So far, no thermodynamically stable diatomic dication has been characterized spectroscopically, primarily because of experimental difficulties associated with their synthesis in sufficient densities in the gas phase. Indeed, such molecules typically involve, as constituents, rare-gas, halogen, chalcogen, and metal atoms. We report here on a new approach to characterize molecular dications based on high-resolution photoelectron spectroscopy of the singly charged parent molecular cation and present the first spectroscopic characterization of a thermodynamically stable diatomic dication, MgAr2+. From the fully resolved vibrational and partially resolved rotational structures of the photoelectron spectra of 24MgAr+ and 26MgAr+, we determined the potential-energy function of the electronic ground state of MgAr2+, its dissociation (binding) energy (D0 = 10 690(3) cm−1), and its harmonic (ωe(24MgAr2+) = 327.02(11) cm−1) and anharmonic (ωexe(24MgAr2+) = 2.477(15) cm−1) vibrational constants. The analysis enables us to explain quantitatively how the strong bond arises in this dication despite the fact that Ar and Mg2+ both have a full-shell rare-gas electronic configuration.Although numerous doubly positively charged diatomic molecules (diatomic dications) are known from investigations using mass spectrometry and  2+.We present a new method to study doubly charged molecules relying on high-resolution spectroscopy of the singly charged parent cation, and report on the first spectroscopic characterization of a thermodynamically stable diatomic dication, MgAr They are encountered in plasmas and play a role in planetary ionospheres1 and more generally in molecular astrophysics.2 Their structure and reactivity are at the focus of numerous studies.3–12 The qualitative aspects of their stability are well known. The condition for their ground state to be thermodynamically stable is that the dissociation limit associated with the products A + B2+ in their ground states lies energetically below  or soft X-ray ranges and are difficult to reach with table-top laboratory radiation sources.Experimentally, the vast majority of DIDIs has been identified by mass-spectrometric methods, which enable their unambiguous detection but do not provide structural information. Only very few DIDIs have been characterized by spectroscopic methods which provide quantitative information on their structure. High-resolution, rotationally resolved spectra have been obtained for only three DIDIs, N2+. To obtain high-resolution spectroscopic information on this dication, we have recorded the photoelectron spectrum of MgAr+ using the techniques of pulsed-field-ionization zero-kinetic-energy photoelectron (PFI-ZEKE-PE)28,29 and mass-analyzed threshold ionization (MATI) spectroscopy.30 Until this work, high-resolution photoelectron spectroscopy of molecular cations had been deemed impossible because of (i) the very limited density of ions that can be generated in the gas phase as a result of space-charge effects and (ii) the high ionization energies of cations. We demonstrate here that high-resolution photoelectron spectroscopy can be applied to samples of less than 1000 state-selected cations and that cations can be efficiently ionized despite their high ionization energies by resonant multiphoton excitation.We present here the first spectroscopic characterization of a thermodynamically stable DIDI, MgAr−1) to obtain information on the rotational contours of the successive vibrational bands of the photoelectron spectrum of MgAr+ and to determine isotopic shifts in the spectra of 24MgAr+ and 26MgAr+. From these observations, we could extract an accurate potential-energy function for the electronic ground state of MgAr2+, prove experimentally that it is thermodynamically stable, and analyze the nature of the bond. We found the binding energy to be more than 1.3 eV (125 kJ mol−1), i.e., comparable to a typical covalent bond, despite the fact that both constituents (Mg2+ and Ar) have full-shell rare-gas electron configurations.The photoelectron spectra we obtained were sufficiently resolved  and low numbers  of MgAr+ ions generated in our experiments.Our approach to obtain spectroscopic information on DIDIs relies on the preparation of the precursor singly-charged molecule BA1/2+ ← A1/2+ designates the transition between the rovibrational level with vibrational and rotational quantum numbers v′ and J′ of the Ω = 1/2 spin–orbit component of the A+ 2ΠΩ electronic state of MgAr+ and the rovibrational level with quantum numbers v′′ and J′′ of the Ω = 1/2 spin–orbit component of the 3dπΩ+ Rydberg state of MgAr+. More generally, we designate the rotational and vibrational quantum numbers of the a state of MgAr with unprimed letters, those of the X+, AΩ+ and 3dπΩ+ of MgAr+ using letters with a plus (+), a prime (′) and a double-prime (′′) superscript, respectively, and those of the X2+ state of MgAr2+ with letters with a double-plus (2+) superscript. The integer quantum numbers N+ and N2+ used to label the rotational levels of the electronic states of Σ symmetry correspond to the standard notation for states following