Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells  infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae  depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored. Mitochondria are multifaceted organelles with an architecture extraordinarily interconnected with cellular functions. Mitochondria can change their structure in coordination with both physiological and pathological cellular processes and these alterations influence a plethora of cell signaling pathways such as bioenergetics  and cellThe impact of mitochondrial dynamics in cells from the immune system and in the modulation of immune responses has been addressed ; Oswaldo Cruz Foundation, Brazil ; and University of Tennessee Health Science Center (UTHSC), United States of America.Six-week-old female BALB/c and Swiss Webster mice were obtained from the animal facilities of the Institute of Biophysics Carlos Chagas Filho , and Oswaldo Cruz Foundation breeding unit . Six-week-old female C57BL/6J mice were purchased from Jackson Laboratories . Mice were housed in environmentally controlled conditions  and monitored daily for survival and wellbeing status . Animals were housed at least 1 week before investigation and all experiments were performed during the light cycle.ad libitum access to standard ovabulmin (OVA)-free laboratory chow and water, were immunized subcutaneously on day 0 with 50 μg OVA  and 5 μg aluminum hydroxide (Sigma) in 200 μL of 0.9% NaCl  as before (n = 5) were euthanized in a CO2 chamber, perfused with 20 ml of sterile PBS to remove blood, and fragments of the right lung lobe immediately fixed and processed for both histology (n = 5) as before  were infected with 1000 TCID50 pH1N1 virus, intranasally under isoflurane anesthesia as previously described , mice received just saline. Bronchoalveolar lavage (BAL) was performed by 2 washes each with 1 ml PBS and recovered cells immediately fixed and processed for TEM, as detailed below.Weight-matched C57BL/6J mice (escribed . The A/Cescribed . The terS. mansoni (100 cercariae/mouse), LE strain as previous work (n = 6) and respective uninfected controls (n = 6) from the same age were euthanized at 55 days of infection (acute phase) (Schistosoma mansoni-infected mice (Swiss Webster mice aged 70 days were injected subcutaneously with a single inoculum of cercariae of ous work . Infectious work . Infectee phase)  by exsane phase) . Fragmene phase)  and TEM ted mice  as previted mice .Bone marrow–derived eosinophils (BMdEos) were generated as described  from the6 cells/well) were incubated with fluorescently tagged antibodies in PBS with 0.1% BSA for 30 min at 4°C. After staining, the cells were washed with PBS with 0.1% BSA and fixed in 4% paraformaldehyde. Data were acquired in a FACS Calibur flow cytometer (BD Biosciences) and analyzed using FloJo software v 7.1 . Positive cells were identified by comparison to the appropriately conjugated isotype control. Antibodies  used included PE-conjugated rat anti-mouse Siglec-F or PE-conjugated IgG2a isotype control.BMdEos  in 0.1 M sodium phosphate buffer in 0.1 M sodium phosphate buffer at 4°C and kept in the same buffer for additional processing. Cell suspensions  were fixed in the same fixative solution as above (while still in suspension) for 1 h at RT or in 1% paraformaldehyde and 1.5% glutaraldehyde) in 1 M sodium cacodylate buffer, pH 7.4, for 1 h at RT and embedded in agar pellets for accurate handling of the eosinophils as previously described (en bloc in 2% uranyl acetate (EMS) in 0.05 M sodium maleate buffer, pH 6.0, for 2 h at RT and washed in the same buffer as before prior to dehydration in graded ethanol and acetone, and infiltration and embedding with a propylene oxide-Epon sequence . After polymerization at 60°C for 16 h, thin sections were obtained using a diamond knife on an ultramicrotome . Sections were mounted on uncoated 200-mesh copper grids (Ted Pella) before staining with lead citrate and viewed with a transmission electron microscope  at 80-120 KV.Tissue fragments (lungs and intestines) were fixed in a mixture of freshly prepared aldehydes [final concentration of 1% paraformaldehyde and 2.5% glutaraldehyde ] in 0.1 M sodium phosphate buffer, pH 7.4, at room temperature (RT). After 2h, fragments were sliced into small pieces of 1 mmescribed . All samn = 204 cells and 1720 mitochondria): 1) Mitochondrial numbers; 2) Mitochondrial size ; 3) Mitochondria proportion/cell section   Cristae morphology; 7) Cristae volume   ; 4) Totaon area) . CristaeElectron tomography and 3D reconstruction were performed as previous works from our group . Eponatep value <0.05 was considered statistically significant. All quantitative analyses were performed using Fiji software .One-way or 2-way ANOVA followed by Tukey multiple comparisons test or the Student’s t test was performed using GraphPad Prism version 6.01 . The normal distribution analysis (Shapiro-Wilk test) was also used to evaluate the shape descriptors. p value <0.05 was considered statistically significant.Multivariate logistic regression analyses were additionally performed using SPSS data analysis statistics software system version 20.0  to estimate the probability (or risk) of a particular outcome (immature or mature eosinophils) given the values of the independent variables, that is, the effect of a group of independent variables on a binary outcome by quantifying each independent variable’s unique contribution . The resex vivo culture system, which generates large numbers of eosinophils at high purity was established from unselected bone marrow progenitors as described  for optimal morphological preservation . ConsideAn important feature of the eosinophil developmental morphology is related to the nucleus, which is initially round, large, and predominantly euchromatic becoming segmented and with condensed peripheral chromatin . Another2 of 64.6 ± 3.8, 43.9 ± 2.7 and 28.2 ± 2.1 for precursor (myeloblast-like), immature, and mature eosinophils, respectively, n = total of 88 cells, mean ± SEM).By correlating the ultrastructural aspects of the cytoplasmic secretory granules and nucleus, we followed the development of mouse eosinophils in cultures, which was also accompanied by a reduction of the cell size . From unHaving established the maturational stages of mouse eosinophils , we nextn = 608 from precursor cells; n = 166 from immature eosinophils; and n = 151 from mature eosinophils), we found that the range of mitochondria numbers per cell section was 5-32, 4-26, and 2-20 in precursor cells, immature, and mature eosinophils, respectively. However, because mitochondria can display different sizes varying from round to very elongated morphology, we consider that the evaluation of the area occupied by these organelles in the eosinophil cytoplasm would be a more accurate parameter for mitochondria quantification than simply enumerating these organelles. Thus, by performing imaging analyses, the mitochondrial area (µm2) was delimited and measured in relation to the cytoplasmic area in each eosinophil section  , mean ± SEM; p < 0.01)  . When eo < 0.01) .2 , while mature cells had a mean size of 0.098 ± 0.007 µm2 . As expected, a diversity of mitochondrial sizes was obtained. However, this range was much higher in immature [14–886 nm2 (n = 166 mitochondria)] compared to mature . Interestingly, while most mitochondria (∼ 65%) in immature eosinophils had sizes higher than 0.100 µm2, mature eosinophils exhibited ∼ 70% of their mitochondrial population with sizes below 0.100 µm2  . In imma.100 µm2 .Mitochondria are organelles with a high level of plasticity, able to undergo morphological changes and interact with other organelles consonant with the cell needs . By examFirst, TEM revealed that mitophagy, the selective degradation of mitochondria by autophagy , was a cSecond, we identified different modes of mitochondrial interactions (inter-organelle membrane contacts) in the cytoplasm. In addition to the recognized mitochondria-ER  and mitop < 0.0001 for all types of interaction)  = 166 for immature and 151 for mature eosinophils; raction) . Remarkaraction) . On the raction) .p = 0.035) or other mitochondria  is associated with an increased chance of a sample being of immature eosinophils while a greater number of the mitochondria in close proximity to cored granules  is associated with an increased chance of the sample being of mature eosinophils (n = 503 mitochondria from immature and 166 from mature eosinophils) with a pseudo R2 (Nagelkerke) of 0.77 and a predictive accuracy of 91.2%. Altogether, our data point out that the mitochondrion-granule interaction is not a random phenomenon.Because this mitochondria-granules interaction particularly drew our attention, we performed multivariate logistic regression analyses to evaluate the proximity of mitochondria with other 7 cell compartments  with independent variables being used to determine the influence of these multiple factors at the same time. Our data confirmed that a greater number of mitochondria in close proximity to coreless granules  (p < 0.0001)  . Moreove 0.0001) . In cont 0.0001) .in situ in eosinophils from the lung parenchyma , large intestine (schistosomiasis mansoni), and in BALs (IAV infection) after cell fixation and processing for TEM following the same protocols that we have been using to conduct pathological analyses of human eosinophils in tissues and cell suspensions, which enable optimal morphology  for cytoplasmic area occupied by mitochondria are: 1) asthma: 2.24 ± 0.28  versus 1.64 ± 0.28 ; p = 0.16; 2) schistosomiasis: 7.12 ± 0.61  versus 5.76 ± 0.3 ; p < 0.05; 3) H1N1 IAV infection: 4.79 ± 0.77  versus 2.94 ± 0.34 ; p < 0.05.Compared to control eosinophils from the same organ/tissue/condition, the cytoplasmic area occupied by mitochondria within eosinophils did not change in allergic inflammation and it was reduced in response to both viral and parasitic infections . Means ±n = number of mitochondria) for cristae numbers are: 1) asthma: 3.62 ± 0.36  and 7.82 ± 1.02 ; p < 0.0001; 2) schistosomiasis: 6.36 ± 0.37  and 7.5 ± 0.31 ; p < 0.0001; 3) H1N1 IAV infection: 20.36 ± 1.46  and 15.32 ± 1.17 ; p < 0.0001. Means ± SEM (n = number of mitochondria) for cristae volume are: 1) asthma: 0.17 ± 0.006  and 0.19 ± 0.005 ; p = 0.006; 2) schistosomiasis: 0.14 ± 0.004  and 0.16 ± 0.004 ; p < 0.001; 3) H1N1 IAV infection: 0.17 ± 0.009  and 0.14 ± 0.007 ; p = 0.032).On the other hand, our imaging analyses at high resolution captured a significant increase in the numbers  and voluWhen in the tissue microenvironment, mitochondria within eosinophils formed mixed cristae, a morphological pattern characterized by tubular and lamellar cristae within the same mitochondrion , not obsp > 0.05), mitochondria-mitochondria interactions increased in asthma (Means ± SEM (n = mitochondria) of 8.74 ± 0.69%  versus 12.22 ± 0.93% ; p = 0.004; (n = mitochondria) of 18.44 ± 0.61%  versus 28.87 ± 0.62 ; p < 0.0001); (n = 110) versus 9.60 ± 0.43% ; p < 0.0001 ; ; and red< 0.0001 .n = 62) versus 9.11 ± 0.49% ; p = 0.01; 2) H1N1 IAV model  versus 19.04 ± 0.96%  shistosomiasis model  versus 21.10 ± 0.60% ; p < 0.0001). These mitochondria-granule associations involved not only membrane contacts, frequently seen as areas with increased electron density, but also tethers , as previously described   or can bThe mitochondrial behavior within eosinophils recruited by the IAV infection particularly drew our attention because several pieces of evidence show that H1N1 IAV target mitochondrial-dependent mechanisms [reviewed in ]. In infMitochondria inter-organelle interactions as a mode to regulate cell functions have been a subject of great interest. While TEM has the limitation of relying on static observations, it enables distinguishing between contiguous membranes and accurate quantification of mitochondrial morphology and interactions . One of In inflammatory tissue eosinophils, mitochondria-mitochondria interactions increase in all conditions except in the H1N1 IAV infection whereas mitochondria-ER interaction was not amplified likely because the peripheral ER in mature eosinophils is not a prominent organelle . While mIn conclusion, we identified that eosinophilopoiesis leads to a significant reduction of the mitochondrial population and that both differentiation and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils. The resulting increased cristae remodeling and inter-organelle contacts, including mitochondria-secretory granules interactions, may potentially influence eosinophil immune responses. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.


