Janthinobacterium, Psychrobacter, and Serratia. These organisms were able to grow on diesel, motor oil, and crude oil at 4 °C. Putative identification showed the presence of sophorolipids and rhamnolipids. Emulsion index test (E24) activity ranged from 36.4–66.7%. Oil displacement tests were comparable to 0.1–1.0% sodium dodecyl sulfate (SDS) solutions. Data presented herein are the first report of organisms of the genus Janthinobacterium to produce biosurfactants and their metabolic capabilities to degrade diverse petroleum hydrocarbons. The organisms’ ability to produce biosurfactants and grow on different hydrocarbons as their sole carbon and energy source at low temperatures (4 °C) makes them suitable candidates for the exploration of hydrocarbon bioremediation in low-temperature environments.Surfactants, both synthetic and natural, are used in a wide range of industrial applications, including the degradation of petroleum hydrocarbons. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants, yet microorganisms from cold environments have been largely overlooked for their biotechnological potential as biosurfactant producers. In this study, four cold-adapted bacterial isolates from Antarctica are investigated for their ability to produce biosurfactants. Here we report on the physical properties and chemical structure of biosurfactants from the genera  Surfactants, also known as surface-active agents, are commonly used in many different industries, including paints, textiles, detergents, health, and personal care products, as well as mineral and petroleum processing ,2,3,4. SBiosurfactants are natural surfactants, produced primarily by microorganisms and plants ,9,10. AsPseudomonas spp., Bacillus spp., Rhodococcus spp., Candida spp., Lactobacillus spp., Arthobacter spp., and Acinetobacter spp. [In particular, bioremediation is an emergent strategy for cleaning up petroleum-contaminated environments ,30,31. PMany untapped oil reservoirs, current drilling efforts, and extensive distribution networks (including offshore drilling and subsea pipelines) are located in cold temperature environments. These regions are particularly vulnerable to spills, as low temperatures not only change the chemical properties and bioavailability of oil, but also slow down its biodegradation. Biosurfactants produced by model organisms, however, have typically been investigated at temperatures >20 °C ,35,36,37Pony Lake (PL) is a small , brackish, and eutrophic lake located at Cape Royds , Ross Island, Antarctica . The lakThe Cotton Glacier stream (CG) is a supraglacial stream in the Transantarctic Mountains , flowing eastward between Sperm Bluff and Queer Mountain in Victoria Land for ~16 km . While tThe bacterial isolates used in this study  were isolated from PL and the CG on R2A agar media (Difco) at 4 °C. Single colonies from each site were selected according to morphological characteristics, including color, size, and colony shape. Colonies were sub-cultured to obtain purified isolates. Isolates were stored in 40% glycerol at −80 °C and archived as part of the Foreman Lab Isolate Collection.Genomic DNA was extracted using the PowerSoil DNA Isolation Kit  and following manufacturer’s recommendations. PCR amplification of the 16S rRNA gene was performed with primers 9F 5′-GAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTACGACTT-3′ . The PCR−1, proteose peptone no. 3 = 0.5 g L−1, casamino acids = 0.5 g L−1, sodium pyruvate = 0.3 g L−1, dipotassium phosphate = 0.3 g L−1, magnesium sulfate = 0.05 g L−1). All liquid cultures were incubated at 4 °C while shaking at 100 rpm for 5–10 days at log phase. Cells were harvested by centrifugation at 4 °C and 4800 rpm for 15 min. Cell pellets were washed twice with 1X M9 Minimal Salts culture media (Difco™). Final cell pellets were resuspended in carbon-free 1X M9 Minimal Salts culture media.Bacterial isolates from freezer stocks were streaked on R2A agar plates and incubated at 4 °C. Single colonies of each organisms were transferred to 50 mL Falcon tubes containing 10 mL of modified R2A broth media . Cell cultures were allowed to grow at 4 °C while shaking at 100 rpm for 10 days. Biosurfactant was extracted by centrifugation at 12,000× w/v), a known chemical surfactant, were used as a positive control.Oil displacement tests were carried out as described by Parthipan et al. . BrieflyE24) was calculated by the following formula:emulsionh and totalh are the height of the emulsion layer and the total solution, respectively. Sterile growth media for biosurfactant production was used as a negative control, and 10% SDS was used as a positive control.An emulsion index test was performed similar to the one originally developed by Cooper and Goldberg . In it, Media agar plates with 1× M9 Minimal Salts culture media were made and coated evenly with 100 µL of crude oil. Then, 1.5 mL of the cell culture growing in the R2A broth media was harvested by centrifugation and washed twice, as previously described. Cell pellets were resuspended in 100 µL of 1× M9 Minimal Salts culture media. The organisms were streaked on 1× M9 Minimal Salts culture media plates coated with 100 µL of crude oil as a sole carbon source and incubated at 4 °C. Plates were visually inspected for growth over a two-month period.To increase the amount of biosurfactants produced, two enrichment flasks containing 140 mL of growth media for biosurfactant production were prepared for each bacterial isolate. Cells were grown as described above. Cell cultures were pelleted by centrifugation at 4800 rpm for 15 min, and the supernatant was collected in pre-combusted (450 °C for 5 h) glass beakers. The beakers were dried in a drying oven at 80 °C for 72 h. The dried biosurfactant was scraped from the beaker into 50 mL falcon tubes. Subsequently, 10 mg of the dried biosurfactant were dissolved in either 1 mL of full-strength acetone, acetic acid, or ethanol at 37 °C for 24 h. To ensure the presence of dissolved biosurfactant in each solution, an oil displacement test, as described above, was performed, along with each solvent as a negative control. Biosurfactants were classified using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and Raman spectroscopy.MALDI mass spectrometry was performed using a Bruker Autoflex MALDI TOF/TOF , with ion source 1 voltage = −20.01 kV, ion source 2 voltage = −17.81 kV, reflector 1 = −21.25 kV, reflector 2 = −10.78 kV, and lens voltage = −8.70 kV. The MALDI TOF/TOF was equipped with a Smartbeam laser (Nd/YAG = 355 nm) capable of operating at a repetition rate of 1000 Hz. The MALDI laser used was capable of optimized delayed extraction time, and the laser beam size was set to small. For each sample preparation, the laser energy was optimized for an optimum signal-to-noise in the samples.3/MeOH . Samples were applied to TLC silica gel plates and developed in a neutral solvent system of CHCl3/MeOH/DIW  and CHCl3/MeOH/DIW , respectively. Glycolipids were stained reversibly with iodine.Thin-layer chromatography (TLC) was used to separate and confirm the presence of polar and non-polar  glycolipids following the method described by Nozomu and Makoto . Briefly2 Raman-grade slide and placed in the drying oven at 40 °C for 1.5 min to evaporate off the deionized water. The samples were analyzed using a 100× objective with an NA of 0.9, and a 532 nm laser with 45 mW of power. The laser was operated at between 1–10% for three accumulations of 10 s for each spectrum. Measurements were recorded using a 600 nm grating. Spectra were fitted with a fourth-degree polynomial line for baseline subtraction. The LabSpec6 fourth-degree data smoothing function was used to reduce the noise-to-signal ratio of the measurements.Raman spectroscopy was performed on a LabRAM HR Evolution Confocal Raman Spectrometer (HORIBA) equipped with the LabSpec6 software package . Dried surfactant samples (10 mg) were dissolved in 100 µL of deionized water. Then 6 µL of each dissolved sample was pipetted onto a clean CaF2 concentrations and CO2 production rates during extended incubations. Data were evaluated by Micro Oxymax software. Briefly, bacterial isolates were grown in R2A broth media, washed, and resuspended in 1X M9 Minimal Salts culture media with no carbon, as described above. The hydrocarbon amendments included diesel, motor oil , and crude oil, at a final concentration of 4.5% (v/v). Hydrocarbon amendments were added to combusted amber bottles in quadruplicate, containing 30 mL of 1X M9 Minimal Salts culture media and cells. One incubation bottle without cells served as an abiotic control for each amendment. All samples were incubated at 4 °C for 30 days, with data collected every 12 h. CO2 production rates were normalized to cell concentrations based on viable plate counts of colony-forming units (CFUs) on R2A agar and control corrected. The abiotic blanks were also investigated for any bacterial contamination at the end of each experiment via plate counts of CFUs on R2A agar. Unpaired t-tests and one-way ANOVA were performed using the statistical software package R [Bacterial isolates were further investigated for their hydrocarbon biodegradation potential at cold temperatures . A Columbus Instruments Micro-Oxymax Respirometer was used to continuously measure COackage R , using aJanthinobacterium svalbardensis. Although Janthinobacteria are commonly known to produce the purple pigment violacein, both isolates from this study did not produce purple colonies when grown on R2A agar plates. It is, however, not uncommon for species within this genus to lack the gene set required for the synthesis of the violacein pigment [Psychrobacter arcticus and was first isolated from Siberian permafrost [Serratia (>99%). Some slightly more distantly related relatives (>97%) were also reported as being psychrotrohic isolates [The phylogenetic characterization of the four bacterial isolates is summarized in  pigment ,51,52. Prmafrost . PL 17 wisolates .E24, were recorded between ~36% (Serratia sp. PL 17) and 67% (Janthinobacterium sp. CG 23.3), which bracketed the E24 index for a 10% SDS solution (~56%). The addition of the surfactant supernatant to a crude oil layer for the oil displacement test formed average clear zones of ~3.5 ± 2.2 cm (Janthinobacterium sp. CG 23.4) to ~6.4 ± 0.4 cm (Serratia sp. PL 17) in size , m/z: 647 (C18 + Na), m/z: 663 (C18:1 1Ac)), and di-rhamnolipids (m/z: 649 (Rha2 C10–C10), m/z: 689 (Rha2 C12–C12)) ) . The pre12–C12)) . TLC als CG 23.4 . As bios CG 23.4 .−1 , 897 cm−1 (Serratia sp. PL 17 and Janthinobacterium sp. CG 23.3), 987 cm−1 , 1058 cm−1 (CG 23.3), 1446 cm−1 (Janthinobacterium sp. CG 23.3), and 1617 cm−1 , which is indicative of (CS) vibration of aliphatic chains, (OO) stretching, (CC) alicyclic/aliphatic chain vibrations, (CC) stretching vibrations, alkyl CH2 bends, and amide I peaks, respectively. In the higher wavenumber range (~2800–3100cm−1), differences in the Raman spectra for each biosurfactant were evident, which can be assigned to the CH stretching of acyl or alkyl chains  . Raman sl chains .2 production continuously monitored over 30 days (2 production (r2 ≥ 0.971). Rates varied markedly over two orders of magnitude between isolates, even within the same species. The highest respiration rates were found for Psychrobacter sp. PL 19 for all three hydrocarbon substrates . It should be noted that when grown on R2A broth at 4 °C, the generation time of the bacterial isolates ranged between 5.0–6.5 h , which further underlies the petroleum hydrocarbon degradation capabilities of Psychrobacter sp. PL 19. However, although CO2 production rates for Psychrobacter sp. PL 19 varied between 344.0 ± 81.5 fg CO2 d−1 cell−1 for diesel, 144.8 ± 59.7 fg CO2 d−1 cell−1 for motor oil, and 271.6 ± 199.1 fg CO2 d−1 cell−1 for crude oil, they were not statistically different  = 2.445, p-value = 0.142).The four bacterial isolates were able to grow on diesel, motor oil, and crude oil as their sole carbon and energy source. No growth was observed in the controls when grown on R2A agar plates after 30 days of incubation. Respiratory activity is shown by the cumulative CO 30 days . The biobstrates ; these rThe four biosurfactant producing bacterial strains were isolated from ephemeral Antarctic environments. It is well-established that many microorganisms produce tensio-active molecules, including biosurfactants, in order to increase the bioavailability of hydrophobic, water-insoluble substances, or the hydrophobicity of the cell surface ,13,17. Hm/z ratios as certain sophorolipids (m/z: 621) and di-rhamnolipids . Hence, caution is advised when generalizing the putative classification of sophorolipids and rhamnolipids in this study. As sophorolipid production has only been observed in yeast to date [12–C12 for all four bacterial isolates, while other rhamnolipid peaks could have been masked by the noise of the measurement. An additional peak indicative of di-rhamnolipids C10–C10 was apparent for the bacterial isolate Serratia sp. PL 17. Naturally-produced