Hund's angular-momentum coupling case (b) and the quantum numbers J, J′ and J′′ used for the rotational levels of electronic states of Π symmetry correspond to the standard notation for states following Hund's angular-momentum coupling case (a), as described, e.g., in The term symbols and quantum numbers used to designate the different states are cumbersome. To define and simplify the notation of states and transitions, we use the nomenclature summarized in 24MgAr and its ions, unless stated otherwise.All results presented below are for 22.1v = 0) ground vibrational state was produced by laser ablation of a rod of natural Mg (24Mg (79%), 25Mg (10%), and 26Mg (11%)) in a supersonic expansion of Ar gas. The molecular beam passed through a 3 mm-diameter skimmer located 8 cm downstream of the ablation source before entering the photoexcitation chamber, where it was intersected at right angles by four co-propagating Nd:YAG-pumped dye lasers  that were frequency doubled or tripled in β-barium borate crystals. We refer to these lasers as lasers 1 to 4 below. The laser wavenumbers were calibrated using a commercial wavemeter with a specified accuracy of 0.02 cm−1. Photoexcitation took place within an electrode stack used to apply pulsed electric potentials to field ionize high Rydberg states and extract the produced photoelectrons and photoions into a linear time-of-flight (TOF) spectrometer. MgAr+ was produced in the lowest vibrational levels (v+ ≤ 5) of the X+ electronic ground state by photoionization of metastable MgAr using laser 1 at  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.ngrt.i" format="PBM" resolution="300" x-size="9" y-size="11" xml:space="preserve">001100100010011000000000000000000000011000100001100110000100010000100100000101100000101000000110000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.454545pt" height="16.000000pt" viewBox="0 0 13.454545 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M160 840 l0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 80 0 80 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -80 0 -80 0 0 40 0 40 -80 0 -80 0 0 -40z M80 520 l0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -200 0 -200 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 80 0 80 40 0 40 0 0 80 0 80 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 200 0 200 -40 0 -40 0 0 40 0 40 -80 0 -80 0 0 -40z"/></g></svg>1 = 39 239 cm−1. The particle density of MgAr+ in the X+(v+ = 5) level was estimated to be ∼104 cm−3 based on the signal intensity in the TOF spectrum, corresponding to ∼200 ions in an interaction volume of ∼0.02 cm3  ← X+(v+) transition  ← X+ at wavenumbers 2 around 31 253 and 31 513 cm−1, respectively. Laser 3 further excited to selected rotational levels of the 3dπ1/2+ states at wavenumbers 3 around 35 597, 35 843, and 35 823 cm−1, respectively. The final ionizing transition to the MgAr2+ X2+ state was induced by laser 4, which was tuned in the range 4 = 44 400–46 500 cm−1. All lasers had the same linear polarization and had a beam diameter of ∼1 mm in the interaction region. The pulse energies of lasers 2, 3, and 4 were typically ∼0.6, ∼0.2, and ∼1 mJ, respectively.For the photoionization of MgAr+ that had been characterized prior to this work are the X+, AΩ+ and B+ states  + Ar(1S0) dissociation asymptote, we identified the lowest vibrational levels of the 3dπ1/2+ state to be ideal intermediate states because of their long lifetimes (>50 ps) and large Franck–Condon factors.471/2+ ← X+ transition recorded by monitoring the yield of Mg+ photodissociation product as a function of the wavenumber 2 of laser 2.42,43 The sticks indicate the positions and relative intensities of individual rovibronic transitions, calculated for a rotational temperature of 4 K using standard expressions37,38 for transitions between rovibrational levels of 2Σ+ and 2Π1/2 states. The red arrow corresponds to the position of laser 2 which selects the J′ = 3.5, 4.5, and 6.5 rotational levels from which the spectrum of the 3dπ1/2+ ← A1/2+ transition depicted in −1, while monitoring the MgAr2+ signal. It consists of three branches characterized by J′′ − J′ = 0, ±1 and the assignments are grouped and colored according to the selected A1/2+ rotational levels.The only electronic states of MgArates see .39,41–46 and the rotational constants  of the 3dπ1/2+ levels were determined from the rotational line positions using the formula for the transition energy,37 denotes the rotational constant of the A1/2+(v′) state determined in v′′ is based on a standard isotopic-shift analysis.47,48 The relative intensities were calculated using well-known expressions for rotational line intensities.37,38 In order to avoid power broadening and obtain well-resolved spectra, the pulse energies of lasers 2 and 3 had to be reduced by factors between 5 and 10, compared to the numbers given above, which were required to record the spectra of MgAr2+ from the selected 3dπ1/2+ levels with a sufficient signal-to-noise ratio.The band origins 2.329 and mass-analyzed threshold ionization (MATI) spectroscopy30 are high-resolution variants of threshold photoelectron