A majority of thereported precursors are dicoordinated with the Sn(II) amidinates,the only tetracoordinated examples. No Ge(II) and Pb(II) amidinatessuitable for vapor deposition have been demonstrated. Herein, we presenttetracoordinated Ge(II), Sn(II), and Pb(II) complexes bearing twosets of chelating 1,3-di- Only a fewtetracoordinated M−N bonded vapor depositionprecursors exist for divalent group 14 elements. Although there area couple of volatile tetracoordinated tin(II) amidinates, no suchGe and Pb compounds are suitable. Herein, we present volatile group14 triazenides as potential vapor deposition precursors. These compoundsare thermally stable, sublime quantitatively between 65 and 70 °C(0.5 mbar), and show single-step volatilization curves by thermogravimetricanalysis. High-quality thin films are commonly grown by chemical vapordeposition (CVD) methods. One subset of CVD is atomic layer deposition(ALD), which is a low-temperature alternative that introduces themetal and nonmetal precursors into the reaction chamber separatelyin alternating pulses.20 This separationallows the deposition process to be governed exclusively by self-limitingchemical reactions on the growth surface. To obtain high-quality filmsby ALD, it is important to have metal and nonmetal precursors thatpossess favorable physical and chemical properties.21 An ALD precursor must be sufficiently volatile for gas-phasetransport to the growth region. It should not decompose in the gasphase in a manner that leads to continuous CVD growth.22 At the surface, the precursor should react ina self-limiting manner to form a stable monolayer and easy-to-purgebyproducts.23 After purging, the monolayershould react with a second precursor (e.g. H2S and H2O) in a second self-limiting reaction. To maximize film growth,the precursor should employ small ligands for effective surface saturationand dense coverage.24 Furthermore, usingprecursors of the same oxidation state as the target material removesthe need for redox chemistry during deposition. Not having to relyon redox chemistry for the deposition process reduces the risk offorming undesired mixed-phase materials. Precursors with M–Nbonds are desirable for ALD because of the high reactivity of thepolarized bond. To date, there is only a small library of exclusivelyM–N bonded Ge(II), Sn(II), and Pb(II) precursors suitable forALD. Group 14 chalcogenidesare of interest because of their outstandingelectrical and optical properties.25 and GeSe26 by ALD, respectively. However,the deposited GeS was X-ray amorphous, while the GeSe films containedunwanted carbon impurities. SnS has been demonstrated by ALD usingthe dicoordinated cyclic diamide and, tetracoordinated Sn(II) formamidinateand amidinate precursors.28 The amidinate ligandsystem provides tetracoordinated, thermally stable, and volatile precursorsthat afford stochiometric SnS with low levels of impurities.The dicoordinated Ge(II) cyclic diamide and hexamethyldisilazidecompounds have been used to deposit GeS29 However, the deposition temperature for ALDgrowth was limited to ≤155 °C due to the poor thermalstability of the deposited surface species. Volatility data have notbeen reported for the tetracoordinated Ge(II) and Pb(II) amidinatesin the literature.31 There are no other exclusivelyM–N bonded tetracoordinated Ge(II) and Pb(II) compounds usedas precursors for ALD, as far as we know.Recently, PbS was deposited using the dicoordinated Pb–Nbonded hexamethyldisilazide and cyclic diamide precursors, renderingnear stochiometric films with low levels of carbon impurities.32 Thereis no mention of their volatility, and these compounds are most likelynot suitable for vapor deposition because of their bulky 1,3-bistriazenideligand. Recently, we explored the 1,3-dialkyltriazenide ligand forgroup 13 metals and used the In(III) and Ga(III) 1,3-diisopropyltriazenideprecursors for ALD of InN and GaN.35 To further explore the1,3-dialkyltriazenide ligand, we envisaged its ability to stabilizedivalent group 14 elements. This would potentially give a new classof tetracoordinated M–N bonded group 14 precursors for ALD.Herein, we present the synthesis, characterization, and thermal studyof Ge(II), Sn(II), and Pb(II) compounds bearing two sets of the 1,3-di-tert-butyltriazenide ligand. Their high volatility and thermalstability make these group 14 triazenides interesting for use as ALDprecursors.A ligand system closelyrelated to the amidinates is the triazenide,where the endocyclic C is replaced by N. There are reports of tetracoordinateddivalent group 14 triazenides.tert-butyltriazenide)lithium with MCl2, whereM = Ge, Sn,or Pb, gave 1–3 in good yields afterrecrystallization .Compounds 1–3 are air- and moisture-sensitiveand low-melting yellow solids. The compounds were characterized byNMR spectroscopy, X-ray crystallography, electron impact mass spectrometry,elemental analysis, and melting point measurement.The reaction of  of 0.65.36 See the Supporting Information for the geometry index calculations for compounds 1–3 and the crystal structures of compounds 2 and 3. Compounds 2 and 3 have crystal structures and molecular geometries analogous to thatof 1. However, the Sn and Pb centers are further distortedtoward tetrahedral geometry . The βangle decreases in the order 1 > 2 > 3. This trend can be explained by a greater p character ofthe M–N bond when moving down the group  computationsare in good agreement with their crystal structures (see the Supporting Information). The stereochemicallyactive lone pair resides in the highest occupied molecular orbital and Pb(II) compounds, making them potentiallyuseful as vapor deposition precursors. When comparing TGA data, compound 2 appears only slightly less volatile than Sn(II) formamidinate.28 This result is noteworthy as Sn(II) formamidinateis the most volatile tetracoordinated M–N bonded compound todate. The TGA data show 1–3 are highlyvolatile, exhibiting 1 Torr of vapor pressures between ∼90and 100 °C (1–3 sublime quantitativelybetween 60 and 75 °C under reduced pressure (0.5 mbar).Thermogravimetric analysis (TGA)showed ual mass 3. Compoud 100 °C 3. Compou1 showedoverlapping exotherms at 181 °C indicated by the irregular peakshape. The compound degraded when heated to 200 °C in a sealedcapillary, suggesting the exotherm is a decomposition event. Compound 2 gave an exotherm at 175 °C, preceded by a small peakat 160 °C. However, it remained intact when heated to 200 °Cfor 24 h, indicating the exotherms at 160 °C and 175 °Care not decompositon events. A third exotherm was found at 280 °Cand is most likely a decomposition event as the compound releasedgas bubbles during melting and changed from a yellow liquid to anoff-white solid when heated to 275 °C in a sealed capillary.Compound 3 showed an exotherm at 85 °C, but themelt resolidified, and the melting point was unaffected by repeatedheating of the compound to 110 °C for hours in sealed capillaries.The heat-treated compound remained unchanged from pure 3, both visually and by 1H NMR analysis, suggesting theexotherm at 85 °C is not a decomposition event. A second exothermof sharp overlapping signals was found at 170 °C. This exothermat 170 °C is most likely a decomposition event as the compounddarkened when heated above 180 °C in a sealed capillary. Uponcooling, the melt resolidified into an inhomogeneous, yellow and graysolid. A third exotherm was observed between 270 and 300 °C.The sublimation temperatures of 1–3 and their TGA and DSC data are summarized in Supporting Information for individual TGA and DSC data.Differentialscanning calorimetry (DSC) of 3, compounds 1–3 have onsets of decomposition fromDSC well above their respective onsets of volatilization from TGA.DSC revealed no thermal events occurring for 1 and 2 at temperatures <100 °C, consistent with the observationsduring melting point measurements.Disregardingthe first exotherm for 1 and 3 are the first examples of tetracoordinatedand exclusively M–N bonded Ge(II) and Pb(II) compounds withhigh volatility and thermal stability. The compounds sublime quantitativelybetween 60 and 75 °C (at 0.5 mbar), and show near-ideal volatilizationby TGA with onsets between ∼130 and 160 °C.Insummary, we have presented volatile divalent Ge, Sn, and Pbtriazenides as potential precursors for vapor deposition. To our knowledge, Caution! Because catenated nitrogen compounds are knownto be associatedwith explosive hazards, tert-butylazide and compounds 1–3 are possible explosive energetic materials.Although we have not experienced any problems in the synthesis, characterization,sublimation, heating, and handling of compounds 1–3, their energetic properties have not been fully investigatedand are therefore unknown. We therefore highly recommend all appropriatestandard safety precautions for handling explosive materials  always be used when working with tert-butylazideand compounds 1–3. In addition, keeptert-butylazide and compounds 1–3 away from other metals (Cu and Ag) and Hg bubblers while using Schlenkequipment.2 atmosphere, either on a Schlenk line usingSchlenk air-free techniques or in a dry box. All anhydrous solventswere purchased from Sigma-Aldrich and further dried over molecularsieves 4 Å. The GeCl2·dioxane complex, SnCl2 (98%) and PbCl2 (98%), and a tert-butyllithium solution (1.7 M in pentane) were purchased from Sigma-Aldrichand used without further purification. The preparation of tert-butylazide was undertaken according to the literatureprocedure.37 All NMR spectra were measuredwith Oxford Varian 300 and AS500 spectrometers at room temperatureunless otherwise stated. Solvent peaks were used as internal standardsfor 1H NMR (300 and 500 MHz) and 13C NMR (75and 125 MHz). Electron ionization mass spectrometry (EI-MS) was performedwith a Kratos Concept–Magnetic sector mass spectrometer inhigh-resolution mode. Melting points were determined in sealed capillarieswith a Stuart SMP10 melting point apparatus and are uncorrected. Elementalanalyses were performed by Mikroanalytisches Laboratorium Kolbe, Germany.All reactions and manipulations were carriedout under a Ntert-Butyllithium  was added dropwise to a solution of tert-butylazide in n-hexane (150 mL) at −78°C. The reaction mixture was stirred for 30 min and then allowedto warm to room temperature for 1 h. The reaction mixture was thenconcentrated under reduced pressure to give a light-yellow solid . This solid was of sufficient purity to be used in the subsequentreactions without further purification. A small amount was purifiedfor characterization by recrystallization from n-hexaneat −35 °C to give a solid. Colorless solid, mp: 247−250°C. 1H NMR :δ 1.28 . 