rhamnolipid biosurfactants are found as mixtures of different congeners, and as such, the chemical composition of the dominant rhamnolipids and the concentration of the congeners largely determines the efficacy of the biosurfactant. Generally, a higher di-rhamnolipid content enhances biosurfactant activity [10–C10 and di-rhamnolipid C12–C12 implies that the physicochemical properties of the biosurfactants produced by the four Antarctic isolates could be up to ~8 and 45 times more effective when compared to mono-rhamnolipids with C10 fatty acids or rhamnolipid mixtures with high proportions of congeners containing unsaturated fatty acids, respectively [Putative identification suggests that all four isolates produced sophorolipids and di-rhamnolipids when grown on canola oil as a sole carbon source . Notewor to date , these b to date . Putativactivity ,61. The ectively . Consideectively , it is vPsychrobacter sp. PL 19 and Janthinobacterium sp. CG 23.3 could significantly emulsify kerosene (E24 > 50%), with a maximum index of 66.7% for Janthinobacterium sp. CG 23.3  isolated from marine Arctic and Antarctic environments [Pseudomonas aeruginosa and Bacillus subtilis, two bacterial species that have traditionally been considered for biosurfactant production, have E24 values of ~70% and ~10% for kerosene, respectively [Psychrobacter sp. PL 19 (13.3 g L−1) and Janthinobacterium sp. CG 23.3 (17.3 g L−1) also indicate biosurfactant production similar to those reported for the yeast Moesziomyces antarcticus (16.3 g L−1). M. antarcticus (previously classified as Pseudozyma antarctica), which was isolated from Lake Vanda, Antarctica [All biosurfactants produced at 4 °C showed emulsification. The bacterial isolates  CG 23.3 . The emuronments ,63. Pseuectively . Dried btarctica , is one tarctica . HoweverPsychrobacter sp. PL 19, Janthinobacterium sp. CG 23.3, and Janthinobacterium sp. CG 23.4 were closely related to Psychrobacter sp. and Janthinobacterium spp., respectively . Further research on petroleum hydrocarbon degradation should include gravimetric methods and calibrated analytical measurements  for optimal quantification [Biosurfactants have emerged as viable solutions in many industrial applications, with the potential to outperform traditional synthetic surfactants due to their unique, environmentally friendly properties. Bioprospecting cold-adapted microbes for new biosurfactants has been limited, with established reports being rather incomplete in terms of identified strains and biosurfactants characterized ,67. Othe


Curcuma mutabilis Škorničk., M. Sabu & Prasanthk., is an endemic Zingiberaceae species from Western Ghats of Kerala, India. Here, we report for the first time, the anticancer potential of petroleum ether extract from C. mutabilis rhizome (CMRP) and a novel labdane diterpenoid, (E)-14, 15-epoxylabda-8(17), 12-dien-16-al (Cm epoxide) isolated from it. CMRP was found to be a mixture of potent bioactive compounds including Cm epoxide. Both the extract and the compound displayed superior antiproliferative activity against several human cancer cell lines, without any display of cytotoxicity towards normal human cells such as peripheral blood derived lymphocytes and erythrocytes. CMRP treatment resulted in phosphatidylserine externalization, increase in the levels of intracellular ROS, Ca2+, loss of mitochondrial membrane potential as well as fragmentation of genomic DNA. Analyses of transcript profiling and immunostained western blots of extract-treated cancer cells confirmed induction of apoptosis by both intrinsic and extrinsic pathways. The purified compound, Cm epoxide, was also found to induce apoptosis in many human cancer cell types tested. Both CMRP and the Cm epoxide were found to be pharmacologically safe in terms of acute toxicity assessment using Swiss albino mice model. Further, molecular docking interactions of Cm epoxide with selected proteins involved in cell survival and death were also indicative of its druggability. Overall, our findings reveal that the endemic C. mutabilis rhizome extract and the compound Cm epoxide isolated from it are potential candidates for development of future cancer chemotherapeutics.Zingiberaceae plants are well known for their use in ethnomedicine.  Classical examples include vinblastine, vincristine, taxol, camptothecin and podophyllotoxin3. However, only about 5–15% of the 250,000 higher plants have been screened for potential drugs4. Despite considerable efforts, clinical use of plant-based, semi-synthetic or synthetic chemotherapeutic agents have been found to be largely inadequate in fulfilling expectations of a safe and affordable cancer cure. Consequently, a constant demand for novel, efficacious and biocompatible anticancer pharmaceuticals bereft of undesirable side-effects, continues to exist5. In this context, it is relevant and logical to exploit the extensive biodiversity of unexplored plants in a tropical country like India. The Western Ghats of India represent one of the world’s ten biodiversity hotspots treasuring more than 700 medicinal plants7.Three quarters of the prescribed anticancer drugs are plant-derivedCurcuma longa), zerumbone (Zingiber zerumbet), gingerol and shogaol  amongst many others13. Curcuma mutabilis Skornick., M. Sabu & Prasanthk. is an unexplored, endemic Zingiberaceae species reported from Western Ghats of Kerala in India14. Essential oil isolated from the rhizome of C. mutabilis, with estrone methyl ether -trien-17-one) as the major constituent, was found to possess potent antiproliferative activity against human cancer cell lines, K562 and HCT11615.The ethnomedicinally important family of Zingiberaceae is credited with the isolation of an impressive array of pure compounds with proven anticancer activity. A few notable examples are curcumin/curcuminoids  was found to possess the most potent activity, which was then investigated further to study its cellular and molecular effects in detail. Subsequent activity-guided isolation of the extract led to the characterization and identification of a labdane diterpenoid, (E)-14,15-epoxylabda-8(17),12-dien-16-al (IUPAC) designated as ‘Curcuma mutabilis epoxide’ by us. An assessment of the in vitro anticancer potential of this compound in comparison to the parent extract, CMRP, was also carried out.The present study aimed to evaluate, for the first time, the cytotoxic and antiproliferative potential of different organic solvent extracts, prepared from C. mutabilis rhizome (CMR) extracts—petroleum ether (CMRP), chloroform (CMRC), acetone (CMRA) and methanol (CMRM) were evaluated by MTT assay using eight different human cancer cell lines such as HCT116 , K562 (chronic myelogenous leukemia), HL60 (acute promyelocytic leukemia), HepG2 (hepatocellular carcinoma), A549 , PC3 (prostate cancer), MCF7, MDA-MB-231 (breast cancer) and embryonic kidney cells, HEK293T. All extracts showed dose-dependent reduction in cell survival percentage , CMRA (6–99 µg/mL) and CMRM (47– > 100 µg/mL) extracts  and K562 (IC50–6.5 µg/mL) which showed the highest sensitivity towards CMRP.The cellular toxicities of cts Fig. . CMRP wa50 values (5–7.5 µg/mL) for majority of the cancer cell lines tested  and MTT assays of CMRP-treated (2.5–15 µg/mL) normal hPBLs revealed that the extract was innocuous with no significant cytotoxicity apparent within the range of IC% and MTT50 value, resulted in severe reduction of about 80–88% in the number of colonies compared to controls  treated controls  cell population increased to ~ 22 and 55% in HCT116 and K562 cells respectively  at cell surface, which is normally restricted to inner membrane layer, is considered as a hallmark of apoptosis. Calcium-dependent binding of fluorochrome-tagged annexin V to externalized PS enables its detection by fluorescence microscopy and quantitation using flow cytometry. Annexin V-Propidium iodide (PI) co-staining also helps to distinguish late apoptotic/necrotic cells from early apoptotic cells, as PI can enter cells and interact with DNA only when membrane integrity is lost2DCF by cellular esterases and later oxidized by ROS into highly fluorescent DCF. Therefore, increase in its fluorescence is directly proportional to the amount of intracellular ROS18. In the present study, both HCT116 and K562 cell types revealed a dose dependent increase in intracellular ROS by fluorescence microscopic  was assessed using DCFH-DA, a cell permeable non-fluorescent probe. Upon entry into the cell, DCFH-DA is converted into membrane impermeable H20. CMRP-induced loss of mitochondrial membrane potential was found to occur in a dose dependent manner as evidenced by both fluorescence microscopy , revealed changes in cell distribution in three major phases of cell cycle, G1, S and G2/M. These changes were dose-dependent and commensurate with a concomitant increase in sub-G1 apoptotic cell population harbouring fractional DNA content Fig. . In othe21. CMRP treated HCT116 and K562 cells displayed increased comet tail lengths, in a concentration dependent manner, indicative of the occurrence of DNA damage , a sensitive, reliable and rapid method for genotoxic assessmentage Fig. a. Likewiage Fig. b.Figure 50 value. Likewise, upregulation of pro-apoptotic BAX gene expression was evident in K562 cells by ~ 6 and 13 folds respectively in cells treated at the IC50 value (6.5 µg/mL) and beyond (12 µg/mL). HCT116 cells, exposed to 5.5 µg/mL and 12 µg/mL showed a 3 and 5-folds increase respectively in BAX expression relative to controls. PUMA expression was also found to be upregulated by 3 folds in HCT116 cells at 12 µg/mL. As expected, the anti-apoptotic Bcl-2 gene expression was found to be downregulated in both cell types by about 0.18 and 0.22 folds at 12 µg/mL, in a concentration dependent manner. Given the fact that survivin and XIAP are inhibitors of apoptosis (IAP), their expression was indicative of promotion of cancer cell survival22. CMRP treatment was found to cause reduction in survivin and XIAP expression in a dose dependent manner. Notably, the effect of extract on survivin downregulation was greater than that on XIAP, especially in K562 cells with only 0.07 folds expression at 12 µg/mL. Overall, CMRP treatment resulted in upregulation of pro-apoptotic BAX and downregulation of anti-apoptotic Bcl-2 and IAPs (survivin and XIAP) gene expressions. The extent of caspase gene expression in both cell types was also found to be upregulated, more so for caspase 8 in HCT116 cells, by CMRP treatment. In other words, CMRP treatment apparently activated expression of genes involved in both intrinsic and extrinsic apoptotic pathways.Molecular analysis of CMRP-induced changes in cancer cells was carried out by RT-qPCR and western blot analysis. The levels of apoptosis-related gene transcripts, caspases -3, -8 and -9, pro-apoptotic BAX and PUMA, anti-apoptotic Bcl-2 and apoptosis inhibitors, survivin and XIAP, were studied. Figure Western blot analysis of CMRP treated HCT116 and K562 cells revealed cleavage of PARP, procaspases -3 and -9, in a dose dependent manner, indicative of apoptosis induction  and tumour cell migration was also observed in CMRP-treated HCT116 cells.RT-qPCR data obtained from HCT116 and K562cells also revealed CMRP-induced variations in the transcript levels of some of the key genes involved in cell survival, cancer progression and migration Fig. c,d. ExprCMRP-treated HCT116 cells were observed to move slower than the untreated / DMSO-treated controls in a dose-dependent manner : Rf = 0.74), UV–visible spectroscopy and HPLC analysis together revealed that SF20 contained a single component, which was identified later as a labdane diterpenoid, namely, (E)-14,15-epoxylabda-8(17),12-dien-16-al (IUPAC) and denoted as ‘Curcuma mutabilis epoxide’ (Cm epoxide) by us.The CMRP extract was sub-fractionated on silica gel columns as described in the Methods section. From a total of 55 sub-fractions (SF)  1166 and 1248.6 (C–O–C stretching within the ether and epoxide groups respectively), 1692.2 (C = O stretching associated with –CHO group), 1649.8 , 2706.6 , 2924.5 and 2839.6 , 1384.6 (gem-dimethyl group), 896.7 (vinylidene C–H group). 1H NMR spectra : δ (ppm) 0.759 , 0.827 , 0.892 , 2.026  13.05, 13.05, 5.14 Hz), 2.417  12.90, 4.34, 2.38 Hz), 2.642  16.84, 11.33, 7.54 Hz), 2.744  16.83, 6.10, 3.05 Hz), 2.872  5.55, 2.79 Hz), 3.035  5.55, 4.39 Hz), 3.657  3.58 Hz), 4.468 , 4.865 , 6.624  7.53, 6.17 Hz), 9.329 ; 13C NMR : δ 193.45, 161.15, 148.19, 137.55, 107.86, 56.89, 55.46, 47.42, 47.09, 42.04, 39.71, 39.18, 37.93, 33.60, 33.59, 24.22, 24.18, 21.74, 19.31, 14.45  in addition to the unsaturated aldehyde group . 