spectroscopy. Spectra are recorded by monitoring the pulsed-electric-field-ionization yield of very high Rydberg states  located just below the ionization thresholds as a function of the frequency of a tunable radiation source. After correction for the shifts of the ionization thresholds induced by the electric fields, the positions of the lines observed in the spectra correspond to energy differences between the levels of the ionized molecule (charge Z) and its precursor (charge Z − 1).Pulsed-field-ionization zero-kinetic-energy photoelectron (PFI-ZEKE-PE) spectroscopyEI of the ionization thresholds induced by a single pulsed field of strength F is given in good approximation by49–51Fi are used (i is the pulse index).52 In first approximation, the widths Γi of the lines obtained by monitoring the field-ionization signal generated by the i-th pulse of the sequence are51E = −hcZ2R/n*2, where R is Rydberg's constant and n* the effective principal quantum number  we used a four-pulse sequence  and, for each laser scan, we recorded the three spectra corresponding to F2, F3, and F4 simultaneously. We recorded the MATI spectra with optimized pulse sequences, e.g., F1 = −0.86 V cm−1, F2 = −1.72 V cm−1, and F3 = +172.4 V cm−1, the spectra obtained from F2 being the high-resolution ones. Because of the small numbers of state-selected MgAr+ ions (∼200 per laser shot), the pulsed-field-ionization signal was very weak (less than 1 count per laser shot) and the spectra had to be measured several times and averaged to improve the signal-to-noise ratio.To record the PFI-ZEKE-PE spectra of MgAr32+(v2+ = 0–8) ← 3dπ1/2+(v′′ = 2–4) photoionizing transitions. Our measurements include an overview photoionization spectrum in the vicinity of the v2+ = 0–2 vibrational levels of the X2+ state, PFI-ZEKE-PE spectra of transitions to the X2+ levels, and MATI spectra of transitions to the X2+(v2+ = 0–8) levels.We have measured the spectra of the X+ from its 3dπ1/2+(v′′ = 2) state to the region of the first three vibrational levels of the ground state of MgAr2+. The spectrum shows two distinct steps that indicate the ionization thresholds associated with the v2+ = 0, 1 levels of the X2+ state. Several sharp resonances are also present in the spectrum, which we attribute to autoionizing Rydberg states of MgAr+. Just above the ionization threshold associated with the X2+(v2+ = 0) state, we could assign several of these resonances to Rydberg states of MgAr+ with principal quantum numbers n = 37–40 belonging to series converging to the X2+(v2+ = 1) level of MgAr2+, as indicated along the lower assignment bar. The blue bars represent the Franck–Condon factors of the X2+(v2+ = 0–2) ← 3dπ1/2+(v′′ = 2) transitions calculated using the potential-energy functions shown in 2+(v2+ = 2) state explains why no step could be detected in the photoionization spectrum at the v2+ = 2 ionization threshold.2+(v2+ = 0) ← 3dπ1/2+(v′′ = 2) transition are presented in 2+ signals that were produced by the different field-ionization pulses. 24MgAr+ and 26MgAr+, respectively, recorded using the two-pulse sequence −0.86, +172.4 V cm−1. We determined the isotopic shift (Δ4 = 9.7 cm−1) of the transition by fitting the error function53 to the high-energy edges of the spectra and extracting the difference in the inflection points shown as black vertical lines. From the isotopic shift of the 3dπ1/2+(v′′ = 2) level  level to be Δ2+(0)X = Δ3dπ − Δ4 = 5.4(20) cm−1. A similar measurement of the isotopic shift of the X2+(v2+ = 1) vibrational threshold yielded Δ2+(1)X = 10.9(20) cm−1. These results, together with the vibrational constants determined for 24MgAr2+ (see below), were used to unambiguously establish the vibrational assignment in a standard isotopic shift analysis.48 The fall of the MATI signal below the ionization threshold (24MgAr+ as it is for 26MgAr+ (High-resolution PFI-ZEKE-PE and MATI spectra of the Xhreshold  is twice 26MgAr+ . This di2+(v2+ = 3) ← 3dπ1/2+(v′′ = 3) transition recorded using the sequence of field-ionization pulses of +0.26, −1.12, −1.72, and +172.4 V cm−1 and collecting the ionization signal from the −1.72 V cm−1 pulse. Although the resolution was not sufficient to resolve the rotational structure in the spectra, we could observe a broadening of the rotational contour when selecting different rotational levels of the 3dπ1/2+(v′′ = 3) state, i.e., J′′ = 1.5  state corresponding to branches with N2+ − J′′ = −1.5,…,+1.5. From simulations (not shown) of the rotational contour using the rotational constants B3′′ = 0.1813 cm−1  cm−1 and ωexe = 2.477(15) cm−1, respectively, which are very close to the values of ωe = 328.2 cm−1 and ωexe = 2.55 cm−1 calculated ab initio by Gardner et al.36 Using the wavenumbers of the A1/2+(v′ = 1) ← X+(v+ = 0)  ← A1/2+(v′ = 1) /(hc) = 111 824(4) cm−1, which, to our knowledge, is the most accurate value for the ionization threshold of a molecular cation obtained to date.The results of our measurements of the vibrational structure of the XD0 of the X2+ state via the thermodynamic cycle1/2+(v′ = 1) state43 (D1(A1/2+) = 5210.7 cm−1) and of the ionization energy of the Mg+ 