13C{1H} NMR : δ 30.7 , 56.7 . Anal. Calcd for C8H18LiN3: C, 58.88; H, 11.12; N, 25.75. Found: C,58.88; H, 11.14; N, 25.67.tert-butyltriazenide)lithium(I)  in THF (20 mL) at−35 °C was added dropwise to a solution of GeCl2·dioxane  in THF (30 mL) at −35 °C.The reaction mixture was held at −35 °C for 10 min andthen stirred at room temperature for 16 h. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue wassuspended in n-hexane, filtered through a pad ofCelite, and concentrated under reduced pressure to give the crudeproduct. The crude product was purified by recrystallization fromTHF/acetonitrile (MeCN) at −35 °C to give 1 as a solid .A solution of . 1H NMR : δ 1.40 . 13C{1H} NMR : δ 30.6 , 59.1. EI-MS (LR): 230.1 (100%), 245.1 (<5%), 287.1(<5%), 329.2 (25%), 386.2 (30%). Anal. Calcd for C16H36GeN6: C, 49.90; H, 9.42; N, 21.82. Found:C, 49.70; H, 9.40; N, 21.78.tert-butyltriazenide)lithium(I) in a 1:4 mixture of Et2O/n-hexane (5 mL) at −35 °C was added dropwise to a suspensionof SnCl2  in n-hexane(5 mL) at −35 °C and then stirred at room temperaturefor 16 h. The reaction mixture was filtered through a pad of Celiteand concentrated under reduced pressure to give the crude product.The crude product was purified by recrystallization from THF/MeCNat −35 °C to give 2 as a solid .Asolution of . 1H NMR : δ 1.35 . 13C{1H} NMR : δ30.6 , 58.9 . EI-MS (LR): 134.9(8%), 274.1 (8%), 373.1 (<5%), 432.2 (25%). Anal. Calcd for C16H36N6Sn: C, 44.57; H, 8.41; N, 19.49.Found: C, 44.04; H, 8.31; N, 19.23.3 was synthesized in thesame manner as 2 using a solution of lithium(I)  in a1:4 mixture of Et2O/n-hexane (20 mL) anda suspension of PbCl2  in n-hexane (20 mL). The crude product was purified by recrystallizationfrom THF/MeCN at −35 °C to give 3 as a solid.Compound 3: Yellow solid, mp: 83−84°C. Sublimation: ∼75 °C (0.5 mbar). 1HNMR : δ 1.30 . 13C{1H} NMR : δ 30.5 , 61.1 . Anal.Calcd for C16H36N6Pb: C, 36.98; H,6.98; N, 16.17. Found: C, 36.56; H, 6.97; N, 16.03.1–3 were obtained byrecrystallizationfrom THF/MeCN at −35 °C. The single crystals were usedfor X-ray diffraction data collection on a Bruker D8 SMART Apex-IIdiffractometer, using graphite-monochromated Mo Kα radiation(λ = 0.71073 Å) at 153 K. All data were collected in ahemisphere with over 95% completeness to 2θ < 50.05°.The structures were solved by direct methods. The coordinates of themetal atoms were determined from the initial solutions and the N andC atoms by subsequent differential Fourier syntheses. The solutiondid not contain much residual electron density, but it remained fora multitude of additional possible positions of light atoms. All non-Hatoms were refined, first by isotropic and then by anisotropic approximationusing Bruker SHELXTL software. The H atoms were addedin a riding approximation and refined isotropically. The solutionsof compounds 1 and 2 gave one B-level alerteach, which was caused by one atom in each structure failing the Hirshfeldtest. These two atoms had significantly bent thermal ellipsoids, potentiallycaused by more than one preferred orientation for these atoms. Theseellipsoids showed no preferred orientation upon manual inspection.Selected crystal data are summarized below.Singlecrystals of 1:C16H36GeN6, M =385.12, monoclinic, space group P2/c, a = 9.702(7) Å, b = 6.055(5)Å, c = 18.549(14) Å, α = 90°,β = 100.971(9)°, γ = 90°, V = 1069.6(14) Å3, Z = 2, Dc = 1.196 g cm−3, μ= 1.440 mm−1, T = 153 K, 6500 reflectionsmeasured, 1877 unique, final R1 [I > 2σ(I)] = 0.0254, wR2  = 0.0672, GOF = 1.040.2: C16H36N6Sn, M = 431.20, orthorhombic, space group Pbcn, a = 12.732(5) Å, b = 13.049(5)Å, c = 13.693(6) Å, V =2274.9(16) Å3, Z = 4, Dc = 1.259 g cm−3, μ = 1.131 mm−1, T = 153 K, 16479 reflections measured,2051 unique, final R1 [I > 2σ(I)] = 0.0256, wR2  = 0.0696, GOF = 0.934.3: C16H36N6Pb, M = 519.70, orthorhombic, space group Pbcn, a = 12.786(4) Å, b = 12.794(4)Å, c = 13.734(4) Å, V =2246.7(12) Å3, Z = 4, Dc = 1.536 g cm−3, μ = 7.518 mm−1, T = 153 K, 15085 reflections measured,1982 unique, final R1 [I > 2σ(I)] = 0.0236, wR2  = 0.0795, GOF = 0.936.2 (99.998 % purity). Pt pans were cleaned by ultrasonication,first in dilute nitric acid (∼3 M), then water, and last 2-propanol.The pans were heated in air by a propane torch until red hot to removeany remaining impurities. During the TGA experiments of 3, if the compound decomposes, then Pb(0) might form and, consequently,alloy with the Pt pans. To avoid potential alloying, pans used forTGA of 3 were coated with a 55 nm Al2O3 layer as a precaution. The coating was grown at 200 °C,using trimethylaluminum and water (0.1 s pulses and 8 s purges each)for 500 cycles.38 All TGA experiments wereperformed under a flow of ultrapure N2 .For ramp experiments, samples were heated to 500 °C at a rateof 10 °C min−1. The Langmuir vapor pressureequations for compounds 1–3 werederived from the TGA data with 10 mg of mass loading using a previouslyreported method39 and employing biscopper(II)as the calibrant.40 Isothermal TGA werecarried out at 90 and 110 °C. A heating rate of 40 °C min−1 was used to rapidly reach the desired temperature.The experiments were carried out with the same mass loadings as theramp experiments. The onset of volatilization was defined as the intersectionbetween the tangent lines of the plateau and slope.TGA was performed on Pt pans with aTA InstrumentsQ50 analyzer housed in an MBraun Labmaster 130 dry box filled withN1–3 were sealedin Al pans. Unless otherwise stated, all samples were heated to 400°C at a rate of 10 °C min−1. N2 (99.998%) was used as the purge gas. Experiments were performedin triplicate with similar mass loadings to ensure validity of therecorded data.DSC experiments were performedusing aTA Instruments Q10 instrument. Inside a glovebox, samples of 0.30± 0.03 mg of Gaussian 16 software.41 Structural optimization andharmonic normal-mode vibrational calculations were done using theB3LYP method43 together with Grimme’sversion 3 dispersion correction44 and thedef2TZVP46 basis set. Minimized structures were confirmedto have no imaginary frequencies.All quantum-chemicalDFT computations were performed using


This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a  N6-methyladenosine (m6A) mRNA methylation in development , which rely on chemical, enzymatic, and/or antibody-based detection of modified ribonucleosides , 2, 3. Telopment , 6, 7, 8neration , 14, 15,hylation , 10. Furhylation , 18, 19.hylation , make itTandem mass spectrometry (LC-MS/MS) is a highly accurate tool for analysis of modified RNAs, which has so far been applied primarily in two approaches to identify and quantify RNA modifications . The fir13C-methyl]-methionine labeling and mass spectrometry (MS) to assess the turnover of base modifications (13C-dynamods) in newly transcribed RNA. With 13C-dynamods, we trace the proportion of newly methylated ribonucleosides and their decay through time, i.e., methylation turnover, from digested polyadenylated RNA and ncRNA. We first showed that polyadenylated RNA and ncRNA were distinguished by the different turnover frequencies (in h−1) of modified ribonucleosides, which are inherently linked to the different half-lives of mRNA, rRNA, and tRNA . We then applied 13C-dynamods in conjugation with abundance measurements of modified ribonucleosides in polyA+ and ncRNA, which resolved their transcriptional versus posttranscriptional sensitivity in response to actinomycin D and metabolic stresses. Thus, the quantitative nature of 13C-dynamods demonstrates its capacity for sensitive characterization of modified ribonucleosides, their origin and dynamics, in multiple RNA classes of interest.Stable isotope labeling is a well-established method to quantify metabolic activity in cultured cells , 42, 43.and tRNA , 44, 45.13C-methyl]-methionine and analyzed the isotopologues  of modified and unmodified ribonucleosides by tandem mass spectrometry (LC-MS/MS) (A and B). The m + 0 isotopologue  represents the mass of the analyzed molecule where all atoms are present as the most common isotope, whereas the m + 1 isotopologue  indicates the mass shift due to 13C incorporation from the 13C-labeled methionine tracer or from the natural abundance of 13C, 15N, 18O, and 2H stable isotopes. With 13C-dynamods, we measure the isotopologue fractions [m + 1/(m + 1 + m + 0)] of modified and unmodified ribonucleosides, which reflect the amount of 13C enrichment (m + 1) in the measured ribonucleoside relative to the total pool of that ribonucleoside (m + 1 + m + 0) (A). Thus, the isotopologue fractions of each ribonucleoside during [13C-methyl]-methionine labeling are internally controlled for the amount of preexisting ribonucleosides  prior to 13C labeling. Here, we analyzed the isotopologues of N6-methyladenosine (m6A), 7-methylguanosine (m7G), 1-methyladenosine (m1A), N6,N6-dimethyladenosine (m62A), 2′-O-methyladensone (Am), 5-methylcytosine (m5C), and unmodified ribonucleosides from digested polyadenylated (polyA+), large (>200 nt) and small (<200 nt) RNA. We observed increased m + 1 and concomitant decreased m + 0 ion counts in modified ribonucleosides from polyA+ and large RNA after 4 and 24 h of [13C-methyl]-methionine labeling, while the m + 1 fractions  of the unmodified ribonucleosides was unaltered during the labeling period . Thus, the change in the “heavy” (m + 1) isotopologue fraction relative to the unlabeled condition  indicates SAM-dependent RNA methylation of newly synthesized RNA, whose turnover we have examined within and across RNA classes. In contrast to singly methylated ribonucleosides, the m62A modification exhibited an enrichment mostly of the m + 2 isotopologue upon 13C labeling (A), and so the m + 2 fraction was used to assess m62A dynamics in subsequent analyses.S-adenosylmethionine (SAM) is the direct substrate of RNA methylation reactions in eukaryotic cells . To tracC-MS/MS) , A and B+ m + 0) A. Thus, ereafter B. Thus, labeling A, and so6A, m7G, and m5C in polyA+-purified RNA when compared with large (>200 nt) and small (<200 nt) RNA (E), in accordance with the faster