1H NMR indicating three protons of a terminal epoxide . The standard application and interpretation of 2D—COSY, HSQC and HMBC NMR spectra  and its structure is depicted in Fig. α, β orientations of the ring protons, the proR, proS relationships of the geminal proton pairs, the E, Z orientations of the geminal proton pair, and the E configuration of the C12 = C13 double bond by the application of NOE (Nuclear Overhauser Effect) and scalar coupling constant data. The configuration of the distal C-14 stereocenter was determined by concerted application of NOE, 3JH,H, and 3JH,C data (by the strong or weak correlations observed in the HMBC spectrum in the latter case). Thus the compound was identified as (E)-14,15-epoxylabda-8(17),12-dien-16-al.f values of 0.30, 0.53, 0.65, 0.73, 0.93 and 0.18, 0.20, 0.29, 0.52 (unlike the single band observed in SF20) , MDA-MB-231 [IC50–2.33 µg/mL (7.7 µM)] and human embryonic kidney cells [IC50–4.25 µg/mL (14 µM)]. It was interesting to note that compared to the well known polyphenol from Curcuma spp., curcumin, which served as positive control, the IC50 values of Cm epoxide (Table 50 concentrations for cancer cell lines (< 2.5 µg/mL), it was found to be rather insignificant with < 5% cell death  of Cm epoxide for 24 h Fig. b. Based ath Fig. c.Table 2Cm epoxide, akin to its parent extract, CMRP, displayed relatively higher potential for clonogenic inhibition Fig. d,e and aSwiss albino mice model was used to evaluate the pharmacological safety of CMRP and Cm epoxide in terms of acute toxicity assessment. Any elicitation of serious behavioural changes or mortality in the animal test groups was not observed. Likewise, no significant deviations were observed in hematological parameters -14,15-epoxylabda-8(17),12-dien-16-al, denoted as Cm epoxide by us, is being reported here for the first time from C. mutabilis. Incidentally, this compound has been reported from another Zingiberaceae plant Alpinia chinensis from Hong Kong earlier as ‘Alpinia epoxide’25, but proof on any bioactivity including anticancer associated with the same was found missing in scientific literature. The observed NMR data of SF20  and thus enable the distinction of C-4 and C-18, the multiplicity assignments of which were otherwise ambiguous. Similarly, chemical shift deviations attributable to decoupling-induced heating in the HSQC spectrum were accounted for by also acquiring the HSQC spectrum without decoupling of the carbon nuclei. Incidentally, it may be relevant to note here that, labdane diterpenoids are bicyclic diterpenes reported with a wide range of bioactivities such as antimicrobial, anti-mutagenic, anti-inflammatory, cytotoxic and cytostatic effects28. Another labdane diterpenoid, (E)-labda-8(17),12-diene-15,16-dial, which displays high structural similarity with Cm epoxide is widely distributed among Alpinia sp. It has been reported to possess antibacterial and antifungal activities in addition to being an inhibitor of α-glucosidase and NO production29. Interestingly, the same compound has also been reported from C. amada (mango ginger) in India and is known to possess antimicrobial activities30. Notably, this compound is structurally different from Cm epoxide due to the presence of an aldehyde (–CHO) group at C-14 position instead of an epoxide group ,13(14)-dien-16-oic acid, [2] (E)-15-ethoxy-15-methoxylabda-8(17)12-dien-16-al, [3] (E)-15α-ethoxy-14α-hydroxylabda-8(17),12-dien-16-olide, and [4] 15-ethoxy-12β-hydroxylabda-8(17),13(14)-dien-16,15-olide. Interestingly, all four were reported with potent antiproliferative activities against various human cancer cell lines31.The quest for novel anticancer drugs without adverse side-effects, sourced from time-tested, experiential ancient wisdom as practiced through use of traditional or ethnomedicinal plants, continue to contribute meaningfully towards cancer cureC. mutabilis rhizome extract (CMRP) as well as that of the labdane diterpenoid, Cm epoxide, isolated from it. A detailed, comparative analysis of their anticancer activities, employing human cancer cell lines with normal human lymphocytes (hPBLs) and erythrocytes as controls, were demonstrative of their therapeutic potential. Interestingly, notwithstanding the potent cytotoxic, antiproliferative and anti-clonogenic activities against several human cancer cell types, both CMRP and Cm epoxide failed to show any adverse effects on normal cells. Both the extract and the compound were equally capable of selective cell killing via induction of apoptosis. Cancer cells exposed to CMRP or Cm epoxide were found to exhibit apoptotic hallmarks such as membrane blebbing, nuclear chromatin condensation, cellular/nuclear fragmentation32, phosphatidylserine externalization, increased intracellular ROS and Ca2+ levels, loss of mitochondrial membrane potential20, genotoxicity and DNA laddering33. Cell cycle analysis also revealed the presence of the apoptotic sub-G1 phase cell population, with fractional DNA content34. Transcript profiling by RT-qPCR provided additional molecular evidence confirming apoptosis induction by way of upregulation of caspases 3, -8 and -9, pro-apoptotic BAX and downregulation of anti-apoptotic Bcl-2 and IAPs such as survivin and XIAP gene expression35. Additionally, transcripts of genes related to pathways regulating cell survival and migration such as mTOR, c-Myc, COX 2, NF-κB (p65), bcr/abl, WT1, TGF-β1 and MMP-936 were also found to be downregulated, excepting PTEN in cancer cells exposed to CMRP. Further, western blot analysis of CMRP-treated cancer cells also bore evidence of apoptosis related caspase 3, -7, -8, -9 activation, PARP cleavage and downregulation of Bcl-237. Additional downregulation of cell cycle related cyclin B1 and chk1 gene expressions were also observed. The upregulation of γ-H2AX expression, indicative of DNA damage, lent further credence to the genotoxicity observed by way of DNA strand breaks seen as comet tails and DNA laddering due to internucleosomal cleavage38. Likewise, scratch and transwell invasion assays also proved CMRP-induced inhibition of adherent HCT116 cancer cell migration and invasion39. Assessment of acute toxicity using Swiss albino mice model also attested to the pharmacological safety of CMRP and Cm epoxide, at the tested dosages, as evidenced by the absence of deviations in hematological parameters, levels of key liver enzymes and histopathological assessment of liver tissue. It may be pertinent to note here that exposure of normal human lymphocytes and erythrocytes to CMRP or Cm epoxide, at cancer cell killing concentrations, did not produce any evidence of cellular damage.To our knowledge, the present study qualifies to be the first report on the anticancer activity of 40, (2) trilaurin, fatty acid derivative recently used in lipid nanoparticle formulation to deliver lipophilic anticancer agents such as curcumin, docetaxel, paclitaxel more effectively for cancer cure41, (3) borneol, a simple bicyclic monoterpene, reported with chemosensitization potential42, (4) γ-sitosterol (phytosterol) which reportedly induces DNA damage, cell cycle arrest and apoptosis in various cancer cell lines43 and (5) β-amyrin, reported to possess antitumour activity against liver carcinoma, Hep-G2 cells, induce apoptosis, disrupt cell cycle regulation and activate JNK and p38 signaling pathways44. Notably, SF27 and SF32 displayed potent cytotoxicity against cancer cells with IC50 values < 30 µg/mL.In addition to SF20, analysis of the other two bioactive sub-fractions of CMRP, SF27 and -32 also revealed the presence of major compounds credited with various bioactivities including anticancer potential. These compounds included (1) 13-apo-beta-carotenone (an eccentric cleavage product of β-carotene)37. RT-qPCR analysis also showed their influence on transcription of various genes involved in cell survival/migration pathways. Being a mixture bioactive compounds, CMRP extract can potentially target multiple cellular pathways, impact physicochemical properties such as solubility thereby affecting bioavailability, antagonize development of drug resistance and reduce or neutralize the toxic/adverse effects of individual component in it45. From this perspective, it is interesting to note that, herbal formulations are in fact an integral part of traditional Chinese, Indian (Ayurveda) and western medicine. There are several reports on the anticancer effects of phytopharmaceuticals administered alone or in combination46.To sum up, it is clear from the foregoing account that in addition to Cm epoxide, CMRP also harbours compounds such as 13-apo-beta-carotenone, trilaurin, borneol, γ-sitosterol and β-amyrin. This potent mixture of bioactive components, was observed to be apoptogenic via both intrinsic and extrinsic pathwaysC. mutabilis, an endemic Zingiberaceae plant confined to Western Ghats of the Indian State of Kerala and that the petroleum ether extract of this plant rhizome (CMRP) is a bioresource of multi-potent bioactive compounds. This study also reports for the first time on successful isolation, identification and demonstration of apoptogenic effects of a labdane diterpenoid, Cm epoxide from CMRP. Further extensions of the present study, in terms of various dosage regimens, combination treatments with other anticancer agents and those involving advanced tumour models of human cancers, are necessary prior to advancement of these agents with promising therapeutic potential into clinical trials.To conclude, the present study demonstrates for the first time the anticancer potential of Curcuma mutabilis was collected from the Nilambur forest (Lattitude–11.2806791°N and longitude–76.1987534°E), Malappuram district of Kerala state, India. The plants were authenticated by Prof. M. Sabu, Department of Botany, University of Calicut, and a voucher specimen (Accession No. 6856 CALI) was deposited in the Calicut University Herbarium. The collected plant rhizomes were washed well with running water, cut into small pieces, shade-dried, powdered using a grinder and then were subjected to serial extractions with organic solvents of increasing polarities such as petroleum ether, chloroform, acetone and methanol by gentle mixing on a gyroshaker for 24 h. The extracts were filtered through Whatman No. 1 filter paper and the filtrates evaporated to dryness. The extract residues were then dissolved in dimethylsulphoxide (DMSO) to prepare a stock solution which was stored at − 20 °C until use.Cell culture media components such as DMEM, RPMI-1640 medium, Ham’s F12 K medium, penicillin, streptomycin, Hi-Sep Lymphocyte separation medium, HiKaryo XL RPMI medium, MTT, acridine orange, BSA, skimmed milk, prestained protein marker and tween 20 were purchased from HiMedia Laboratories, Mumbai, India. DMSO, trypan blue, Hoechst 33258, DCFH-DA, Rhodamine-123, Tris base, TRI reagent, Oligo(dT)-18 mer primers, Protease/phosphatase Inhibitor Cocktail and KBr were purchased from Sigma-Aldrich, USA. Fetal bovine serum, Dulbecco’s Phosphate Buffered Saline (DPBS), Trypsin–EDTA, Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, Fluo-3 AM, propidium iodide and Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix were purchased from Thermo Fisher Scientific, USA. M-MuLV reverse transcriptase enzyme, RNase A, dNTP mix and agarose were purchased from Genei Laboratories Pvt. Ltd, Bangalore, India. Triton X-100, EDTA, glycine, ethidium bromide, BCIP, NBT, silica gel for column chromatography (60–120 meshes), vanillin, sulphuric acid and solvents such as petroleum ether, chloroform, acetone, methanol  were procured from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Antibodies were procured from Cell Signaling Technology, Inc., USA and Sigma-Aldrich, USA. Primers sets were purchased from Eurofins Genomics India, Bangalore, India.2. hPBL cells were separated from whole blood collected from healthy donors and cultured in HiKaryo XL RPMI media according to manufacturer’s protocol. Briefly, the isolation and culture of hPBLs was carried out as follows: 5.0 mL of human blood was mixed with an equal volume of phosphate buffered saline (PBS) and then carefully overlaid on 6.0 mL of Hi-Sep Lymphocyte separation medium in a centrifuge tube and spun at 400 × g for 30 min at room temperature. The buffy coat obtained at the fluid interface, aspired using a sterile Pasteur pipette, was then washed twice with PBS. Finally, cells were seeded at a density of 1 × 105 cells/mL into 5.0 mL HiKaryo XL RPMI medium and incubated at 37 °C for 48 h for growth.The cell lines used in the present study were chronic myelogenous leukemia—K562, acute promyelocytic leukemia—HL60, colorectal carcinoma—HCT116, hepatocellular carcinoma—Hep G2, non-small lung alveolar carcinoma—A549, prostate cancer—PC3, breast cancers—MCF7 and MDA-MB-231, embryonic kidney—HEK293T, purchased from the National Center for Cell Science (Pune), India. K562 and HL60 were maintained and propagated in RPMI 1640 medium, whilst HCT116, HepG2, MCF7, MDA-MB-231 and HEK293T were cultured in DMEM; A549 and PC3 cells were cultured in Ham’s F12K medium. All media were supplemented with 10% (v/v) fetal bovine serum, streptomycin (100 μg/mL) and penicillin (100 U/mL). Cell cultures were maintained at 37 °C in a humidified atmosphere of 5% COThe blood samples used for hemolysis assay and isolation of lymphocytes for lymphocyte culture and MTT assay was willingly self-donated by the