3p 2P1/2 state54 (EI(Mg+ 3p 2P1/2)/(hc) = 85598.33 cm−1) as well as the wavenumber of the X2+(v2+ = 0) ← A1/2+(v′ = 1) transition, calculated from D0(X2+) lies within ∼40 cm−1 of the theoretical value of 10730.5 cm−1 reported by Gardner et al.36We determined the dissociation threshold 2+ ground state we used a model potential of the formA, B, and b are adjustable parameters, Z = 2, and α is set to the known value of the polarizability volume of Ar.55 In 2+ and Ar. Several authors have used similar model potential functions to describe singly-charged diatomic molecular ions dominated by electrostatic interactions  kJ mol−1) appears surprisingly large at first sight given the rare-gas electron configurations of Ar and Mg2+. The attractive part of the potential is dominated by the long-range term, with a minor contribution from the Morse term, which highlights the electrostatic nature of the bond. However, considering that both Mg2+ and Ar are chemically hard species, we expected the Morse term to be even less significant. We attribute this weak “chemical” contribution to the binding energy to a charge-transfer interaction with the repulsive MgAr2+ A2+ state  + Ar+(2P3/2). This asymptote lies 5842 cm−1 above the dissociation limit Mg2+ + Ar, corresponding to the difference between the known values of the ionization energies of Ar and Mg+.54 The strong Pauli repulsion between the full-shell constituents Mg2+ and Ar dominates the potential energy at distances below the LeRoy radius60RLeRoy  of the MgAr2+ X2+ ground state using the techniques of PFI-ZEKE-PE and MATI spectroscopy and were able to observe the effect of the rotational structure on the spectra as well as to determine the isotopic shifts of the X2+ levels of 24MgAr2+ and 26MgAr2+. From our measurements we could determine accurate values for the ionization energy of MgAr+ and the dissociation energy of the ground state of MgAr2+. We also derived a potential-energy function that accurately describes the interaction between Mg2+ and Ar and unravels the binding mechanisms. The bond is dominated by electrostatic interactions. The double charge of the Mg2+ (Z = 2) constituent leads to a dissociation energy that is comparable to that of a covalent bond. This behavior follows from the Z2 (= 4) dependence of the charge-induced-dipole interaction (see Z2 = 1) an increase in binding energy of much more than a factor of four is expected because of the reduction of the internuclear separation when increasing Z. From 2+. In the case of MgAr2+ and the isoelectronic singly charged NaAr+ one finds η = 4.07 using  and R+e,NaAr = 2.78 Å from In this article, we have reported the first spectroscopic characterization of a thermodynamically stable DIDI. We measured the photoionizing transitions to the first nine vibrational levels  the measurement of high-resolution photoelectron spectra does not require high-density samples (here 5000 ions per second at 104 cm−3 density) and is ideally suited for the study of charged species, as is well known for anions  = 121267.64 cm−1 in the case of Mg+ 54), which is low as explained in Section 1. The metallic nature of B further guarantees that B+ and therefore also BA+ have low-lying electronic states that can be used as intermediate states to efficiently ionize BA+ in resonant multiphoton processes. (iii) The parent cation BA+ can be produced in selected rovibrational levels by photoexcitation from the ground state of BA using threshold ionization techniques (see 2+.In this article, we have demonstrated the successful use of high-resolution photoelectron spectroscopy of the singly-charged parent ion BAques see , which c+, however, are more stringent because the energies are higher in this case and adequate multiphoton ionization sequences may be more difficult to find. Nevertheless, the list of metastable DIDIs presented in the review of Sabzyan et al.9 makes one optimistic that several of them can be studied with the method presented here. Studies of He22+, O22+, and CO2+ would be of particular importance for fundamental reasons and also for applications in atmospheric chemistry and astrophysics. In the future, we expect that progress in the development of powerful narrow-band VUV radiation at free-electron laser facilities will offer the possibility of efficiently ionizing molecular cations in single-photon processes and recording their high-resolution single-photon photoelectron spectra.Photoelectron spectroscopy of singly charged cations can also be used to study metastable DIDIs. The requirements for the efficient photoionization of BAThe experimental method presented here to study molecular dications is not restricted to DIDIs, but can of course be equally well applied to polyatomic systems.There are no conflicts to declare.