life cycle of mRNA in mammalian cells , suggesting that a large portion of the signal for these modifications in the polyA+ fraction might derive from contaminating ribosomal RNA (rRNA). Of note, the isotopologues are analyzed on all ribonucleosides from the same polyA+ pool (C), so the purity of polyA+ enrichment is the same in all cases. This indicates that the relative abundance in mRNAs versus contaminating ncRNA is much higher for m6A, m7G, and m5C compared with m1A, m62A, and Am. Interestingly, we detected m62A in small RNAs from 786O cells, and its kinetics was different when compared with large RNA (E).Quantification of isotopologue fractions showed a faster kinetics for m nt) RNA E, in accan cells . In contractions E, suggesyA+ pool C, so thearge RNA E.13C-methyl]-methionine followed by replacement (chase) with unlabeled methionine and analyzed the isotopologues over time. In accordance with the preceding findings (E), we observed an exponential decay of the m + 1 isotopologue fraction for m6A/m7G but not for Am/m1A/m62A in polyA+ RNA (A). Conversely, the modifications of total/large RNAs exhibited uniformly slow turnover (B), consistent with the high stability of rRNA and tRNA in growing cells  = m(0) + kt) fits well the turnover of m1A/m62A/Am but not m6A/m7G in polyA+ RNA, in which the “U-shaped” curve supports a nonlinear model (m(t) = m(0) e(−kt)) (C and S2A). Conversely, the linear regression fitted well the turnover of ncRNA modifications (B and C). The turnover frequency determined for Am/m1A/m62A in polyA+ was similar to ncRNA modifications (k = 0.031 h−1 on average) and significantly slower than m6A (k = 0.244 h−1) or m7G (k = 0.089 h−1) in polyA+ RNA (A and B). Thus, the turnover of m1A/m62A/Am from polyA+ fractions is incompatible with the exponential turnover of mRNA modifications ) C and S2Aications , B and ClyA+ RNA , A and BA k = 0.2 h−1 or mications , confirm7G was in between that of m62A and m1A/Am in polyA+ RNA (D), and the sensitivity of MS detection was similar across all measured modifications except m5C, as determined from equimolar injections of pure compounds (D). Thus, the distinct turnover of RNA modifications in the polyA+-purified fraction was not explained by differences in normalized abundances alone or analytical sensitivity of ribonucleoside detection. Instead, the modified ribonucleosides were more abundant in large RNA relative to polyA+ RNA by a factor of 17/37/97/9 for Am/m1A/m62A/m7G, respectively, while m6A level was greater in polyA+ RNA by a factor of 2 (E). From the determined turnover frequencies (h−1) of ncRNA (B), we estimated that a 50% contribution of ncRNA signal to the ribonucleoside pool in polyA+-purified RNA would lower the detected turnover of the bona fide m6A mRNA modification, with a turnover frequency of 0.242 h−1, to that of m7G, with a turnover frequency of ∼0.09 h−1 (E). That is, reliable turnover-based detection of a hypothetical mRNA modification that is more abundant by a factor of 17 to 97 in ncRNA relative to mRNA is attainable with a maximal ncRNA contamination of 0.5% (50%/97) to 2.9% (50%/17), but even an optimal performance of available methods to purify polyA+ RNA commonly contains 2 to 3% of contaminating ncRNA (F)  of mlyA+ RNA D, and thompounds D. Thus, tor of 2 E. From tof ncRNA B, we est0.09 h−1 E. That ing ncRNA F , we reasoned that free ribonucleosides derived from metabolic extracts would be derived mainly from endogenous degradation of mRNAs, rather than ncRNAs. Thus, we employed 13C-dynamods to examine the methylation turnover of free ribonucleosides, thereby enriching for mRNA-derived ribonucleosides and decreasing interference from rRNA-derived ribonucleosides. We found a fast kinetics following 13C labeling that was similar for m6A, m7G, and Am, indicating that they are primarily derived from the same RNA type  (F–H). Interestingly, the m62A modification also exhibited a nonlinear, faster kinetics than m1A or 1-methylguanosine (m1G) (F–H), which are known tRNA modifications and were present in the metabolic extracts at high levels (G) , which compromises the quantification under conditions of partial 13C labeling. Thus, while the turnover analysis of the polyA+ fraction could not be used to validate lowly abundant modifications due to rRNA contamination (E), the turnover of Am and m62A in the free pool suggests that these modifications indeed partly derive from rapidly decaying RNAs (F), which may include non-ribosomal RNAs. This was supported by the normalized levels of Am and m62A in the free pool being more similar to those of polyA+ than rRNA (H). These results confirm the presence of Am in mRNA  , F–H. Inne (m1G) , F–H, whh levels G 47). W. W6A wasmination E, the tuing RNAs F, which han rRNA H. These  in mRNA  and suggin mRNAs , 50.6A, N6,2-O-dimethyladenosine (m6Am), and m1A (versus ncRNA to metabolic stresses linked to RNA (de)methylation. First, we confirmed that actinomycin (ActD), a pan inhibitor of eukaryotic transcription, completely inhibited the methylation turnover of polyA+ and large RNA (A), in accordance with the cotranscriptional deposition of mRNA and most rRNA modifications (6A (m6A/A) in polyA+ RNA (B), but not of m7G, which supports the role of m6A in global mRNA destabilization, as initially reported (C and D). In contrast, the abundance of modified ribonucleosides in large RNA was unaltered under these forms of stress (E), whereas glutamine deprivation led to increased m6A levels in polyA+ RNA (F), which was in contrast to ActD treatment (B). Thus, the reduced methylation turnover of large RNA simply results from an inhibited transcription of the rRNAs themselves, while the altered abundance of m6A without significant changes in its turnover indicates its posttranscriptional lability to stress conditions via altered (i) decay of m6A-enriched versus m6A-depleted mRNAs or (ii) dynamics of the m6A modification itself. These data show that measurements of methylation turnover and ribonucleoside abundances resolve transcriptional versus posttranscriptional effects on RNA modifications in nonstationary (stimulus-dependent) experiments.The maintenance of methylation reactions requires SAM and serine/glycine, which feed one-carbon metabolism and SAM synthesis . Convers and m1A , 53. We arge RNA A, in accications , 56, 57.lyA+ RNA B, but noreported . Deprivareported , C and Df stress E, wherealyA+ RNA F, which reatment B. Thus, 13C labeling informs on the distinct dynamics of polyA+ and ncRNA modifications and their sensitivity to metabolic perturbations of mammalian cells. 13C-dynamods presents several advances in the application of MS approaches to study RNA turnover and dynamics. First, in contrast to approaches that rely on prior/in-parallel labeling with 15N/13C-enriched nucleosides to distinguish between preexisting and newly modified RNA  (B)   of ribonucleotide (NTP) synthesis  , 43 of v(in h−1) B (43). Manalogs) , 59 or tynthesis , 63, 64.ynthesis , 66. Als13C incorporation into newly methylated RNA does not assess nonmodified RNAs and hence does not capture the removal of RNA modifications. That is, any RNA demethylation activity can only affect the abundances of modified ribonucleosides as a whole (sum of m + 1 and m + 1), not the m + 1 or m + 0 isotopologues individually, which would be required to cause a differential m + 1 fraction. Since RNA transcription and decay rates are constant at metabolic steady state, we propose that the methylation turnover is an accurate readout of RNA turnover, which allowed us to assess the contributions of short-lived (mRNAs) and long-lived (noncoding) RNAs to a pool of modified RNAs and free ribonucleosides. Indeed, this information could not be drawn if labeled ribonucleoside themselves or its precursors were used to trace ribonucleotide synthesis  or transcription. Likewise, the source RNA cannot be pinpointed if the abundance (levels) of modified ribonucleosides is measured in isolation.Reliable study of mRNA modifications by MS analyses of ribonucleosides is challenging due to the high abundance of heavily modified ncRNAs that invariably contaminate the polyA+ fraction , 37. To 1A/m62A) in polyA+-purified RNA as being derived from mRNAs. Based on methylation turnover, we estimated that these modifications in the polyA+ fraction could be detected with a maximal ncRNA contamination of 0.5 to 2.9%, since Am/m1A/m62A levels were found higher in ncRNAs by a factor of 17–37–97, respectively. In contrast, a ∼6% of ncRNA contamination would be required to fully account for the slower m7G turnover detected in polyA+ RNA, since m7G levels were found only approximately nine times higher in ncRNAs. Since the m7G turnover reflected that of a bona fide mRNA modification, its slower kinetics in polyA+-purified RNA suggests a temporal delay relative to m6A turnover. A further evidence for the distinct turnover of m7G relative to m6A in mRNAs is the response to metabolic stresses, where the m7G turnover changed in large RNA but not polyA+ RNA following 4 h of 13C labeling (C and D). In this respect, while m6A is cotranscriptionally deposited in mRNA    and largin vitro , but we e.g., a stress condition, may affect the afferent  or efferent  turnover parts of methylated RNAs. Combined with abundance measurements, this allowed us to assess transcriptional versus nontranscriptional sensitivity of modified RNAs to stress conditions. This was evidenced by the reduced methylation turnover of ncRNAs following serine deprivation and ActD treatment, wherein the abundance of modifications was unaltered in the former  but m6A levels decreased in the latter . The observed inhibited turnover of methylated ncRNAs under serine deprivation is in line with the expected inhibition of mTOR activity and thereby of rRNA biogenesis (6A levels in polyA+-purified RNA without significantly changing (not increasing) its turnover. These data suggest that m6A is posttranscriptionally sensitive to glutamine levels through either (i) an altered decay of m6A-enriched versus m6A-depleted RNAs or (ii) inhibition of m6A demethylation itself. The demethylation of m6A is a stepwise conversion into adenosine through the formation of N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) intermediates  and preexisting methylated (m + 0 fraction) RNAs differently are exposed differently to RNA