitoxine production. The major respiratory quinone is ubiquinone 8. The major cellular fatty acids are C T (= LMG 32774 T = DSM 114812 T), was isolated from pollen of birch in Giessen, Hesse, Germany (50° 34′ 10.755″ N 8° 40′ 17.859″ E). The genome of the type strain has a size of 5.04 Mbp with a G + C content of 65.31 mol%.The type strain, Bb-Pol-6Supplementary file1 (PDF 1384 KB)Below is the link to the electronic supplementary material.