A techno-economicand sensitivity analysis for the bioconversion of biogas into 10 tectoine·y–1 was conducted in two stages: (I)bioconversion of CH4 into ectoine in a bubble column bioreactorand (II) ectoine purification via ion exchange chromatography. Thetechno-economic analysis showed high investment (4.2 M€) andoperational costs (1.4 M€·y–1). However,the high margin between the ectoine market value (600–1000€·kg–1) and the estimated ectoine productioncosts (214 €·kg–1) resulted in a highprofitability for the process, with a net present value evaluatedat 20 years (NPV20) of 33.6 M€. The cost sensitivityanalysis conducted revealed a great influence of equipment and consumablecosts on the ectoine production costs. In contrast to alternativebiogas valorization into heat and electricity or into low added-valuebioproducts, biogas bioconversion into ectoine exhibited high robustnesstoward changes in energy, water, transportation, and labor costs.The worst- and best-case scenarios evaluated showed ectoine break-evenprices ranging from 158 to 275 €·kg–1, ∼3–6 times lower than the current industrial ectoinemarket value.The capacity of haloalkaliphilicmethanotrophic bacteria to synthesizeectoine from CH A techno-economic and sensitivity assessmentof biogas valorizationinto ectoine, the highest added-value bioproduct produced with methanotrophicbacteria. Particularly,different European consortia have included in their biorefinery conceptsthe biological transformation of biogas into: biomethane (INCOVERand URBIOFIN), PHA (URBIOFIN), biostimulants (CIRCULAR BIOCARBON)or ectoine (DEEP PURPLE). These demo-scale projects, together withan extensive investigation work at laboratory scale, have consistentlyevidenced the high economic and environmental potential of these technologiesat industrial scale.10In this context, alternative biogas valorization pathways suchas the production of biomethane  and platformchemicals such as methanol, polyhydroxyalkanoates (PHA), or singlecell protein from biogas components (mainly methane (CH4-based bioproducts struggle to compete inprice against their oil-based or sugar-based counterparts mainly dueto the high energy demand required for CH4 and oxygen (O2) gas–liquid mass transfer and the low productivityof methanotrophic fermentation processes.12 These technological barriers are especially relevant for the productionof low added-value products such as PHA (4–20 €·kg–1), single cell protein (0.5–1 €·kg–1), or methanol (0.5–2 €·kg–1), whose operational costs often exceed their marketselling prices, thus hindering their production in waste treatmentfacilities.8Today, CH–1), a bacterialosmotic protector with a high industrial interest in the cosmeticindustry, has opened the door to the production of high added-valueproducts from biogas.14 Ectoine, with a market priceranging from 600 to 1000 €·kg–1 andan annual demand in the range of 20 t, has been traditionally producedvia sugar-based fermentation with Halomonas elongate in a process called “bio-milking”.16 This biotechnological process presents high production costs dueto the use of high quality carbon sources  and sterileconditions, and an expensive downstream processing.18 Therefore, the use of CH4-biogas as a widely availableand low-cost substrate might contribute to the reduction of ectoineproduction costs and can be regarded as an opportunity for waste managementcompanies to invest in circular economy concepts. However, the techno-economicfeasibility of upscaling the production process and the influenceof commodity prices and capital costs on biogas-based ectoine productionremain unknown.However, the recentdiscovery of the capacity of haloalkaliphilicmethanotrophic bacteria to accumulate high amounts of ectoine  and to the ectoinepurification process via ion exchange chromatography (IEX). In addition,a sensitivity analysis was performed to assess the influence of laborand transportation costs, commodity prices, capital costs, and theinterest and tax rates on the final market price of ectoine.To the best of the authors knowledge, this study constitutes thefirst comprehensive techno-economic and sensitivity analysis focusingon the production of high added-value products from biogas.Supporting Information . A detailed compilation of the equipment designcalculation has been included in the SI. The text includes all the relevant parameters for a reliable reproductionof the process design, economic calculations, and sensitivity analysisherein performed.All calculationswere performed in Excel Sheets and have been corroboratedwith global and elemental mass balances. Mass and energy balanceswere performed assuming an ideal gas behavior given the low pressureand temperatures of the streams. Mass and energy balances as wellas stream tables can be consulted in the 3·h–1 was selected as a model centralized anaerobic digestion plant constructedin medium and large municipalities. In this type of plant, 40% ofthe total biogas production is typically used for internal energyprovision via CHP and therefore, the remaining 600 N m3·h–1 are available to be further valorized.In this study, 67 N m3·h–1 of theremaining biogas stream was considered as a carbon source for theproduction of 10 t·y–1 ectoine. The processwas divided into two different stages: (I) ectoine biosynthesis frombiogas and (II) ectoine extraction and purification .A waste treatmentplant with a continuousbiogas production of 1000 N mfication 1. A more2S removal efficiency(H2S-RE).19 In