degradation, e.g., through nuclear/cytosolic compartmentalization.A nonstationary condition, ogenesis . In contmediates , whose pthylases , 53, it ependent . With exOur study demonstrates how quantification of methylation turnover and abundance can be used to examine the presence of RNA modifications in RNA classes, their temporal dynamics, and sensitivity to stress conditions. These insights open new directions to be further explored by MS and orthogonal approaches to obtain information on particular RNAs.2 in high-glucose DMEM medium  supplemented with 10% fetal bovine serum (FBS) . For 13C labeling experiments, 786O cells were grown in high-glucose DMEM medium without glutamine, methionine, and cystine  supplemented with 10% dialyzed FBS  to minimize contributions of unlabeled amino acids typically present in regular FBS, 2 mM glutamine, 0.1 mM cystine, and 0.2 mM [13C-methyl]-methionine (CK Isotopes Limited). Cells were maintained at 50 to 60% confluence (or 30–40% in chase experiments) and washed with PBS before switching to 13C-labeled medium (or regular medium in chase) for the indicated time periods.786O cells were obtained from the Crick Cell Services and cultured at 37 °C with 5% COvia two rounds of polyA tail hybridization with Oligo-dT magnetic Dynabeads .At the conclusion of metabolic labeling, the medium was aspirated and extraction of total RNA was performed with the mirVana isolation kit according to manufacturer’s instructions . Large (>200 nt) RNAs were purified by adding 1/3 volume of 100% ethanol to the aqueous phase recovered from the organic extraction before loading into the filter cartridge of the mirVana Kit. Small (<200 nt) RNAs were purified by collecting the total filtrate, addition of 2/3 volume of 100% ethanol, and loading into the filter cartridge. Polyadenylated RNA was purified from total/large RNA 2, and 10 mM NaCH3COO pH 5.3 and incubated for 2 h at 37 °C. Subsequently, NH4HCO3 (100 mM) and 5 units of alkaline phosphatase (CIP)  were added and the sample incubated for 2 h  at 37 °C. Formic acid was added at 0.1% v/v in a final volume of 50 μl, samples were filtered  and 15 to 20 μl analyzed in duplicate by LC-MS. Ribonucleosides were resolved with a C18 reverse-phase column  and eluted with a gradient of 0.1% v/v formic acid (solvent A) and 80% acetonitrile in 0.1% formic acid (solvent B) at a flow rate of 0.2 ml/min and 40 °C: 100% solvent A for 3 min, 12% solvent B for 12 min, and 100% solvent B for 2 min after which the column was re-equilibrated with 100% solvent A for 3 min . The ribonucleoside separation was performed using U3000 HPLC (Thermo Scientific) and the detection by a TSQ Quantiva Triple Quadrupole mass spectrometer  controlled by the Xcalibur software version 4.0.27 (Thermo Scientific). The HPLC was coupled to the TSQ Quantiva using a HESI (heated electrospray) ion source (Thermo Scientific) operating in positive ionization mode with the following parameters: capillary voltage, 3500 V; sheath gas flow, 7.35 l/min; gas temperature, 325C. The first and third quadrupoles (Q1 and Q3) were stringently fixed to 0.2 units of resolution and set to detect the mass of the precursor ribonucleoside ion (Q1) and of the base and ribose product ions (Q3). The ribonucleosides were identified by comparison of the retention time and detected mass transitions to commercially available standards. The collision energies were experimentally defined based on the fragmentation pattern of each ribonucleoside standard and chosen based on the maximum intensity of the base product; the ribose ring was used only as a qualifying transition. The retention time, mass transitions (m/z), and collision energies of each ribonucleoside were: adenosine, ∼4.1 min, 268.1 -> 136.1 m/z, 20 V; guanosine, ∼5.4 min, 284.1 -> 135.0 m/z, 35.5 V; cytidine, ∼1.4 min, 244.2 -> 112.05 m/z, 12 V; m6A, ∼8.7 min, 282.1 -> 150.1 m/z, 20 V; m1A, ∼1.8 min, 282.1 -> 150.1 m/z, 20 V, m7G, ∼2.3 min, 298.05 -> 166.1 m/z, 20 V; m5C, ∼1.8 min, 258.2 -> 126.1, m/z, 13 V; m62A, ∼11.9 min, 296.2 -> 164.1 m/z, 22 V. Each mass transition above corresponded to the m + 0 isotopologue and increased by one (m + 1), two (m + 2), and three (m + 3) units for detection of the other isotopologues, e.g., m62A: 297.2 -> 165.1 (m + 1), 298.2 -> 166.1 (m + 2), 299.2 -> 167.1 (m + 3). The dwell time for each transition was 30 ms for a duty cycle of 930 ms (31 transitions), and 8 to 20 data points per chromatographic peak were obtained for “short” and “long” peaks, respectively. A mix of ribonucleoside standards containing 0.5, 1, 5, 10, 50, 100, 500 fmol, 1, 5, 10, 50, or 100 pmol of each ribonucleoside was run in parallel after the biological samples for absolute quantifications, a subset of which is shown in Fig. S2D. Data were recorded using the Xcalibur 3.0.63 software (Thermo Fisher Scientific) and analyzed using Skyline (version 19.1) (Purified RNA (100–250 ng) was digested into ribonucleosides using one unit of nuclease P1  in 25 μl of buffer 25 mM NaCl, 2.5 mM ZnClon 19.1)  (Supplemon 19.1) .13C-methyl]-methionine, metabolic activity quenched by adding ice-cold PBS. Metabolites were extracted by addition of 600 μl ice-cold 1:1 (vol/vol) methanol/water to the cell pellets, samples were transferred to a chilled microcentrifuge tube containing 300 μl chloroform and 600 μl methanol . Samples were sonicated in a water bath for 8 min at 4 °C and centrifuged  for 10 min at 4 °C. The supernatant containing the extract was transferred to a new tube for evaporation in a speed-vacuum centrifuge, resuspended in 3:3:1 (vol/vol/vol) methanol/water/chloroform  to phase separate polar metabolites (upper aqueous phase) from nonpolar metabolites (lower organic phase), and centrifuged. The aqueous phase was transferred to a new tube for evaporation in a speed-vacuum centrifuge and resuspended in 100 μl water for LC-MS acquisition. LC-MS analysis was performed using a Dionex UltiMate LC system (Thermo Fisher Scientific) with a ZIC-pHILIC column , as described previously (m/z using a Q Exactive Orbitrap instrument (Thermo Fisher Scientific) with heated electrospray ionization and polarity switching mode at a resolution of 70,000 (at 200 m/z). MS parameters were as follows: spray voltage 3.5 kV for positive mode and 3.2 kV for negative mode; probe temperature, 320 °C; sheath gas, 30 arbitrary units; auxiliary gas, five arbitrary units. Parallel reaction monitoring (PRM) was used at a resolution of 17,500 to confirm the identification of metabolites; collision energies were set individually in HCD  mode. Data were recorded using the Xcalibur 3.0.63 software and analyzed using Tracefinder 4.1 (ThermoFisher Scientific) according to the manufacturer’s workflows.At the end of cell culture with [eviously . A 15 mi62A) relative to the total ion counts of the m + 0 plus m + 1 isotopologues. The kinetics of isotopologue fractions and goodness of fit were determined using the Curve Fitting toolbox of Matlab R2020a (MathWorks) either by a linear regression [f(x) = p1∗x + p2] or an exponential fit: a one-term function in the chase experiments , and a two-term function to fit the isotopologue fractions of free ribonucleosides .The isotopologue fractions were defined as the total ion counts of the m + 1 isotopologue (except for m + 2 in mThe mass spectrometry.raw data and Skyline files pertaining This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Lemna aequinoctialis) is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives for livestock. It is also an ideal system for basic biological research and various practical applications. In this study, we attempt to establish a micropropagation technique and Agrobacterium-mediated transformation in L. aequinoctialis. The plant-growth regulator type and concentration and Agrobacterium-mediated transformation were evaluated for their effects on duckweed callus induction, proliferation, regeneration, and gene transformation efficiency. Calli were successfully induced from 100% of explants on Murashige and Skoog (MS) medium containing 25.0 μM 2,4-dichlorophenoxyacetic acid  and 2.0 μM thidiazuron (TDZ). MS medium containing 4.5 μM 2,4-D and 2.0 μM TDZ supported the long-lasting growth of calli. Fronds regenerated from 100% of calli on Schenk and Hildebrandt (SH) medium containing 1.0 μM 6-benzyladenine (6-BA). We also determined that 200 μM acetosyringone in the cocultivation medium for 1 day in the dark was crucial for transformation efficiency (up to 3 ± 1%). Additionally, we propose that both techniques will facilitate efficient high-throughput genetic manipulation in Lemnaceae.Duckweed ( Lemna is a member of the Lemnaceae or duckweed family. In nature, there are five genera within this family: Landoltia, Lemna, Spirodela, Wolffiella, and Wolffia [®, a recombinant IFN-α2b synthesized by a duckweed-based expression system that uses Lemna minor, for the large-scale production of recombinant pharmaceuticals and interferons [The genus  Wolffia . Duckwee Wolffia ,3. The b Wolffia ,5,6. In  Wolffia ,8,9,10 a Wolffia . Moreoveerferons .Agrobacterium-mediated transformation system has been developed for several duckweeds, including Lemna gibba [Lemna minor [Lemna turionifera [Landoltia punctata (formerly Spirodela oligorrhiza) [Spirodela polyrhiza [Wolffia arrhizal [Wolffia globosa [Lemna aequinoctialis 6002, which is one of the most widely distributed duckweed species in Asia [L. minor [Agrobacterium and consequently the transformation frequencies were not optimized in detail. The plant material of this research was a Taiwanese isolate of L. aequinoctialis. It is known that different duckweed isolates might vary in their physiological properties, including hormonal requirements, regeneration potential, and susceptibility to A. tumefaciens [To date, an na gibba ,14, Lemnna minor ,19,20,21ionifera , Landoltorrhiza) ,23, Spirolyrhiza , Wolffiaarrhizal ,25, and  globosa . In addi globosa  reported in Asia ,29. HoweL. minor ,15. The Agrobacterium-mediated transformation methodology to better define the optimal basal-medium formulation, plant-growth regulator type and concentration, light, cocultivation time, and acetosyringone (AS) concentration for callus induction, proliferation, regeneration, and genetic transformation in the Taiwanese isolate of L. aequinoctialis.To facilitate efficient high-throughput genetic