first author (Soumya T.) and the second author (Lakshmipriya T.) of the manuscript submitted. It may be noted that according to the Indian Council for Medical Research, New Delhi, India  the ethical approval for use of self-donated blood by the researcher was not deemed to be necessary. According to this guideline, proposals which present less than minimal risks are exempted from the ethical review process.C. mutabilis rhizome extracts or column sub-fractions from CMRP, prepared in DMSO and incubated for 24 h, followed by MTT assay as previously described47.For cytotoxicity evaluation, all cancer and normal cell types were exposed to varying concentrations (1–100 μg/mL) of different 48. Briefly, cells (1 × 105cells/mL) were incubated with varying concentrations of individual extract(s) or the compound for 24 h. The cells were harvested, washed with PBS (pH-7.4), stained with 0.4% trypan blue solution and counted using a haemocytometer. The result was expressed as percentage dead cells relative to control.Cell viability of extract(s) / Cm epoxide-treated cancer cells was also determined using trypan blue dye exclusion assay as previously described49.Cells treated with CMRP or Cm epoxide for 24 h were washed with PBS and counted. Aliquots of 500 cells were then plated onto a 6-well dish containing complete medium in the case of HCT116; for K562, a cell suspension in RPMI medium containing 0.3% low melting agar was used. Following 10 days of incubation, the colonies were stained with crystal violet  and counted manuallyCytomorphological changes induced by CMRP or Cm epoxide, if any, in cancer cell lines HCT116, K562 and MDA-MB-231 were observed and photographed using light (phase-contrast), fluorescence and scanning electron (CARL-Zeiss Gemini 300 SEM) microscopic techniques.The control and treated cells were collected, washed twice with PBS, re-suspended in Hoechst 33258 staining solution (1 mg/mL) and incubated for 10 min at room temperature in the dark. The cells were then observed for changes in nuclear morphology under a fluorescence microscope.5 cells/mL using PBS. The AO—EB solution  was added to the cell suspension at a final concentration of 100 μg/mL prior to observation under a fluorescence microscope.The control and treated cells were harvested, washed with cold PBS (pH—7.4) and adjusted to a cell density of 1 × 10Externalization of PS in the treated cancer cells was evaluated by staining with Alexa Fluor 488—annexin V  in accordance with the manufacturer’s instructions. In addition to fluorescence microscopy, the intensity of cellular fluorescence was also measured by flow cytometry on a BD FACS ARIA II machine with BD FACS Diva software 6.1.3.18. Briefly, the control and treated cells were stained with 10 μM DCFH-DA for 30 min at 37 °C, washed twice with PBS and observed under fluorescence microscope. The quantification of ROS was carried out using flow cytometry as mentioned earlier.Cellular ROS levels of treated cancer cells were studied by using the cell permeable fluorescent probe, 2′,7′ dichlorofluorescin diacetate (DCFH-DA)2+ levels were determined by deploying a fluorescent dye, Fluo-3 AM19. The control and treated cells were washed twice with PBS, following incubation with 5 μM Fluo-3 AM for 30 min at 37 °C. The cells were then washed and subjected to fluorescence microscopy and flow cytometric analysis.Alterations in the intracellular Ca20. Cells were then washed with PBS, centrifuged, resuspended in PBS, mounted on slides for fluorescence microscopy. Flow cytometric analysis of fluorescence due to rhodamine 123 within the cells was analysed as described earlier.Mitochondrial membrane potential of control and treated cells were determined by staining with the lipophilic cationic dye, rhodamine 123 (5 μg/mL) for 30 min at 37°CFlow cytometric assessment of cell cycle distribution was carried out by staining cells with DNA intercalating dye, propidium iodide (PI). Based on DNA content, the percentage of cells distributed within the sub-groups of the total cell population representing sub-G0/G1, G0/G1, S and G2/M phases of cell cycle were quantified on a BD FACS-ARIA II cytometer using BD FACS Diva software version 6.1.3. Briefly, control and treated cells were separately harvested, washed twice with PBS, fixed in 70% ice cold ethanol and preserved at − 20 °C until use. The cells were stained with 20 µg/mL PI solution containing 200 µg/mL RNase A (DNase free) for 30 min prior to data acquisition and analysis.50.The comet assay was used to detect DNA strand breaks within single cells subjected to gel electrophoresis under alkaline conditions. Genotoxic evaluation of the CMRP extract was carried out essentially as previously described33, electrophoresed on 1.8% agarose gel and observed under gel documentation system for DNA fragmentation.The control and treated cells were washed with PBS, resuspended in lysis buffer  and incubated for 15 min at room temperature. DNA was then isolated from this cell lysates by standard procedures as previously described51.Total cellular RNA from the control and treated cells were isolated using TRI Reagent in accordance with the manufacturer’s protocol. The RNA samples were treated with RQ1 RNase-free DNase  to digest any residual chromosomal DNA and then quantified using UV spectrophotometer . For first strand cDNA synthesis, initially 5 μL of reaction containing 2 μg of total RNA, 1 μL of 10 mM dNTP mix and 1 μL of 100 μM oligo dT primer were incubated at 65 °C for 10 min, spun briefly and placed in ice. To this, 2 μL of 5 × M-MuLV reverse transcriptase buffer, 0.25 μL of 20 U/μL M-MuLV Reverse transcriptase, 0.25 μL of Ribolock RNase Inhibitor  were added and made up to 10 μL with sterile nuclease free water. The reaction mixture was incubated at 37 °C for 60 min and then heated to 95 °C for 10 min and stored at − 20 °C until use. Later, 2.0 µL of the cDNA, along with SYBR Premix Ex Taq (Tli RNaseH Plus)  and 0.25 µM of individual primer sets  of proteins were subjected SDS-PAGE profiling on a 4–12% gradient gel. The fractionated polypeptides were blotted onto Immobilon PVDF membrane  blocked with 5% (w/v) skimmed milk prepared in TBST (TBS with 0.1% Tween 20) for 1 h at room temperature. Immunostaining was carried out overnight at 4 °C separately with mouse/rabbit-derived primary antibodies (dilution 1:1000) against β-actin, caspase -3, -7, -8, -9, PARP, Bcl-2, phospho-H2AX, cyclin B1 and Chk1. The membranes were then washed in TBST and incubated for 1 h at room temperature with either anti-mouse or anti-rabbit ALP or HRP-conjugated secondary antibodies (1:2000). Following another wash with TBST, the blots were exposed to BCIP/NBT or chemiluminescene methods to visualize the immunostained polypeptides.SDS-PAGE and western blotting were performed essentially as described previouslyC. mutabilis rhizome (CMRP) which displayed potent antiproliferative activity, was subjected to further analysis of bioactive fraction/compound(s). Column chromatography of CMRP was carried out using a glass column (300 × 18 mm) of silica gel 60 (60–120 meshes). The gel was mixed well with hexane to degas and then packed into the column. The sample (CMRP—2.5 g) was mixed with a small amount of silica gel applied to the packed column without trapping any air bubbles. Using individual solvents or combinations thereof of increasing polarities, fractions were eluted using hexane , hexane : chloroform , chloroform , chloroform : acetone , acetone: methanol  and methanol . The sub-fractions obtained as of 20 mL aliquots were analysed using TLC followed by assessment of their anticancer activities. Depending upon lowest IC50 values and separation pattern of bands observed in TLC, three fractions, SF20, -27 and -32 were chosen for analysis of compounds present therein.Among the various extracts tested, the petroleum ether extract of 254 TLC plates  using toluene: ethyl acetate  solvent system for SF20 and toluene: ethyl acetate: methanol  for SF27 and -32, followed by derivatization with vanillin-sulphuric acid reagent. SF20 was further characterized with UV–visible spectroscopy (Perkin-Elmer spectrophotometer) and HPLC (Shimadzu Prominance system) under standard conditions. The mass of the purified compound was analysed by HPLC-HRMS system (Thermo scientific Q Exactive orbitrap mass spectrometer fitted with HypersilTM C18-column). The mobile phase consisted of a gradient of 97% methanol and 3% water with 0.1% formic acid for 5 min at a flow rate of 150 µL/min. The sample injection volume was 2 µL and detection was carried out at 235 nm. Electrospray ionization source was operated in both positive and negative mode with an ion spray voltage of 3KV. Oven temperature was set to 30 °C and m/z scan range was kept between 100−2000 Da.The sub-fractions were then analysed on aluminium backed, pre-coated, silica gel 60 F−1. NMR spectra were acquired using a Bruker Avance NMR spectrometer equipped with a 5 mm inverse-configuration probe of triple-axis-gradient capability with a field-strength of 14.1 T operating at 600.1 and 150.9 MHz for 1H and 13C nuclei, respectively. Spectra were acquired at 25 °C in samples containing CDCl3. The chemical shifts of 1H and 13C nuclei are reported relative to TMS (δ = 0 ppm for both 1H and 13C). The chemical shifts of 1H nuclei are reported to three decimal places when the multiplet was amenable to first-order analysis or to two decimal places when the multiplet was beyond such interpretation; for overlapped signals, chemical shifts were taken from 2D NMR spectra and are reported to three decimal places to distinguish them. General NMR experimental and acquisition details for 1D 1H, 13C, and DEPT observation and standard gradient-selected 2D COSY, HSQC, HMBC,and NOESY spectra and routine chemical shift assignment using 2D NMR have been previously described54.For FTIR analysis, 1 mg of SF20 was finely ground with about 100 mg of KBr, pressed into a thin disc by using hydraulic press and then connected to a vacuum chamber to dehydrate the sample. FTIR spectroscopy of this KBr disc was then carried out using Jasco 4100 IR spectrometer within the frequencies ranging from 4000–400 cmGC–MS analysis of sub-fractions was carried out separately in an Agilent gas chromatography 6850 Network GC system , fitted with a HP-5MS capillary column . Initially, the oven was maintained at 60 °C for 1 min and the temperature was then gradually increased to 250 °C at a rate of 5 °C /min. Injector temperature was kept at 280 °C. Helium was used as a carrier gas, adjusted to column velocity flow of 1 mL/min with a split ratio of 10:1 and a split flow at 10 mL/min. The sample (1 μL) was injected into the system. Identification of components was based on comparison with mass spectral fragmentation patterns of those stored in the MS library [NIST databases—NIST08 and NIST08.L ].All animal experiments were conducted in accordance with the CPCSEA guidelines and all experimental protocols were approved by ethical committee for animal care, Kannur University as stipulated by Indian National Law on animal care and use. The experimental design was approved by the Institutional animal ethics committee (KULS/IAEC/2019/28), Kannur University. Swiss albino mice, 6–8 weeks old and weighing approximately 18–22 g were maintained in well-ventilated cages under standard conditions of room temperature, pressure, and humidity and fed with mice standard diet  and water ad libitum, at 12 h light/dark cycle. To determine acute toxicity, a single dose of CMRP (50 and 200 mg/kg body weight) and Cm epoxide (25 and 100 mg/kg body weight) were administered orally to groups of 6 animals each. The mice were then observed for the next 7 days for any gross behavioural change and death. Following euthanization on the 8th day, hematological parameters  and liver function tests were carried out by SI methods, along with histopathological analysis of liver tissue by H-E staining.Molecular docking was carried out using Schrodinger maestro Glide software as manufacture instructions. Native 3D protein structures of EGFR, HER2, VEGFR, B-RAF, ABL kinase, CDK2, HDAC, PI3K and AKT were downloaded from PDB database followed by refining for Glide docking using protein preparation wizard tool. Known inhibitors of these proteins were chosen from PubChem database to enable a comparison with that of Cm epoxide’s docking efficiency. Ligand docking was carried out using prepared ligand(s) and receptor-grid-generated protein in XP (extra precision) mode. Docking scores representing the sum of all interactions between the amino acid residues at the binding site and the ligand molecule as well as overall binding energy of whole protein–ligand complex predicted by Molecular Mechanics/Generalized Born Surface Area (MMGBSA) scores were analysed, for choosing best targets of Cm epoxide.All experimental data are expressed as mean ± SD from three independent experiments. Results were analysed for significance by one-way ANOVA using SPSS software version 16.0.Supplementary Information.