this process,sulfur-oxidizing bacteria use nitrate (NO3–) instead of oxygen as the electron acceptor for the oxidation ofH2S into SO42–.20 A 4 m3  = 1.3 m) biotricklingfilter packed with a mixture of activated carbon and inert materialwas designed with an empty bed residence time (EBRT) of 3 min andan H2S-RE of 99%. A molar nitrogen-to-sulfur ratio of 2.5was guaranteed by continuously spraying the packing media with a sodiumnitrate (NaNO3) and micronutrients solution at a tricklingliquid velocity of 10 m·h–1.21 A constant pH of 7 was maintained via addition of 5 M sodiumhydroxide solution (NaOH).Prior to biogas valorizationinto ectoine, a biogas desulfurization stage was designed for preventingcorrosion in downstream piping and equipment. Biological anoxic desulfurizationwas selected as the model technology given its low operational costs,reduced environmental impact, and high H4-biogas. The use of mixed methanotrophic cultureshas been demonstrated to be an efficient method supporting long-termoperation and process robustness, given its inherent prevention ofculture contamination.22 Despite the factthat the bacterial population structure might be highly variable dependingon the inoculum and the culture and environmental conditions, previouswork at lab-scale under nonsterile conditions has shown a predominanceof ectoine producing methanotrophs such as Methylomicrobiumburyatense and Methylomicrobium japanense species in these mixed cultures.23 ANaCl concentration of 6%w·w–1 has been reportedin the literature as the optimal salinity for the accumulation ofectoine in haloalkaliphilic methanotrophic cultures.24 The bioreactor was operated under continuous mode at a dilutionrate of 0.4 d–1. A mineral medium solution containing41.0 g NaNO3·L–1, 82.5 g NaCl·L–1, and trace concentrations of micronutrients was continuouslyadded to support haloalkaliphilic methanotrophic bacteria growth andectoine synthesis. Recent studies have shown that high copper concentrationspromote ectoine excretion from the cell in haloalkaliphilic bacteria.24 In this study, ectoine excretion under highsalinity conditions was considered negligible, given the trace levelsof copper in the mineral medium. A tungsten concentration of 0.07mg·L–1 was supplemented to the liquid mediumin order to prevent the formation of formic acid, which typicallyexhibits a significant inhibitory effect on the elimination capacityof methane (CH4-EC).25 A specificbiomass production yield of 0.4 g biomass · g CH4–1 and an ectoine accumulation of 70 mg ectoine·g biomass–1 were chosen according to previous resultsat laboratory scale.23 The stoichiometricformulas of biomass and ectoine were C4H8O2N and C6H10N2O2, respectively. A mineralization ratio of 0.7 mol CO2·molCH4–1 and an oxygen demand of 1.5 molO2·mol CH4–1 were usedaccording to A mixed culture of haloalkaliphilicmethanotrophic bacteria was selected for the production of ectoinefrom CH3  and a gas EBRT of 1.2 h wascalculated as the model bioreactor to support an effective gas–liquidmass transfer of CH4 (biogas) and O2 (air).The calculation process has been detailed in the SI. Perfect mixing in the BCB was assumed given the high turbulenceinduced by the high biogas/air gas flow and the high height-to-diameterratio (H/D) (10) of the bioreactor,resulting in a CH4 removal efficiency (CH4-RE)of 90%. CH4-RE was defined as the percentage of methanebeing eliminated in the bioreactor by the action of methanotrophicbacteria according to Q stands for the volumetric gas flow inthe inlet and outlet streams, and YCH4 stands for the molar fraction of CH4 in the gas phasein the inlet and outlet gas streams.A BCB with a total volume of 196 m4/CO2/N2/O2, respectively.Therefore, further valorization of the outlet gas stream was not consideredas an energy vector given the high amount of inert compounds (N2 and CO2) and the concentration of CH4 and O2 below the explosion limits (CH4 5–15%and O2 > 13%). Additionally, recycling the outlet streaminto the bioreactor would result in a decrease in the gas–liquidgradient and a high energy cost for the recompression of the gas stream.The calculated outlet gas composition was 1.4/17.1/79.3/2.1%forCH4-EC constitutes the main biotechnological limitationgiven its large influence on the capital investment costs (TIC).7 CH4-EC has been defined as the CH4 mass flow eliminated by volumetric unit of bioreactor accordingto Q stands for the inlet and outlet volumetric flow of gasstreams, YCH4 refers to the molar fractionof CH4 in the inlet and outlet streams, and V represents the liquid volume in the bioreactor.Previous studies on biogas valorization in BCBs have shown thatCH4-EC in large-scale BCBs. For the purposeof this study, a CH4-EC value of 148 g CH4 m–3 h–1 was extrapolated from commercialBCBs treating 30%v·v–1 CO streams, which canreach up to 1 kg CO·m–3·h–1.26 A CO volumetric mass transfer coefficient(klaCO) was calculated with CCOin represents the inlet COgas concentration, HCO refers to the dimensionlessHenry’s law constant of CO (29.81 at 288.15 K and 3.78 atm),and CLCO for the bulk aqueous CO concentration,considered negligible under mass