manipulation in Lemnaceae, our study focuses on micropropagation and Lemna aequinoctialis (p < 0.05) effect on callus induction (w/v) sucrose, 0.3% (w/v) Phytagel, and 2,4-D (25.0 μM) combined with TDZ (2.0 μM) at pH 5.7.Two to three-week-old fronds of octialis A were usnduction . Frond pnduction . The calnduction . Calli cnduction B. The canduction C. Thus, w/v) sucrose, 0.3% (w/v) Phytagel, and 2,4-D (4.5 μM) combined with TDZ (2.0 μM) at pH 5.7.Because callus proliferation was very slow on CIM, six combinations of 2,4-D with TDZ were tested to determine if they would enhance the rate of callus growth. Nodular callus-proliferation medium (NCPM) with 4.5 µM 2,4-D and 2.0 µM TDZ supported the significant growth rate of calli , which pL. aequinoctialis regenerating from callus tissue is shown in w/v) sucrose, 0.3% (w/v) Phytagel, and 6-BA (1.0 μM) at pH 5.7.Both SH and MS basal media supplemented with 6-BA were evaluated for their ability to regenerate fronds using nodular calli derived from the most effective NCPM . The resLemna aequinoctialis, we elucidated the effect of altering the concentrations of acetosyringone (AS), coculture days, and photoperiods on transformation efficiency  were evaluated in the three transgenic lines with the strongest GFP fluorescence . First, Lemna gibba, but only ~10% of fronds produced calli. In this study, we found that L. aequinoctialis calli could be induced on MS medium containing 25.0 µM 2,4-D and 2.0 µM TDZ at a rate of 100%, which was better than the results from Liu et al. [L. minor. In addition, Huang et al. [−1 2,4-D (~67.9 µM) and 2 mg L−1 6-BA (~9.01 µM) for Landoltia punctata; Yang et al. [Spirodela polyrhiza; and Khvatkov et al. [−1 2,4-D (~22.6 µM) and 0.5 mg L−1 6-BA (~2.25 µM) in Wolffia arrhiza.Previous studies have implemented callus induction on species within Lemnoideae and have revealed that both the type and concentration of PGRs can significantly enhance the callus induction rate ,30,31. Au et al. . Our resu et al.  found thv et al.  reportedL. minor, Cantó-Pastor et al. [−1 2,4-D (~9.04 μM) for subsequent growth and proliferation in L. gibba. Therefore, we found that FRM with 1.0 μM 6-BA in SH medium had a 100% rate of regeneration. For L. gibba, a 100% plant regeneration rate was observed on FRM with 1.0 μM 6-BA in SH medium [Spirodela punctata, a 95–98% plant regeneration rate was attained on FRM with 1.0 mg L−1 2ip in McCown Woody Plant medium [L. gibba G3 and W. arrhiza fronds could also regenerate on PGR-free SH medium [Our results showed that nodular callus-production medium (NCPM) with 4.5 μM 2,4-D and 2.0 μM TDZ could significantly improve callus proliferation. For r et al.  transferr et al.  transferH medium . For Spit medium . It has H medium ,35,36. TAgrobacterium-mediated transformation in L. aequinoctialis. In addition, the stable transformation efficiency obtained was approximately 3.8% [L. minor, a different species of the same genus, or 0.5–5% in Spirodela oligorrhiza [W. arrhizal [Agrobacterium-mediated transformation in L. aequinoctialis. Acetosyringone (AS) has previously been shown to aid in the transformation of several crops, such as cotton, rice, and wheat [Agrobacterium transformation of L. minor. In addition, Vunsh et al. [S. oligorrhiza with 100 µM AS at pH 5.6. These conditions had similar results to those found in the transformation protocol of S. oligorrhiza, L. gibba and L. minor [L. aequinoctialis and Agrobacterium. Vunsh et al. [S. oligorrhiza and S. polyrhiza. We know that longer cocultivation periods of approximately 4–5 days could cause the over-proliferation of Agrobacterium around the plant materials, ultimately leading to the death of the plant tissue [L. minor cocultured for 3 days under a 16 h light/8 h dark photoperiod. They suggested that the possible reason for the difference was due to different physiological properties among duckweed species. Eventually, we successfully regenerated fronds from the transgenic calli. These fronds stably expressed green fluorescence through vegetative proliferation for several generations (up to 1 year). To date, transgenic fronds have shown uniform GFP activity, which suggests their ability to stably inherit transgenes through mitotic divisions.Here, by establishing the transformation protocol, we achieved a 3 ± 1% stable transformation success rate for ely 3.8%  and 4% [ely 3.8%  in L. migorrhiza  and 0.4%arrhizal ,38. Thusnd wheat . Researcnd wheat  used 200h et al.  supplemeL. minor ,14,15. OL. minor . In our h et al.  and Yangh et al.  showed tt tissue . InteresA. tumefaciens strain, binary vector types, infection methods, surfactant addition, and so on [Agrobacterium infection. In comparison to previous studies of Agrobacterium-mediated transformation in duckweeds [L. aequinoctialis in the future.However, several factors greatly affect the transformation efficiency, including plant species, type of explants, nd so on . In sevend so on , chickpend so on , and cownd so on , it has uckweeds ,15,16,17uckweeds  also sugLemna aequinoctialis was collected from a pool at National Pingtung University of Science and Technology (NPUST), Pingtung, Taiwan. The duckweed species was identified by an atpF-atpH noncoding spacer [w/v) Phytagel  at pH 5.7. All cultures were maintained at 27 ± 1 °C under a 12 h photoperiod of cool-white fluorescent light of 80 μmol photons m−2 s−1 intensity. An axenic clone was produced and maintained in our laboratory for 4 years. Two to three-week-old axenic fronds were used for callus induction.g spacer . After sw/v) sucrose and 0.3% (w/v) Phytagel at pH 5.7. Different combinations of plant-growth regulators were added in the medium. For cytokinins, we used either 6-benzyladenine (6-BA) or thidiazuron (TDZ) at 0, 0.5, 1 and 2 μM; for auxins, we used 2,4-dichlorophenoxyacetic acid  at 5, 10, 25 and 50 μM. Thirty-two fronds were cultured in sterilized 150 × 20 mm Petri dishes containing 100 mL solid medium for callus induction. The explants were maintained under a 12/12 h photoperiod at 27 ± 1 °C (80 μmol photons m−2 s−1). Each treatment was performed in triplicate. The efficiency of callus induction on each plate was calculated after 10 weeks of cultivation.To evaluate the optimal conditions for callus induction, fronds (~3.5 mm in length) were grown on MS medium containing 3% (w/v) sucrose, 0.3% (w/v) Phytagel, and either 2,4-D at 4.5 or 9.0 μM combined with TDZ  at pH 5.7. Five calli were cultured in sterilized 90 × 20 mm Petri dishes containing 50 mL solid medium under a 12/12 h photoperiod at 27 ± 1 °C (80 μmol photons/m2/s). Each treatment was performed in triplicate. The relative growth rate (RGR) of calli during 4 weeks of cultivation was calculated with the following equation [t − lnW0)/(tt − t0), where Wt is the fresh weight at time tt and W0 is the initial fresh weight at the beginning of the treatment. The nodular calli derived from the most effective nodular callus-proliferation medium (NCPM) were then used for frond regeneration and gene transformation.To evaluate the optimal conditions for callus proliferation, calli (~5 mm in diameter) derived from the most effective callus-induction medium (CIM) were cultured on MS medium containing 3% (equation : RGR = (w/v) sucrose, respectively, 0.3% (w/v) Phytagel, and 6-BA  at pH 5.7. Five calli were cultured in sterilized 90 × 20 mm Petri dishes containing 50 mL solid medium under a 12/12 h photoperiod at 27 ± 1 °C (80 μmol photons m−2 s−1). Each treatment was performed in triplicate. The efficiency of frond regeneration on each plate was calculated after 4 weeks of cultivation.To determine the optimal conditions for frond regeneration, calli (~5 mm in diameter) derived from the most effective NCPM were cultured on both MS and SH media containing 3% and 1% . GFP was driven by the maize ubiquitin1 promoter together with its intron (Ubi1) promoter and terminated by the Nos poly (A) signal.The green fluorescence protein (GFP) expression vector pD-clone1  was intrLemna aequinoctialis, various parameters influencing transformation efficiency were evaluated. Three days prior to the transformation experiment, Agrobacterium tumefaciens (pD-clone1) from a freshly streaked plate was inoculated in 3 mL of liquid YEP medium  containing ampicillin (100 mg L−1) and kanamycin (50 mg L−1) and grown overnight on a rotary shaker (180 rpm) at 27 °C. The next day, 0.5 mL of this primary culture was inoculated in 50 mL subculture medium  containing ampicillin (100 mg L−1) and kanamycin (50 mg L−1). The culture was grown at 27 °C with shaking (180 rpm) until the OD600 reached 0.6–0.8. The bacteria were harvested by centrifuging at 5000 rpm for 7 min at 4 °C and resuspended in 10 mL MS liquid medium. After soaking for 2 min, the calli  were blotted dry on sterile filter paper and cocultivated on the most effective NCPM (pH 5.2) supplemented with 0, 50, 100, and 200 µM acetosyringone (AS) for 1–3 days at 27 ± 1 °C under a 12/12 h photoperiod or dark conditions.To develop a genetic transformation protocol for −1 cefotaxime once per hour for five hours on a shaker at 110 rpm. The mixture was left to culture overnight. After washing, the calli were blotted dry and cultured on selection medium (the most effective NCPM containing 50 mg L−1 hygromycin and 275 mg L−1 cefotaxime) (NCPM-CH) under a 12/12 h photoperiod at 27 ± 1 °C (80 μmol photons/m2/s).After cocultivation, nodular calli from each treatment were collected and washed with sterile distilled water at least 20 times, followed by washing with MS liquid medium containing 275 mg L−1 hygromycin for frond regeneration.After culturing on the selection medium for 3 days, we started to screen calli showing GFP signals under a fluorescence stereomicroscope  until the nodular calli began to proliferate, which were regarded as putative transformants. The frequency of GFP expression was calculated as the number of calli showing GFP out of the total number of treated cells. Hygromycin-resistant calli could be found at approximately 10 weeks. The putative transformants were transferred to regeneration medium containing 50 mg LGFP using the following specific primers: GFP-F: 5′-CAAGGGCGAGGAGCTGTT-3′; and GFP-R: 5′-CTTGTACAGCTCGTCCATGC-3′. The chloroplast marker atpF-atpH was amplified as an endogenous control [w/v) agarose gel and stained with SafeView™ Classic .Genomic