Paenibacillus strain isolated in previous research exhibited antimicrobial activity against relevant human pathogens including Staphylococcus aureus and Listeria monocytogenes. In this study, the genome of the aforementioned strain, designated as MP1, was shotgun sequenced. The draft genome of strain MP1 was subject to multiple genomic analyses to taxonomically characterize it and identify the genes potentially responsible for its antimicrobial activity.A Paenibacillus strain, MP1. Average Nucleotide Identity (ANI) analysis established strain MP1 as a new strain of the previously characterized Paenibacillus alvei. The genomic analysis identified several putative secondary metabolite clusters including seven Nonribosomal Peptide Synthetase clusters (NRPS) , one bacteriocin or other unspecified Ribosomally Synthesized and Post-Translationally modified Peptide Product (RiPP), one lanthipeptide, and six hybrid clusters (NRPS-Type I Polyketide synthase (T1PKS) and NRPS-trans Amino Transferase Polyketide Synthase (AT-PKS)).Here we report the draft genome sequence of an antimicrobial producing  Paenibacillus spp. produce a variety of peptide and non-peptide-containing antimicrobial compounds. These compounds include hydrolytic enzymes, lantibiotics, polymyxins, paenibacterin, various organic molecules, and others [d others . Of thesd others . HoweverPaenibacillus spp. for the discovery of novel antimicrobials, the genomic information of these bacteria remains insufficient [Paenibacillus strain isolated from buckwheat honey [S. aureus and L. monocytogenes. The goal of this study was to conduct whole-genome sequencing of this strain, hereafter designated MP1, to taxonomically characterize it and identify genes possibly responsible for the production of the compound(s) that yield strain MP1 its antimicrobial activity.Although there is increasing interest in fficient . Previouat honey  was idenStrain MP1 was maintained at − 80 °C in 15% (vol/vol) glycerol Luria–Bertani medium . Prior to extraction, strain MP1 was streaked and grown on Luria–Bertani agar. Genomic DNA was isolated from a culture of strain MP1 grown by inoculating 9 mL Luria–Bertani medium with a single colony and incubating at 37 °C for 18 h with continuous shaking at 200 rpm. DNA was extracted from 1.8 mL culture using the QiaAMP DNA Minikit  following a slightly modified protocol. In brief, cells were pelleted and subjected to lysozyme (20 mg/mL) treatment  at 37 °C for 60 min; DNA was extracted from this lysate using the QiaAMP DNA Minikit following manufacturer instructions for RNA-free genomic DNA using RNase A . Following extraction, DNA was spectrophotometrically quality checked using a NanoDrop  to ensure the 260/280 and 260/230 nm absorbance ratios were greater than 1.8 and 2.0 respectively. Library preparation, quality control, and sequencing were conducted by the Cornell University Veterinary Molecular Diagnostics Laboratory utilizing the Nextera XT DNA library preparation and indexing kits  and an Illumina MiSeq  to obtain 2 × 250 bp paired-end reads; this yielded 3,467,242 raw reads (866 Mb).50, G + C content), and BBmap (v37.50) [Raw reads were trimmed and paired using Trimmomatic v0.39)  with the  with th(v37.50)  and Samt(v37.50)  were use(v37.50) .Paenibacillus type strains deposited in the National Center for Biotechnology Information (NCBI) assembly database. Following this, all-v-all ANI analysis was conducted among all strains with whole genomes deposited in the NCBI assembly database for the species of the closest related type strain. ANI analysis was conducted via the OrthoANI method using OAT (v1.40) [The species identification of strain MP1 was determined by ANI analysis of strain MP1′s genome versus the whole genomes of all available  (v1.40)  with BLA (v1.40) . The spe (v1.40)  via the Genome annotation was conducted by the NCBI using the Prokaryotic Genome Annotation Pipeline (PGAP) , 18. Bec50 value of 129,056 bp, 46.11% G + C content, and 6,511,289 bp; average sequencing coverage was 106.03 ×  , TS-15 , and A6-6i-x  . Hierarchical cluster analysis revealed two distinct clusters above 95% ANI; one cluster contained strains MP1 and DSM 29, and the other contained strains B-LR, TS-15, and A6-6i-x , subtle differences were revealed. Strain MP1 contained 626 less genes than the whole genome of type strain DSM 29 . When visualized using Mauve, it became clear that divergent regions were related to the presence of mobile genetic elements  within each genome, specifically plasmids and prophage. This was confirmed when the individual genomes were analyzed using Phaster. The results from Phaster indicate a difference of 594 genes attributed to the presence of 13 prophage regions in strain MP1  vs 20 in type strain DSM 29  , one hybrid cluster containing a lasso peptide and NRPSs, and one hybrid cluster containing a sactipeptide and resorcinol. Notably, strain MP1 contained one large operon with five putative NRPS genes spanning 71,579 nt with no similarity to genes involved in the biosynthesis of other known NRPs; type strain DSM 29 contained a similar cluster that displayed synteny with strain MP1 , one bacteriocin, one lanthipeptide, four hybrid NRPS clusters containing NRPSs and polyketide synthetases ; the population of phage in communities is dynamic, and geographic origin likely affected the composition of microbial communities, including phage, each isolate was exposed to. Nonetheless, due to the high ANI value between MP1 and DSM 29, strain MP1 is a new representative of the P. dendritiformis C454 was reported in 2016 as the novel lasso peptide tailored by a new class of kinases [Paenibacillus, including strain MP1, could lead to the discovery of medically relevant (or otherwise) compounds for use against pathogenic organisms.AntiSMASH analysis of the PGAP annotated MP1 genome confirmed its potential to produce a variety of nonribosomal peptides and polyketides. Some of the identified clusters were related to previously known compounds including polymyxin, paenibacterin, and paenibactin; others were not—such as the lasso peptide cluster. The most recent results support that the structure of paenibacterin as a cyclic lipodepsipeptide antibiotic might be useful for creating new antibiotics via synthetic routes , which i kinases . The potFinally, this study demonstrates several antimicrobial compounds are yet to be characterized and highlights the necessity to experimentally verify the function of synthesis genes. Such efforts will result in a better understanding of the structure and synthesis of antimicrobial compounds and will lead to better genome-based predictions in the future.The acknowledged analysis is based on the draft genome of strain MP1. Therefore, regions of the complete genome may be duplicated or missing from the assembly.Additional file 1: Data 1. The QUAST Summary table.Additional file 2: Data 2. OrthoAni_Paenibacillus_type_strains.Additional file 3: Data 3. OrthoAni_Paenibacillus_alvei.Additional file 4: Data 4. DSM29_prophage_supplement.Additional file 5: Data 5. MP1_prophage_supplement.