transfer limitation scenarios.27However, to date there is not available information in theliteratureconcerning CH4 mass transfercoefficient (CH4-kla) was calculated according to Vm standsfor the molar volume at the normal boiling point of CH4 (35.05 m3·kg–1) and CO (32.74m3·kg–1).28The volumetric CH4-EC was calculated accordingto i stands for the volumetricmass transfer coefficient of substance i, CCH4,in stands for the inlet CH4 gas concentration, HCH4 for the dimensionless Henry’s law constantof CH4 (43.03 at 288.15 K and 3.78 atm), and CL,CH4 for the bulk aqueous CH4 concentration,considered negligible under mass-transfer limitation scenarios.27CH–1. An aliquot of the liquid fraction of 10% was daily wasted fromthe system to avoid the accumulation of secondary metabolites. Theconcentrated biomass stream was subjected to a hyposmotic shock ina nonsaline medium to promote the excretion of 85% of the total intracellularectoine.30 The process was carried out in a 104.1 L continuousstirred tank reactor (CSTR) with a hydraulic retention time (HRT)of 5 min.26 Biomass containing only 15%of the initial intracellular ectoine was subsequently centrifugedto a concentration of 200 g biomass·L–1 andrecirculated to the bioreactor. A fraction of the biomass was continuouslywasted to maintain an average biomass residence time of 9 d, preventingbiomass activity decay, similarly to the industrial biomilking processwith Halomonas Elongate.11 The aqueous solution containing the extracted ectoine was desalinizedvia subsequent ultrafiltration and electrodialysis. A 58.2 m2 ultrafiltration membrane system was designed with a typical permeateflux of 15 L·m–2·h–1, a pressure drop of 300 mbar, and a biomass recovery of 99%. Accordingly,a typical permeate flux of 45 L·m–2·h–1, a pressure drop of 200 mbar, and a concentrationfactor of 25 were selected for desalination with a 17.5 m2 electrodialysis system.Haloalkaliphilicmethanotrophic bacteria containing ectoine was harvested and centrifugedto reach a biomass concentration of 200 g biomass·L31 This process was simulated in this paper with a product recoveryand purity of 62% and 97%, respectively. Prior to the isolation ofectoine via IEX, the liquid stream containing ectoine was acidifiedto pH 2 by addition of 10 M HCl in a 1.7 m3  CSTR operated at a HRT of 1h.18 Then, the ectoine broth was pumpedinto a column packed with an ion-exchange resin and selectively adsorbed.A resin bed volume (BV) of 500 L  was needed to achieve an ectoine recovery of90%. A high performance ion-exchange resin (DOWEX 50w × 8) withan adsorbing capacity of 0.1 kg ectoine·kg resin–1 and a density of 800 kg·m3 was selected.32 The adsorbed ectoine was washed with 2 BV of98%w·w–1 H2SO4 and 2BV of distilled water to remove impurities. Finally, the ectoine waseluted with 6 BV of 1.3 M NaOH, of which 4 BV were discarded. Subsequently,the liquid was neutralized to pH 7 via addition of 98%w·w–1 H2SO4 in a 40.4 L  CSTR with an EBRT of1 h.A two-step IEX and methanol crystallizationprocess has been identified as the most efficient and scalable methodfor ectoine concentration and purification, capable of providing ahigh recovery and purity of the product.–1 in a 400 L spray dryer operated at0.3 atm and heated via low pressure steam (2 bar). The spray dryingsystem was designed with a specific evaporation rate of 100 kg water·m–3·h–1. Prior to ectoine crystallization,the solid product was dissolved into methanol (10 kg methanol·kgectoine–1) in a 16.4 L  CSTR operated at an HRT of 1 h. Anadditional ultrafiltration (1.1 m2) step was included beforeectoine crystallization to remove insoluble matter, especially theNa2SO4 produced during the previous neutralizationstep. Ectoine crystallization was performed in a 12.3 L  CSTR operated at a HRT of 1 h.99% of the methanol was evaporated at 65 °C with the use of lowpressure steam (2 bar) and subsequently recovered in a 3.6 m2 condenser using cooling water as the refrigerant (15 °C). Theectoine crystallized was then centrifuged to remove the remainingmethanol. The final product was obtained after a second drying stepin a 0.9 m2 tray dryer with warm air (20 °C).In a later step, the product was dried to a moisture contentof5%w·w20), the payback period (PP), and the internal rate of return(IRR). The NPV20 was calculated from the free cash flow(FCF) according to t represents the financialperiod in years, and r stands for the interest rate(5%). For the calculation of FCF, the TIC were attributed to year0 and a circulating capital over the TIC of 5% was included in year1. A linear depreciation of 20 years and a tax rate of 30% were selectedfor the calculation. A median selling price of 600 €·kgectoine–1 was used to estimate the NPV20. The IRR was calculated as the value of r thatmakes NPV20 = 0. Finally, the PP was estimated as the firstperiod in which the accumulated FCF is positive. The break-even pricewas used for estimating the ectoine production costs as the valueof sales that guarantees a NPV20 equal to zero.The economic analysis was performedusing as indicators the net present value evaluated at 20 years .TIC were calculated according