DNA was extracted from wild-type and putative transformants by using an RBC Real Genomics Genomic DNA Extraction Kit  and used as a template for PCR analysis to test the presence for  control . The PCRGFP and Lemna aequinoctialis Actin  expression using the following specific primers: the same primer pairs of GFP; LaActin-F: 5′-GACGCAGATCATGTTCGAGA-3′; and LaActin-R: 5′-TGGTTCCACCACTAAGCACA-3′. PCR amplification was performed as mentioned above. Three batches of independently isolated total RNA samples underwent three rounds of RT-PCR. All tests were repeated at least three times, and the results from one repeat are shown in the figures. LaActin was used as the internal control.Total RNA was isolated from wild-type and putative transformants using TRIzol reagent , and cDNA synthesis involved the use of an MMLV reverse transcriptase first-strand cDNA synthesis kit . cDNAs were used as a template for RT-PCR analysis to determine Total protein extracted from the fronds of wild-type plants and putative transformants (15 μg each lane) was subjected to SDS-PAGE using a 12% polyacrylamide gel and Western blot analysis with a monoclonal antibody against GFP  as described in . Immunorp < 0.05.A one-way analysis of variance (ANOVA) was used to analyze the data. When the ANOVA identified differences among the groups, Duncan’s multiple range test was conducted to examine significant differences among the treatments in SAS software . The level of significance was Lemna aequinoctialis (Agrobacterium tumefaciens strain EHA101 harboring pD-clone1. A stable transformation frequency (3.13 ± 1.08%) was observed in calli cocultivated with Agrobacterium on NCPM containing 200 μM AS for 1 day in the dark. Therefore, the protocol for micropropagation and Agrobacterium-mediated transformation could be a good foundation for the transgenic engineering of L. aequinoctialis.In conclusion, we developed an efficient micropropagation technique and achieved stable transformation in octialis . Our res


Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The mK and maxV of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium  Gracilaria sp. and Gelidium sp. [d-galactose and 3,6-anhydro-l-galactose [Agar is the main glycan component that is segregated from the cell wall of red algae, for instance, dium sp. ,2. As thdium sp. ,4. Agaroalactose .l-galactose at the reducing ends of products, and β-agarase (EC 3.2.1.81), which cleaves the β-1,4 glycoside bonds to generate NAOS with β-d-galactose at the reducing ends of products [Agarases that depolymerize agarose into agarooligosaccharides (AOS) or neoagarooligosaccharides (NAOS) have been found in several gram-negative bacteria isolated from submarine sediments, seaweeds, marine organisms, and seawater . Agaraseproducts ,6.Compared to α-agarases, β-agarases have a wider distribution in nature . β-agaraUntil now, many agarases have been thermostable but lacking in activity at low temperatures . TherefoShewanella sp. WPAGA9 for three seaweed polysaccharides [Shewanella sp. WPAGA9 had been further investigated and characterized. The results showed that the recombinant AgaW1540 (rAgaW1540) can maintain 85.4% of maximum enzymatic activity at 0 °C, indicating an outstanding level of cold-adaptation. The current study provides a potential bio-tool for NAOS production at low temperatures, and an ideal model for researching the cold-adapted mechanisms of agarases.Our former study demonstrated the degradation activities of a deep-sea bacterium charides . In thisShewanella sp. WPAGA9 was predicted to be a putative β-agarase gene due to its maximum identity of 74.64% with the β-agarase genes from Agarivorans sp. HZ105 (GenBank accession number: HQ625023.1) and Pseudoalteromonas sp. CY24 (GenBank accession number: AY150179.3). In the phylogenetic trees constructed by the methods of maximum likelihood . The annotation results from the dbCAN database assigned AgaW1540 into the GH16 family. The gene agaW1540 encoded 453 amino acids with a theoretical molecular weight of 50.96 kDa. AgaW1540 possesses an N-terminal signal peptide of 21 amino acids with a possible cleavage site between position Ala21 and Ala22. The gene agaW1540 was expressed in E. coli BL21(DE3) cells using a pEAZY®-Blunt E2 expression vector . The recombinant protein was released by ultrasonication, and was purified by Ni-nitrilotriacetic acid (NTA) Sefinose™ Resin . A single distinct band of rAgaW1540 was displayed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular weight of ~50 kDa  in the genome of kelihood a, neighbkelihood b, and mikelihood c, the nu ~50 kDa d, which The optimum pH of rAgaW1540 was 7.0. Meanwhile, rAga1540 exhibited the maximum activity at an extensive pH coverage, and maintained 98.2%, 93.6%, and 98.4% of the maximum activity at pH 4.0, 5.0, and 6.0, respectively a. The op2+ and Zn2+ had negative effects on rAgaW1540; meanwhile, Ba2+, Mn2+, Ni2+, K+, Ga2+, Sr2+, Ag+, Cr2+, and EDTA slightly promoted the enzymatic activity of eAgaW1540. Fe2+ exhibited a promotion that increased the relative activity of rAgaW1540 up to 126.99% , agarose was firstly degraded into NA12, NA8, NA6, and NA4 after degradation for 10 min, which suggested rAgaW1540 was an endo-type agarase. Subsequently, NA12 and NA8 were further degraded into NA4 and NA6. The main products of rAgaW1540 were NA4 and NA6 after degradation for 24 h a. To furd-thiogalactopyranoside  and dbCAN  databases. Based on the nucleotide sequences, the agarase genes were aligned by using ClustalW and the phylogenetic analysis was performed by the methods of neighbor-joining, maximum likelihood, and minimum evolution in MEGA 5.2 software  with a bootstrap value of 1000. The prediction of signal peptide was performed by using SignalP-5.0 . Shewanella sp. WPAGA9 was cultured in marine broth 2216E  at 30 °C for 12 h. The genomic DNA of Shewanella sp. WPAGA9 (GenBank accession number: CP062058) was extracted by using a TIANamp Bacteria DNA Kit . The gene agaW1540 was amplified through PCR with a forward primer (5′-AAACCTAGTATGAATGTTATCTATGCTACTAC-3′) and a reverse primer (5′-CTCTACTTTAAATTGTTGATTGGTTCCG-3′). PCR was conducted using a C1000™ Thermal Cycler  and the PCR condition was as follows: 3 min at 95 °C; 25 cycles of 95 °C for 25 s, 55 °C for 25 s, 72 °C for 1 min/kb; and extension at 72 °C for 6 min. The PCR products were purified by Universal DNA Purification Kit  and then were ligated with pEAZY®-Blunt E2 expression vector . The recombinant vector harboring agaW1540 gene was transformed into E. coli BL21(DE3) pLysS cells.The nucleotide sequence of agaW1540 had been deposited in the GenBank database under the accession number MZ398234. The sequence of agaW1540 was annotated against NCBI nr (E. coli BL21(DE3) pLysS cells harboring the recombinant vector were cultured at 37 °C in LB broth  including 100 μg/mL ampicillin, and the culture broth was induced by adding 0.1 mM IPTG at 16 °C until the OD600nm reached 0.6–0.8. The cells were collected by centrifugation at 10,000 rpm for 10 min at 4 °C, and then were subsequently resuspended in a Tris-HCl buffer . The resuspended cells were fractured using a sonicator  at a rate of 3s/2s impulse frequency for 20 min in ice. The Ni-nitrilotriacetic acid (NTA) Sefinose™ Resin  was used to further purify rAgaW1540 with 6×His-tagged from the crude enzyme solution. The molecular weight and purity of rAgaW1540 were subsequently estimated by SDS-PAGE and Coomassie brilliant blue staining.The w/v) with different pHs including 3.0–8.0 (50 mM sodium citrate-dibasic sodium phosphate), 8.0–9.0 (50 mM Tris-HCl), and 9.0–10.6 (50 mM glycine-NaOH), and the mixture was incubated at the temperatures of 0, 10, 20, 30, 35, 40, 45, 50, 60, 70, and 80 °C for 10 min, respectively. Then, 750 μL 3,5-dinitrosalicylic acid (DNS) solution was added, and the resulting sugar was measured by the DNS method [2+, Cd2+, Mg2+, Mn2+, Cu2+, Ni2+, Na+, K+, Ga2+, Sr2+, Zn2+, Ag+, Fe2+, Cr2+, and EDTA at a final concentration of 10 mM. The effect of NaCl concentrations on rAgaW1540 activity was measured by determining the activity at the NaCl concentrations of 0–2.5 mol·L−1. The kinetic parameters of rAgaW1540 were measured by determining the activity with the agarose at final concentrations of 0.1–0.6 mg/mL without any metal additive, and the mK and maxV values were subsequently calculated by the Lineweaver–Burk plot. All determinations were repeated three times.The agarase activity (U) was defined as the amount of the enzyme that released 1 μmol of reducing sugars per minute under certain conditions. In detail, 10 μL enzyme solution was mixed with 240 μL agarose  at 25 °C. The products were developed on a silica gel 60 plate  with the developing solvent containing n-butanol, acetic acid, and water . The products on the plates were visualized by heating with 20% H2SO4 at 130 °C for 10 min. To further identify the degradation products, the hydrolysates of rAgaW1540 were analyzed by IC. A Dionex CarboPac PA-100 column (4 × 250 mm) was used to separate the oligosaccharides. The mobile phases were 100 mmol/L NaOH and 150 mmol/L NaAc, and the flow rate was 0.25 mL/min. The injection volume was 25 μL. The commercial neoagaro-oligosaccharides, including NA2, NA4, NA6, and NA8, were used as standards for TLC and IC analyses.TLC analysis was conducted to investigate the hydrolysis modes and products of rAgaW1540 in 0, 10, 20, 30, and 40 min. The agarolytic reaction of rAgaW1540 were conducted in a 50 mM Tris-HCl buffer (pH 7.0) with 0.1% agarose  of IPTG solution were added into the fermentation broth to reduce the recombinant proteins. Then, the agarose activity was measured by the method in section “Characterization of enzymatic properties”. The induction time was future optimized by determining the agarase activity in the fermentation broth after 4, 8, 12, 16, 20, and 24 h inductions by IPTG. The induction temperatures, including 12, 16, 20, 24, and 28 h, were also optimized to increase the production of rAgaW1540.To enhance product yields, fermentation optimization was performed to improve the expression condition of rAgaW1540 in