Caeculus Dufour  contains 19 previously described species, most of which are found in North America, and for which no comprehensive phylogenetic treatment exists. Here, one new species from Alberta, Canada, is described: Caeculuscassiopeiae Bernard & Lumley, sp. nov., and another caeculid known to be present in Canada is documented. The new species is characterized within the genus with a character state matrix, from which an updated key is produced. A systematic analysis of all 20 species based on morphological and geographical distribution traits obtained from literature represents the first phylogenetic review of the genus.The genus  Caeculidae contains 108 previously described species of large (750–3000 µm) prostigmatic mites in seven genera distributed worldwide, with 19 species in the genus Caeculus Dufour  collected the other species in Alberta’s Writing-on-Stone Provincial Park in 1978, and the same species in Alberta’s Waterton Lakes National Park in 1980, which are the only previously known collections from Canada and are deposited in the CNCI. He identified this species only to Caeculus and did not publish it, but herein we identify it as C.cremnicolus Enns. The only published record of caeculoids in Canada is by Acari still lists only one previously recorded caeculid species  Gaertner) and alfalfa (Medicagosativa L.). We revisited the same locality on June 26, 2017 and using a paintbrush collected additional specimens, which were found only on open, exposed soil surfaces of the arid SW slope during the hottest part of the day . The soil surfaces had a thin hardened crust, possibly created by drying after rainfall.On 27 July 2014 in Medicine Hat, Alberta, Canada, we collected two female caeculid specimens in yellow pan traps filled with soapy water , which fBSk)   with a mBSk)  and the BSk) .EtOH) and cleared the other in 85% lactic acid  and dissected it before mounting on a slide in a solution of 1.66 g polyvinyl alcohol, 10 mL lactic acid, 1 mL glycerol, and 10 mL distilled water  for analysis under dissection and compound microscopes, both of which contributed to creating the free-hand illustrations. With the 2017 specimens, we kept two alive for observation and stored the remainder in 95% EtOH.After photographing the specimens collected in 2014 with a K2 DistaMax long-distance microscope , we stored one in 95% ethyl alcohol  for our phylogenetic analysis. Note that the ingroup of this study includes only species currently in the genus Caeculus and excludes those that have been reassigned to other genera  Invertebrate Zoology Collection, Edmonton, Alberta, Canada.AD adanal sclerite;AG aggenital sclerite;CH chelicera;IL idiosomal length;IW idiosomal width;PA palp;PG progenital valve;PS pseudanal sclerite.Taxon classificationAnimaliaTrombidiformesCaeculidaeBernard & Lumleysp. nov.DE2AB6DE-B683-5DCF-ADA4-A1678DF6EAA5http://zoobank.org/BC876F53-B89C-4AEA-ABC7-EA8CA18B7F91Holotype. Canada • 1 ♀; Alberta, Medicine Hat, near Gas City Campground; 50°2.23'N, 110°43.56'W; elev. ca 700 m; 26–27 Jul. 2014; M. Buck leg.; yellow pan traps; arid SW slope and adjacent disturbed plain of Agropyroncristatum and Medicagosativa; det. J. Bernard and L. Lumley, 30 Aug. 2016; cleared in 85% lactic acid, dissected, and slide-mounted; PMAE M00019466.Paratype. Canada • 1 ♀; ibid.; stored in 95% EtOH; PMAE M00030967.EtOH; PMAE M00030972 to M00030979.Canada • 8 ♀♀; ibid.; 26 Jun. 2017; L. Lumley leg.; collected with paintbrush; hard dry soil surface; det. L. Lumley; stored in 95% b setae on the centrodorsal opisthosoma are arranged in an M, three pairs of Pp setae are aligned on the posterior third of the aspidosoma, and trochanter III has three setae along anterolateral surface.The five N = 2, all measurements in micrometres, μm).; 1.920 × longer than greatest width  (718–829 = IW). Sclerites tawny with pale ridges, ochre to raw sienna between sclerites, translucent white setae , one median eye immediately inferior to base of naso  with barbed distal end , width 425–491 (0.592 × IW) anterior to eyes, posterior margin of sclerite 309–357 (0.430 × IW); pronounced furrow along length of medial axis containing three or four shallow longitudinal reticulated ridges, anterior furrow width 135–157 (~0.189 × IW), posterior furrow width 102–118 (~0.142 × IW), posterior furrow depth 35–41 (~0.050 × IW); two pairs procurved clavate Pa setae on anterior margin; none or one pair clavate Pm setae near corners of anterior margin; three pairs clavate Pp setae medial to eyes on posterior lateral margins, aligned parallel to mid-sagittal plane; area around eyes dark, posterior eyes 1.077 × diameter of anterior eyes , posterior margin 477–552 (0.665 × IW); one pair clavate a1 setae near anterior margin of sclerite midway between mid-sagittal plane and lateral margins; 2.5 pairs clavate b setae: b1 pair anterior to mid-transverse plane and third of distance between mid-sagittal plane and lateral margins, b2 pair posterior to mid-transverse plane and midway between mid-sagittal plane and lateral margins, one unpaired median bs seta present between anterior and posterior pairs, the five setae together forming M-shape; one or two pairs clavate c setae along posterior margin   near anterior margin, one or two b setae at middle of sclerite , and two c setae near posterior margin (c3 and c4); lyrifissure ia between a3 and b3; lyrifissure im between b4 and c3  Fig. . Laterod c3 Fig. . Mediodotes Fig. . Pluripoeta Fig. .Idiosoma venter , epimere II with three clavate setae along anterior margin; epimeres III and IV fused (separate from I + II), epimere III with three clavate setae along anterior margin and one clavate seta midway along proximal margin, epimere IV with one clavate seta at anteroproximal margin, one clavate seta on midline a third epimeral length from proximal margin, and three clavate setae along posterior margin  each with seven smooth acuminate simple setae; aggenital sclerites (AG) each with three clavate setae; surrounding ventral cuticle bearing nine pairs of clavate setae, including pair ag1 between epimeres IV  each bearing two clavate setae; pseudanal sclerites (PS) each with three clavate setae; one unpaired medial clavate seta posterior to anus; lyrifissure ih laterad to anterior pseudanal sclerite  five-segmented, setal formula, trochanter-tarsus, solenidia ω and eupathidia ζ in brackets: 0-2-1-5-10(1ω+1ζ); trochanter without setae; femur bearing two dorsal clavate setae midway along length, with proximal seta a third length of distal seta; genu bearing longest subclavate seta on tubercle at distal laterodorsal margin; tibia bearing five setae: one proximal anteroventral barbed spiniform seta, one laterodorsal spiniform seta with barbed distal end, one proximal dorsal elongated clavate seta, one distal dorsal barbed spiniform seta, and one posterolateral clavate seta  each with fixed digit regressed to lobe and movable digit uncinate  than n –21(2ζ)–22(1φ+1ζ+1κ”)–12(1ω+5ζ+1ε); leg II 5–10+4–16–17(1φ)–14(1ω+1ε); leg III 7–5+2(2ζ)–9(1ζ)–15(1φ+1ζ)–9(1bt+1ζ); leg IV 7–2(1ζ)+3(2ζ)–9(1ζ)–13(2ζ)–13(1bt+1ω). Leg I length 1301–1502 ; tarsus IV with one solenidion ω a third the length from distal end on dorsal surface , and C.gertschi Mulaik sister to C.hypopachus Mulaik (100%). Despite lower branch support for the B clade, there is high support for C.kerrulius Mulaik being sister to C.echinipes (100%). Sister to clade B is the strongly supported C clade, which represents C.lewisi McDaniel & Boe as sister to C.cassiopeiae sp. nov. (100%), and this pair is sister to C.valverdius Mulaik (100%).The phylogeny reveals three morphological clades, termed A, B, and C Fig. . CaeculuCaeculuscassiopeiae sp. nov. is morphologically most similar to C.lewisi McDaniel & Boe and C.valverdius Mulaik, several noteworthy differences exist  C.valverdius bears six b setae on the centrodorsal opisthosomal sclerite, but five b setae are arranged in an M-shape in the new species; (ii) each laterodorsal opisthosomal sclerite has a single c seta in C.valverdius whereas the new species has two c setae; (iii) the posterior opisthosomal sclerite has five e setae in C.valverdius whereas the new species has seven; (iv) C.valverdius has six setae on each progenital valve whereas the new species has seven; (v) the adanal sclerites each have three setae in C.valverdius and two in the new species; (vi) genu I bears a single posteroventral rake seta in the new species whereas C.valverdius has four; and (vii) C.valverdius has two anterolateral setae on the proximal half of trochanter III, but the new species has three that are more evenly distributed along the length.Although st Table . The dicC.dorotheae and C.janetae both have six anteroventral rake setae on tibia I, which is a unique character. Caeculusgertschi and C.hypopachus each bear four rake setae in that location, a character shared with some members of clade B. Of the species in clade A, most are not recorded as having tarsal bothridial setae bt, which occur in most other Caeculus as well as in the outgroups, indicating possible plesiomorphy for the rest of the genus. One exception is C.gertschi  like Medicine Hat, whereas Newell has a humid continental climate (Dfa)  . The soiC.cremnicolus are 1911 km from the closest published location in Easley, Missouri (Cfa) by the Köppen-Geiger system, but the climates in the Alberta locations are incongruent; Writing-on-Stone Provincial Park is classed as cold semi-arid (BSk) yet Waterton Lakes National Park is continental subarctic (Dfc)  . Soils aC.cremnicolus and C.cassiopeiae sp. nov. are 126 km apart and in the same climate, their ranges could potentially overlap if C.cremnicolus can inhabit the soil at Medicine Hat. These species are nevertheless in separate clades . The other clades also contain representatives from the North American southwest as well as those from other areas, and there is a diversity of locations represented by the taxa that are not in clades. Caeculusechinipes is the only species of Caeculus described to date from Europe. The results suggest that the ancestor of C.echinipes spread to Europe from North America. Further work to include molecular data would be helpful to clarify the weaker branch support shown in the topology.Our phylogenetic analysis suggests that the common ancestor of Caeculuscassiopeiae sp. nov. elevates the number of known Canadian caeculids to two. Based on a maximum parsimony analysis of a character state matrix for all members of the genus, it is most closely related to C.lewisi. The phylogeny also suggests multiple introductions of caeculids into Canada, and that the origin of the genus is the American southwest. Further collection of caeculids in North America is required to determine to what extent ranges overlap, examine ecology or additional morphological traits  that were not described in previously published taxonomic accounts, and to enable genetic analysis. Population studies can further describe the degree of intraspecific variation and clarify species boundaries. Our phylogeny provides the first analysis of the genus, which can be useful for future systematic studies that integrate taxonomy and genetics to develop a better understanding of the genus Caeculus and its family.Our description of


To describe acute kidney injury (AKI) natural history and to identify predictors of major adverse kidney events (MAKE) within 1 year in patients supported by veno-arterial extracorporeal membrane oxygenation (VA-ECMO).Retrospective observational study.Medical French intensive care unit between January 2014 and December 2016.Consecutive patients implanted with VA-ECMO ≥ 16 years, VA-ECMO for at least ≥ 48 h, and without end-stage chronic kidney disease (CKD).None.2 and MAKE at day 30 and day 90 defined as one of the following criteria within day 30 or day 90: death, receipt of renal replacement therapy and serum creatinine ≥ threefold increase.Multivariate logistic regression of factors associated with MAKE at 1 year defined as one of the following criteria within day 360: death and receipt of renal replacement therapy (RRT) or persistent renal dysfunction, i.e., CKD ≥ stage 3 corresponding to an estimated glomerular filtration rate (eGFR) ≤ 60 ml/min/1.73 mp 0.02), Kidney Disease Improving Global Outcome (KDIGO) stage at cannulation (p = 0.03), e.g., stage 3 vs. reference stage 0 OR 10.20 [1.77–58.87], and number of red blood cell (RBC) packs received while under ECMO . At 1 year among the 51 survivors, almost half of the alive patients (n = 20/51) had a decline of estimated glomerular filtration (eGFR) > 30% mL/min/1.73 m2. Their median eGFR decline was − 26.3% .158 consecutive patients were included . Among them 145 (91.8%) developed an AKI during the intensive care unit (ICU) stay and 85 (53.8%) needed renal replacement therapy (RRT). 59.9% (91/152), 60.5% (89/147) and 85.1% (120/141) evaluable patients had a MAKE-30, MAKE-90 and MAKE-360, respectively. Factors significantly associated with MAKE-360 were eGFR at baseline (odds ratio (OR) 0.98, confidence interval 95% (CI) , n = 20/51) had a decline eGFR > 30%.Patients undergoing VA-ECMO had a high risk of AKI during the ICU stay. Factors associated with MAKE 360 were mainly eGFR at baseline, KDIGO stage at cannulation and, number of RBC packs received while under ECMO. Among survivors at 1 year, almost half of the alive patients ( Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is a technique to provide circulatory assistance for patients suffering from cardiogenic shock . Acute kAcute and long-term outcomes of AKI occurring in intensive care unit (ICU) are well-described. AKI is associated with early and late mortality rising with AKI severity –5. FurthLong-term risk of CKD in patients treated by VA-ECMO was never analyzed. Because of extended use of this technique, early identification of factors associated with better renal outcome of survivors is urgently needed. This information may help to better define the patients who would benefit the most from ECMO or may inspire prompt prevention initiatives and improve the outcome.The objectives of this study were to describe the natural history of renal function within 1 year and to identify independent predictors of major kidney events (MAKE) at day 30, day 90 and at 1 year in patients who underwent VA-ECMO.We retrospectively analyzed the charts of consecutive patients who received VA-ECMO during their ICU stay in our 20-bed ICU  between 1st of January 2014 and 31st of December 2016. We included patients older than 16 years, and who were under VA-ECMO for at least ≥ 48 h. Patients who received veno-venous ECMO and patients with pre-hospitalization end-stage renal disease (ESRD) requiring renal replacement therapy (RRT) were excluded. Patients who met inclusion criteria multiple times in different ICU stays within a year were included only once.Data were mainly collected from electronic medical records or otherwise by asking general practitioner of the patients.The following data were recorded at ICU admission: age, sex, body max index (BMI), Simplified Acute Physiology Score (SAPSII) , the SOFWithin the ICU stay, we collected ECMO-associated bleeding complications: number of red blood cell (RBC) transfusions, hemopericardium defined as early (within 3 days) or otherwise late or other haemorrhagic shock needing surgery.We collected also: ICU and hospital length of stay, duration of VA-ECMO support, renal function, need of RRT and survival at each time point .2) or we imputed SCr for a MDRD of 75 ml/min/1.73 m2 as recommended in the KDIGO guidelines on AKI.AKI stages were defined according to the current  KDIGO guidelines based on elevations in serum creatinine (SCr) from the baseline reference value or requirement of RRT . The ref2. If eGFR by MDRD was over 90 ml/min/1.73 m2, CKD stage was labeled 0/1 as no urinalysis was available to distinguish further between these two stages.Baseline renal function at admission and at 1 year were calculated using the simplified MDRD equation based on age, gender, race, and calibration for Scr. Normal renal function was defined as an estimated glomerular filtration rate (eGFR) > 90 mM/min/1.73 mIndividuals with decreased levels of eGFR were classified according to the consensual stages of CKD .2.The main goal of the present study was to describe the natural history of renal function within 1 year and to identify the risk factors for major adverse kidney events at day 360 (MAKE-360) defined as one of the following criteria within 1 year: death and receipt of renal replacement therapy (RRT) or persistent renal dysfunction, i.e., CKD ≥ stage 3 corresponding to an eGFR by MDRD ≤ 60 ml/min/1.73 mThe secondary outcomes were to identify the risk factors for MAKE at day 30 (MAKE-30) and at day 90 (MAKE-90) defined as one of the following criteria within day 30 or day 90: death, receipt of RRT and SCr ≥ threefold increase.This study was approved by the ethical committee of the French Society of Intensive Care (SRLF).The ethical committee requested that attending physician received a mail with an information letter to deliver to the patient or to a family member.The database was declared to the French National Commission of data processing (CNIL).Quantitative variables are reported as median  and qualitative variables as number (%). For all the analyses the date of origin (day 0) was the of ECMO cannulation.We performed a univariate analysis with a logistic regression to determine factors associated with MAKE-360, MAKE-30 and MAKE-90 multivariate analysis included medically relevant items:eGFR at baseline, KDIGO stage at cannulation, SOFA score without hemodynamic and renal items, activated partial thromboplastin time (aPTT) at cannulation, duration of cardiopulmonary bypass (CBP), number of RBC packs while under ECMO, ECMO duration.Between 2014 and 2016, 203 patients received VA-ECMO during their ICU stays. 39 patients (19%) died within 48 h after implementation and 6 other patients had an exclusion criterion. One hundred and fifty-eight patients were included , baseline eGFR 78 [62–101], Simplified Acute Physiology Score II: 55 [39–66], Sepsis-related Organ Failure Assessment Score: 9 [7–12], time on VA-ECMO: 7.5 [4–12] days). Among them 145 (91.8%) develop an AKI during the intensive care unit (ICU) stay and 85 (54%) needed renal replacement therapy (RRT). Finally 152, 147 and 141 patients were included for the D30, D90 and 1-year analysis, respectively. At 1 year, 17 patients (10.1%) patients were lost to follow-up at 1 year (none undergoing RRT at last news) . The median baseline eGFR was 78.3 [62.2–101] ml/min/1.73 m2. The repartition of CKD stages at baseline is described in Fig. At baseline, 51 32.3%) patients had no significant CKD (eGFR over 90 ml/min/1.73 m.3% patieOnly 13 patients 8.2%) did not develop any AKI during ICU stay according to KDIGO classification and 85 (53.8%) patients needed RRT within ICU stay , KDIGO stage at cannulation (p = 0.03), e.g., stage 3 vs. reference stage 0 OR 10.20 [1.77–58.87], and number of red blood cell (RBC) packs received while under ECMO . At 1 year among the 51 survivors, almost half of the alive patients (n = 20/51) had a decline of estimated glomerular filtration (eGFR) > 30% ml/min/1.73 m2. Their median eGFR decline was − 26.3% . The only factor independently and constantly associated with MAKE whatever the evaluation timeline was the number of RBC packs received while under ECMO, OR 1.09 CI 95% [1.03–1.15], p < 0.01 at day 30 and OR 1.08 CI 95% [1.02–1.15], p < 0.01 at day 90.Factors significantly associated with MAKE-360 were eGFR at baseline (odds ratio (OR) 0.98, confidence interval 95% (CI) , Ninety-two (60.5%), 70 (47.6%), and 51 (38.9%) of patients were alive at D30, D90 and at 1 year, respectively. Among patients who died in ICU, 31 (43%) were not weaned of RRT at the time of death. Only 4 (6.7%) of the alive patients at ICU discharge were on RRT after the 1-year follow-up, three of them did not require RRT at ICU discharge but experienced recurrent AKI during follow-up.n = 20/51) had a decline of estimated glomerular filtration (eGFR) > 30% ml/min/1.73 m2. Their median eGFR decline was − 26.3%  corresponding to a median absolute decline of − 19.8  ml/min/1.73 m2.The repartition of CKD stages at 1 year is described in Fig. 2) at 1 year and half of those patients had a decline > 30%. Those results are of paramount importance since this technique is now commonly used worldwide.To date, only a few studies have evaluated the prevalence of AKI in patients undergoing VA-ECMO and long-term renal function of these patients was never reported. Among 158 patients included in our study, 145 (91.8%) develop an AKI during the intensive care unit (ICU) stay and 85 (53.8%) needed renal replacement therapy (RRT). 59.9% (91/152), 60.5% (89/147) and 85.1% (120/141) evaluable patients had a MAKE-30, MAKE-90 and MAKE-360, respectively. Factors significantly associated with MAKE-360 were eGFR, at baseline, KDIGO stage at cannulation and, number of RBC received while under ECMO. Our results documented also that among survivors of VA-ECMO therapy, long-term renal impairment is major with a median eGFR decline rate of 26.3% , and in matched controls without AKI , respectively. Given the small number of patients who undergo VA-ECMO annually in each center and the lack of long-term follow-up for these patients, such an analysis is not suitable. Consequently, we used a surrogate marker, i.e., decline in eGFR to assess the risk of ESRD. This surrogate outcome has been described by Coresh et al. [We found that baseline renal function was associated with MAKE at 1 year. In a large cohort of patients older than 67 years with hospital-associated AKI who survived to discharge, Ischani et al. , found th et al. , who anah et al. .The magnitude of the eGFR decline allows understanding that any second renal hit can precipitate patients to ESRD. As a matter of fact, in our study, ESRD at 1 year was rare, but occurred in patients with recurrent AKI. This pattern, i.e., relapsing without recovery, was previously described in a study that examined the different patterns for AKI reversal in critically ill patients. It represents 14.7% of the 16,968 patients studied .Extra-corporeal Life Support Organization) [An unexpected finding of this study was that RBC packs transfusion was associated with MAKE whatever the evaluation timeline. Aubron et al.  found thization)  and depeization) . Excessiization) . In adulization)  and in oization) .Results obtained in our study should be tempered by several limitations. First, our data reflect the experience of a single center potentially limited external validity. Second, we evaluated a mixed population of patients, who have received VA-ECMO following medical, postcardiotomy or post-cardiac arrest cardiogenic shock. A detailed evaluation of each specific population might be the focus on future studies. Third, although these analyses are based on the totality of our experience over 3 years, the total number of patients who underwent VA-ECMO is not high and our analyses could have been underpowered for some analyses. Fourth, many patients were lost to follow-up at 1 year and taking into consideration the high mortality rate of this population, these losses may have had a great impact on the results. Fifth, we did not collect data on anticoagulation and hemoglobin levels prior to transfusion, we did not monitor plasma free hemoglobin or other hemolysis parameters and due to retrospective data collection, we might lack information on potential unmeasured confounders.Patients receiving VA-ECMO are at risk to develop AKI within the ICU stay and had a severe eGFR decline at 1 year.