This study aims to taxonomically identify and characterise the phylogenetic relationships of spiny lobsters based on mitochondrial cytochrome c oxidase I (COI) and 16S rRNA genes from Bangladesh waters. A total of 19 barcode sequences  were successfully generated from 12 collected spiny lobster samples representing four species belonging to the family Palinuridae. The average genetic distances within and between species were 0.834 ± 0.427 and 17.810 ± 0.830, respectively, in COI and 0.107 ± 0.255 and 8.401 ± 2.547, respectively, in 16S rRNA genes. The successful amplification rate of 16S rRNA was higher than that of the COI marker. In the maximum likelihood (ML) tree, the sequences of the same species were clustered together under a single clade for both COI and 16S rRNA, which supports the efficacy of both marker genes in differentiating lobster species. •Panulirus homarus, P. ornatus, P. polyphagus and P. versicolor, were morphologically described.Four Lobster species, •Molecular characterization was confirmed based on two markers, COI and 16S rRNA.•This is the first comprehensive taxonomic description of spiny lobsters from Bangladesh. Spiny Lobster, COI, 16S rRNA, DNA Barcoding, Bangladesh. The higThere is no published work thus far on the detailed taxonomic description and molecular characterization of spiny lobsters from Bangladesh. The present study aims to validate the morphologically identified lobster species based on mitochondrial cytochrome c oxidase I (COI) and the 16S rRNA gene.22.1The target lobster specimens were collected as dead from Teknaf (20°46′37.6″N 92°15′20.0″E), Cox's Bazar (21°24′15.3″N 91°53′10.1″E), and St. Martin Island (20°36′49.3″N 92°19′51.6″E) of Bangladesh from December 2020 to March 2021. Immediately after collection, the specimens were preserved in ice and transported to the laboratory for morphological identification. Taxonomic identification of the specimen was performed based on morphometric and meristic characteristics following the guidelines of 2.2DNA was extracted from a 5-mg tissue sample collected from the lower abdomen of the specimen using a Promega Wizard® Genomic DNA Purification kit. The quality and quantity of the extracted DNA were measured using a NanoDrop™ spectrophotometer. COI and 16S rRNA gene sequences were amplified by polymerase chain reaction with the primers LCO-1490 (forward) 5′-TCAACAAATCATAAGGACATTGG-3′ and HCO-2198 (reverse) 5′- TAAACTTCAGGGTGTCCAAAGAATCA-3′ for COI  and the 2.3The assembled contigs of the gene sequences were prepared by the CAP3 DNA assembly program using Unipro Ugene . Each se3Panulirus homarus, P. ornatus, P. polyphagus and P. versicolor.A total of 12 specimens were examined during the study from the Bangladesh coast: Teknaf, Cox's Bazar and St. Martin's Island. Morphometric identification and molecular characterization confirmed four species of spiny lobsters, 3.13.1.1English Name: Scalloped spiny lobsterMaterial examined: Two females, Bangladesh, Teknaf, the southernmost point in mainland Bangladesh. 20°46′37.6″N 92°15′20.0″E, ID DUZM_CR_144B- DUZM_CR_144B.2Habitat: Inhabits shallow waters among rocks, often in the surf zone. Maximum depth of 90 m  in turbid waters near river mouths at depths from 3 to 90 m but usually less than 40 m deep  .We generated barcodes consisting of 19 partial COI and 16S rRNA sequences of the 4 inuridae . The aveinuridae . Wide ba4P. homarus, P. ornatus, and P. polyphagus have been nationally assessed as vulnerable (VU) with P. versicolor assessed as endangered (EN)  Act 2012 as ‘protected animals’  . Howeverred (EN) . Moreoveanimals’ . P. poly species . Unfortui.e., high AT content in Palinuridae, similar to that in a previous study .https://www.ncbi.nlm.nih.gov/genbank/) under accession number MW514205-06, MW514208-11, MW832710-13, MW504991-92, MW504994-96, and MW832552-55.Data associated with this study has been deposited at NCBI Gen-Bank databases (The authors declare no conflict of interest.No additional information is available for this paper.


GOS with sizes of 11 μm, 1 μm, and 0.45 μm are prepared. Based on the optical limiting and Z-scan technology systems under 532 nm and 1064 nm nanosecond laser excitation, the nonlinear optical properties of GOS are investigated. It is found that GOS exhibits outstanding optical limiting properties. In addition, the optical limiting response of GOS is size-dependent. Concerning the largest particle size, GOS has the best nonlinear optical response, while the precursor shows no nonlinear optical performance. Meanwhile, GOS doped gel glass also displays excellent optical limiting properties with high transmittance, which preliminarily validates the application of GOS and other scintillators in nonlinear optics and encourages more research to better realize the potential of GOS.With the advancement of ultra-fast and high-energy pulsed laser output, lasers have caused serious harm to precision instruments and human eyes. Therefore, the development of optical limiting materials with a fast response, low optical limiting threshold, and high damage threshold are important. In this work, for the first time, it is reported that phosphors Gd Optformance . Zhou etependent . Lu et aoperties .2O2S:Tb3+(GOS) phosphors have excellent fluorescence properties with a hexagonal crystal system. GOS has attracted wide attention in TV screens, cathode-ray tubes, and X-ray intensifying screens because of its excellent physical properties [As a typical scintillator, Gdoperties ,14, suchoperties ,16. Theroperties ,18,19. HIn this work, GOS of different sizes and high quality were synthesized via the precursor method and the high-temperature solid-phase method. The morphology and microstructure were characterized. In addition, the OL properties and size effect were explored. It was found that the GOS exhibited excellent optical limiting performance, and the larger size GOS possessed a more superior OL response. The mechanism included nonlinear absorption and nonlinear refraction. Finally, GOS was then introduced to the gel glass to form a solid sample and the OL performance was investigated. The wide range of sizes of GOS and the high transmittances of GOS/gel glass endow GOS with great potential for application in laser protection.2O2S:Tb3+(GOS) with average diameters of 11 ± 1 μm (GOS-11 μm), 1 ± 0.1 μm (GOS-1 μm), and 0.45 ± 0.1 μm (GOS-0.45 μm). 2O(CO3)2·H2O: Tb3+ precursor indicates that spherical particles  ground multiplets as assigned in the literature [5D4 → 7F5 transition) being the strongest. Among the three samples, GOS-1 μm had the strongest luminescence intensity, GOS-0.45 μm was the next, and GOS-11 μm was the weakest. 7FJ J = –6 groundterature , with th2O(CO3)2·H2O: Tb3+ shows no signal at the same condition  and limiting threshold  are two vital parameters to evaluate the optical limiting performance. Materials with low FON and FOL are predicted to be promising candidates in optical limiting applications. The variations of normalized transmittance with incident intensity are shown in ON of GOS-11 μm, GOS-1 μm, and GOS-0.45 μm were 0.99, 1.43, and 1.69 J·cm−2, and the FOL were 2.17, 2.90, 3.53 J·cm−2, respectively. For 1064 nm excitation, the FON of GOS-11 μm, GOS-1 μm, and GOS-0.45 μm were 2.8, 3.98, and 4.46 J·cm−2, and the FOL were 4.18, 5.59, and 5.59 J·cm−2, respectively. The GOS-11 μm has the lowest FON and FOL at two wavelengths , TbCl3·6H2O (99.99%) urea (99.5%), S (99.99%), Tb4O7 (99.999%), and Gd2O3 (99.9%); all were purchased from Aladdin. Na2CO3 of analytical purity were obtained from Sinopharm Chemical Reagent Co., Ltd. All reagents were analytically graded and used without further purification. Deionized water was used as a solvent throughout the experiment.The following chemicals were used in this work: GdCl3·6H2O, TbCl3·6H2O, and urea were added to deionized water with constant stirring. Next, the transparent solutions were maintained at 91 °C for a period of 4 h, leading to the formation of white precipitates. Subsequently, the white precipitates were collected by suction filtration and washed three times with deionized water. This was followed by drying in a vacuum oven at 80 °C overnight. Finally, precursor Gd2O(CO3)2·H2O: Tb3+ powders were obtained.First, GdCl2O(CO3)2·H2O: Tb3+, S, and Na2CO3 were mixed thoroughly. Then, the the mixed raw materials were placed in a crucible, which was calcined at 900 °C with 5 °C·min−1 rate for 4 h in a reducing atmosphere (in 8 vol%H2/Ar). The product obtained was washed with deionized water and dilute hydrochloric acid. The phosphor powders were finally obtained after drying at 80 °C overnight.Precursor Method: The precursor Gd2O3, Tb4O7, S, and Na2CO3 were mixed thoroughly. The following experimental steps are the same as Precursor Method.High-temperature solid-phase method: The GdGOS gel glass was prepared by the sol-gel method, which was via the hydrolysis and polycondensation of MTES in acidic conditions . The molar ratio of MTES:water:ethanol was 1:3.5:12.05. The mixture was stirred overnight and the solvents were removed by rotary steaming. Then the GOS was added to the sol and was stirred evenly. Lastly, the GOS doped gel was cast into polypropylene cells and dried to obtain the GOS gel glass.X-ray diffraction (XRD) was performed on a D8 Advance X-ray diffractometer  with Cu Kα radiation (λ = 1.54056 Å) at 40 kV and 40 mA. Scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 Scanning Electron Field Emission Microscope. The emission spectra of the products were recorded by a Carry Eclipse . All measurements were performed at room temperature (RT). The NLO properties at 532 and 1064 nm were investigated using a Z-scan setup . The beam waist radius of 532 and 1064 nm are 13.5 and 27 μm for the Z scan setup, respectively. The optical limiting performance of GOS with different sizes in the solution state and the gel glass state were performed at 532 nm and 1064 nm excitation with the waist radius of 25 μm and 27 μm.In summary, we have successfully prepared different lateral sizes of GOS  via the precursor method and the high-temperature solid-phase method. The study reported the first nonlinear optical properties of GOS and their size effects. GOS exhibited excellent optical limiting performance, and the larger GOS performed better. Subsequently, the GOS was doped into gel glass. The obtained solid sample possessed high transmittance at a dope concentration of 0.5 wt% and showed an outstanding optical limiting response, revealing their potential in developing nonlinear and optoelectronic materials and devices.