Planktochlorella nurekis with elevated cell size and DNA levels compared to wild type strain that was achieved by a safe non-vector approach, namely co-treatment with colchicine and cytochalasin B (CC). A slight increase in growth rate was observed in twelve clones of CC-treated cells. For biochemical profiling, several parameters were considered, namely the content of proteins, amino acids, lipids, fatty acids, β-glucans, chlorophylls, carotenoids, B vitamins and ash. CC-treated cells were characterized by elevated levels of lipids compared to unmodified cells. Moreover, the ratio of carotenoids to chlorophyll a and total antioxidant capacity were slightly increased in CC-treated cells. We suggest that Planktochlorella nurekis with modified DNA levels and improved lipid content can be considered to be used as a dietary supplement and biofuel feedstock.Microalgae are freshwater and marine unicellular photosynthetic organisms that utilize sunlight to produce biomass. Due to fast microalgal growth rate and their unique biochemical profiles and potential applications in food and renewable energy industries, the interest in microalgal research is rapidly increasing. Biochemical and genetic engineering have been considered to improve microalgal biomass production but these manipulations also limited microalgal growth. The aim of the study was the biochemical characterization of recently identified microalgal strain  Spirulina (Arthrospira) and Chlorella vulgaris, Dunaliella salina a natural source of β-carotene, Haematococcus pluvialis a producer of astaxanthin for aquaculture feed, Crypthecodinium a producer of long chain polyunsaturated fatty acid (LC-PUFA) docosahexaenoic acid (DHA) and Nannochloropsis a producer of eicosapentaenoic acid (EPA)  of microalgae may be several times higher than the oil yield of oil crops, e.g., microalgae with 70% and 30% oil of total biomass may achieve 23- and 10-fold higher oil yield compared to oil palm, respectively . Severalkessleri  and nitrkessleri ,26. Howevulgaris . Thus, avulgaris . For exanditions . The gro2 day−1) . Moreoveeudonana . The abieudonana . Howevereudonana ,18. Thuslyploidy . Two diplyploidy .Planktochlorella nurekis cells were established to be about 60% of dry weight  [Planktochlorella nurekis amino acid profile (Chlorella amino acid profile [p < 0.05), methionine , threonine , valine  and serine  and DNA content  ,5. In ge profile  was foun content . Our datPlanktochlorella nurekis were characterized by the levels of β-glucans of 8.5% of dry weight  and BHT (butylated hydroxytoluene) [Planktochlorella nurekis were comparable to TAC values of different Chlorella sp. samples [Chlamydomonas reinhardtii were also accompanied by cold and oxidative stress resistance [Spirulina platensis [The levels of chlorophyll a  and chlorophyll b  of WT cells  . Thus, ttoluene) . TAC val samples . CC co-tsistance . It has latensis . HoweverChlorella may also contain other beneficial components such as vitamins  and macro- and microelements  [As calcium) , we deciPlanktochlorella nurekis WT cells  and the amount of obtained DNA was quantified using a NanoDrop 2000 Spectrophotometer . The sequences of ITS regions were obtained by PCR amplification using Mastercycler gradient thermal cycler , and a pair of primers  [Planktochlorella nurekis species.The microalga samples were provided by Bioorganic Technologies sp. z o.o. . Species identification was based on DNA sequencing. Briefly, nucleic acids were extracted from the algae using 5% Chelex-100 as described elsewhere . The samACTA-3′) . Each rePlanktochlorella nurekis strains were grown in 120 L photobioreactors in liquid medium containing the following macro- and microelements: KNO3 (5 g/L), MgSO4 (2.5 g/L), KH2PO4 (1.25 g/L), (NH4)2SO4 (0.6 g/L), FeSO4x7H2O (3.7 mg/L), MnCl2x4H2O (2.2 mg/L), H3BO3 (1.5 mg/L), CoCl2x6H2O (15 µg/L), ZnSO4x7H2O (0.25 mg/L), CuSO4x5H2O (0.25 mg/L), NiSO4x6H2O (0.5 mg/L), NH4VO3 (23 µg/L), MoO3 (18 µg/L), Na2WO4x2H2O (0.16 mg/L), KI (0.05 mg/L) and EDTA (0.04 g/L) , pH 5.85–5.95 at 30 ± 1.0 °C under continuous artificial LED red light (645 nm) and blue light (460 nm) (ratio 3:1) and 16,000 LUX. Three independent cultures were considered.The 6/mL, 6-well plate, a total volume of 2 mL) were treated with colchicine  and cytochalasin B  (CC treatment) at 30 °C for 24 h with shaking (500 rpm/min) and continuous red and blue light (P.426103). Then, the cells were selected without colchicine and cytochalasin B for further sub-culturing in 6-well plates for 96 h and 250 mL flasks for 10 days. When the cell culture reached 5 × 105 cells/mL, the cells  were transferred to 2.5 L photobioreactors and grown in cell culture medium with CO2 flow (8%), continuous red and blue light at 30 °C for 7 days. Typically, the cell density of 6 × 106 cells/mL and 2 g of wet biomass per liter of culture were achieved at the end of the culture. To ensure that 24 h co-treatment with colchicine and cytochalasin B resulted in stable phenotype of increased cell size and DNA content compared to untreated cells, cells were additionally cultured without colchicine and cytochalasin B for 30 days and DNA levels were compared (time 0 and after 30 days) and cells were then propagated in 120 L bioreactors. During all selection steps, the cells were monitored using a light microscope (objectives 40× and 100×). The cell number and cell viability were monitored using a TC10™ automated cell counter and trypan blue staining  and measurements of optical density (OD) using a TECAN microplate reader at 500 nm. After CC treatment and cell selection in 2.5 L bioreactors, twelve clones were considered for further analysis based on the most potent proliferative activity as judged by OD values (OD measurements at 500 nm using a microplate reader). Wet biomass was obtained after centrifugation of the cell culture  and dry mass was obtained using thin layer drying method using a moisture analyzer at 42 °C for 24 h.For long-term selection, microalgal cells . 10,000 events were analyzed for each sample triplicate using two channels (bright field), namely Ch01 (435–480 nm) and Ch09 (570–595 nm) with the 488 nm and 642 nm lasers. Five subpopulations of cells were considered, namely single cells sized 5–10 µm (R1), single cells sized 1–5 µm (R2), large single cells sized 10–15 µm and autosporangia (R3), cell aggregates sized over 15 µm (R4) and dividing cells with autospores (R5). Representative dot plots and cell images are presented.The size of microalgal cells was analyzed using an Olympus BX61 differential interference contrast microscope equipped with a DP72 CCD camera and Olympus CellF . Moreover, the subpopulations of cells in terms of their cell size and formation of cell aggregates were investigated using Amnisv/v/v) in a total volume of 1 mL to 50 mg of cell pellet (wet biomass). Cell suspensions were mixed and centrifuged . These steps were repeated three times.DNA content analysis was performed as comprehensively described elsewhere  with min2SO4) and liberation of reduced nitrogen as ammonium sulphate. After alkalization of the mixture, the produced ammonia was distilled off with water vapor and bound in a boric acid solution. The distillate was titrated with a standard solution of hydrochloric acid. Protein content was calculated by multiplying the total Kjeldahl nitrogen by nitrogen-to-protein conversion factor of 5.95 that is recommended for Chlorella sp. [The protein levels were determined according to Kjeldahl method in laboratory accredited according to ISO/IEC 17025:2005 . This method is based on the mineralization of the sample using concentrated sulphuric acid .N-hydroxysuccinimidyl carbamate (AQC) as the reactant followed by ultra-performance liquid chromatography (UPLC) analysis with spectrophotometric detection (PDA) at 260 nm and using the AccQ* Tag column Ultra, C-18, 2.1 × 100 mm, 1.7 μm.Tryptophan was determined by high performance liquid chromatography with fluorescence detection (HPLC-FLD) method (Commission Regulation (EC) No. 152/2009 of January 27, 2009, Annex III G). The method for the determination of total tryptophan consisted of hydrolysis in alkaline medium using a saturated solution of barium hydroxide and heating the sample at 110 °C for 20 h. After hydrolysis, an internal standard was added, namely ISTD α-methyl-tryptophan. The following conditions were considered: column C18 ; column temperature: room temperature; isocratic elution; mobile phase: 3.00 g acetic acid + 900 mL water + 50.0 mL solution of 1,1,1-trichloro-2-methyl-2-propanol in methanol (1 g/100 mL) adjusted pH to 5.00 using ethanolamine and made up to 1 000 mL with water; flow rate: 1 mL/min; total run time: 34 min; detection wavelength: excitation: 280 nm, emission: 356 nm; injection volume: 20 μL. The other amino acids were determined using ultra-high performance liquid chromatography with spectrophotometric detection (UPLC-UV) method (test procedure PB 59 KLP). The method consisted of hydrolysis with 6N hydrochloric acid solution and the extraction of hydrolysis products with water. The obtained extract was subjected to pre-column derivatization using 6-aminoquinoline-Total fat (lipids) was determined in laboratory accredited according to ISO/IEC 17025:2005 . The method is based on hydrolysis of the sample with a hydrochloric acid solution to release fat from protein-fat and sugar-fat complexes, then separating the fat from the hydrolyzate by filtration through a filter paper, drying the filter and extracting the fat in a Soxhlet apparatus and determining the weight of fat content in the sample.v/v) and 300 μL of 0.6 M HCl in methanol were added to the sample. The vials were sealed (PTFE caps) and the content of the vial was mixed and placed in a laboratory oven  heated to 85 °C ± 3 °C for 1 h. The vials were then cooled to room temperature. After cooling, 1 mL of petroleum ether was added to a vial and the content was mixed for 1 min and allowed to separate for 1 h. 100 μL of the upper phase was transferred to a 2 mL chromatographic vial and 400 μL of petroleum ether was added. The analysis was carried out using a gas chromatograph  with a mass detector  in full scan mode. The following parameters were considered: ions from 50 m/z (mass to charge ratio) to 400 m/z were monitored, source temperature—230° C, ionization type—electron (EI), temperature program: 40–260 °C, column HP-5 MS (Ultra Inert/ 30 m × 0.25 mm I.D. × 0.25-μm). For identification and quantification of FAs external standard was used . The linearity was determined on the basis of six point calibration curves (R2 from 0.925 to 0.999).For determination of SAFA, MUFA, PUFA and selected fatty acids, Laboratory Analytical Procedure (LAP) was used . BrieflyThe analysis of β-glucan content was performed according to manufacturer’s instructions  and ,32 with v/v). The samples were then mixed (30 min), sonicated in an ultrasonic bath (5 min), incubated at 40 °C for 30 min with shaking (400 rpm) and centrifuged . The obtained supernatant was dissolved using 80% acetone (v/v) to give an absorbance ≤ 1. The measurements were performed using quartz cuvettes in a spectrophotometer Evolution™ 300 UV-Vis  at λ = 646.8 nm, 663.2 nm and 470 nm against 80% acetone as a blank. The samples were protected from light during the analysis. The following formula was used to calculate the content of chlorophyll a, chlorophyll b and carotenoids (µg/mL) using 80% acetone (v/v) as a solvent [a is a concentration of chlorophyll a, Cb is a concentration of chlorophyll b and Cx + c is a concentration of total carotenoids.The content of pigments  was assayed in dry biomass (50 mg) after extraction using 1 mL of 80% acetone . The test organism was provided with a liquid nutrient medium that contained an adequate quantity of all required growth nutrients except niacin that is essential for its growth. Addition of sample extract with hydrochloric acid or standard solution, containing niacin enabled growth of the organism. After sample extraction, graded amounts of sample or standard solutions were given to tubes with nutrient medium and were inoculated with the specific test organism. After incubation, the response was measured turbidimetrically and compared to that of calibration solutions with known concentrations (LOQ: 0.04 mg/100 g). Biotin was determined according to microbiological activity U.S. Pharmacopeia, USP 21, 3rd supplement, 1986, Method 88 . The test organism was provided with a liquid nutrient medium that contained an adequate quantity of all required growth nutrients except biotin that is essential for its growth. Addition of sample extract with sodium hydroxide or standard solution, containing biotin enabled growth of the organism. After sample extraction, graded amounts of sample or standard solutions were given to tubes with nutrient medium and were inoculated with the specific test organism. After incubation, the response was measured turbidimetrically and compared to that of calibration solutions with known concentrations (LOQ: 1 µg/100 g). Folic acid was determined according to microbiological activity modified EN 14131 . The test organism was provided with a liquid nutrient medium that contained an adequate quantity of all required growth nutrients except folic acid that is essential for its growth. Addition of sample extract or standard solution containing folic acid enabled growth of the organism. After sample extraction, graded amounts of sample or standard solutions were given to tubes with nutrient medium and inoculated with the specific test organism. After incubation, the response was measured turbidimetrically and compared to that of calibration solutions with known concentrations (LOQ: 5 µg/100 g).Vitamins were determined according to JARS, Poland, certificate of the Polish Center for Accreditation no AB 1095, test procedure the USP 34, method 441 (mod.), microb. Act. Riboflavin was determined using HPLC method . The sample was extracted using a methanolic solution with Titriplex V. The extract was separated by HPLC on a reversed phase column. The quantification of vitamin B2 was performed using UV-detection at 268 nm (Limit of Quantification (LOQ): 0.05 mg/100 g). Niacin was determined according to microbiological activity USP 30, 2007, Meth. 441 (with test organism: 1 is a crucible mass (g), m2 is a mass of algae (g) and m3 is a mass of the crucible with the residue after burning (g).For ash content analysis, the crucibles were pre-conditioned in a muffle furnace at 575 °C for 20 h, cooled to a temperature of about 100 °C and transferred to a desiccator. After reaching room temperature, the crucibles were weighed on the analytical balance with an accuracy of 0.1 mg. Samples of microalgae were weighed into the crucibles (100 mg ± 5 mg). Samples were burned in a muffle furnace at 575 °C for 6 h. After cooling to a temperature of about 100 °C, the crucibles were transferred to the desiccator and after reaching room temperature, the crucibles and ashes were weighed. Ash content was calculated from the formula:p-value < 0.05 was considered as a statistically significant.The mean values ± SD were calculated on the basis of at least three independent experiments. Box and whisker plots were also considered. Statistical significance was evaluated using GraphPad Prism 5 using one-way ANOVA and Dunnett’s test. Correlation analysis of the data was performed using a Linear Correlation (Pearson r) test. A We strongly believe that established protocol of a co-treatment with colchicine and cytochalasin B (CC) to manipulate DNA levels and modulate functional bio-components may be widely used to obtain microalgal cells of different species with improved biochemical features that may have potential applications in food and renewable energy industries. CC-modified microalgal cells may be considered as a more attractive source of dietary supplements, nutraceuticals and as a biofuel feedstock compared to untreated cells. More studies involving other microalgal species are needed to confirm such assumptions.