to Lang’sMethod. This method is based on a series of factors for the estimationof the TIC from the sum of the individual prices of equipment (PEC).A Lang factor of 4.09 has been calculated as optimum for solid–liquidprocesses like the one evaluated in this paper 1.33 EquiTables S1–S7; Figures S1–S3). Typicalvalues for energy requirements in centrifuges (1 kWh·m–3), mixers (0.2 kW·m–3), and electrodialysis(7 kWh·m–3) were selected according to theliterature.37 Energy requirements for pumps were calculated accordingto Ppump represents the power in kW, Q stands for the volumetric flow expressed in m3·s–1, ΔP is the pressure drop inkPa, and 0.7 is the electrical efficiency of pumps and compressors.Operational costswere calculatedas the sum of consumables ,transportation cost of raw materials and products, maintenance costs,labor costs, and wastewater treatment cost. Given the high geographicalvariability in commodity prices, Madrid (Spain) was selected as themodel city for the estimation, presenting a worldwide average costfor energy and water selling prices 2. ConsumPblower representsthe power in kW, Pis stands for the isentropicpower in kW, 0.7 is the electrical blower efficiency, γ is theadiabatic coefficient, Tout refers to the gas isentropic outlet temperature, Tin represents the gas inlet temperature, Pm is the gas molecular weight, and Q stands for the inlet volumetric flow.Energy requirementsfor blowers and compressors were calculatedaccording to –1). The cost of ectoine transportation was equallydistributed between production and extraction costs. Yearly maintenancecosts of 3.5% over the TIC were selected as recommended by industrialwaste operators. To the best of the authors’ knowledge, thereis not a standard method for evaluating the labor cost of biogas upgradingprocesses integrated in larger facilities. Therefore, labor costswere evaluated assuming a total of 192 person-h·week–1, as recommended by industrial waste operators. Two full-time operatorswith 8 h-shift during week days (2 person · 1 shift · 8h·shift–1 · 5 d·week–1 = 80 person-h·week–1) and 2 part-time operatorsduring the evening and night shifts during the whole week (2 person· 2 shift–1 · 4 h·shift–1 · 7 d·week–1 = 112 person-h·week–1) were herein considered. A wage of 14.5 €·person-h–1 was considered as average salary in Madrid (Spain).38 Wastewater treatment costs were considered comparableto domestic wastewater (0.2 €·m–3) giventhe low organic load of these waste streams.Transportation costs for rawmaterials and ectoine products wereconsidered comparable to other petrochemical products  and liquid (6 m3 water·h–1) streams processed at similarHRT, which incurred a great variability in equipment size. The applicationof the Lang’s Method resulted in a TIC of 4.21 M€, with2.69 M€ and 1.52 M€ for the ectoine production and downstreamprocessing, respectively  was due to the need for high-quality materials that resist thehigh corrosion induced by the high salinity of the mineral mediumand the additional safety measures required for the ATEX applicationderived from mixing biogas and air.39 Theseresults are in good agreement with previous techno-economic analysesstudying the bioconversion of CH4 into PHA, which identifiedBCBs as the most expensive pieces of equipment.40 Thebiotrickling filter dedicated to biogas desulfurization constitutedthe second most expensive equipment in the biogas bioconversion processwith 53 048 €.The commissioning of the BCB,with an individual volume of 195mThe equipment showing the highestPEC in the downstream processingstage were the drying units with 0.22 M€, accounting for 59.6%of the downstream PEC and 21.6% of the overall process PEC. Ectoineexcretion, ultrafiltration, and electrodialysis, IEX, and crystallizationrepresented 8.0%, 8.3%, 8.9%, and 15.2% over the total downstreamPEC, respectively.–1, with 0.3 M€·y–1 and 1.1 M€·y–1 associated with ectoine production and purification,respectively. The purchase of consumables  was the most significant operational cost (0.9 M€·y–1), accounting for 65.7% of the total operational costs and to their limited lifespan (80 d). In this context,the bulk purchase of this raw material (1825 kg·y–1) could help in reducing the effective selling price of ectoine.The ectoine retention performance was used as resin selection criteriain this study, while the maximum NPV20 might be achievedusing resins with a lower performance but a longer lifespan.The operationalcosts for biogas-basedectoine production accounted for 1.4 M€·yal costs3. The ioMaintenance and labor costs representedthe second and third largestoperational cost, with 10.3% and 10.2% of the total cost share 3. Mainte–1 and 121.0t·y–1 of NaCl and NaNO3, respectively).However, this cost was marginal compared to those reported by Shazadand co-workers (2013) during the production of PHA from slaughterwaste, which accounted for almost 50% of the total production costs.41 The transportation cost of products representedonly 2.5% of the total transportation costs given the reduced amountof ectoine produced (10.0 t·y–1). These resultssuggest that the use of in situ produced biogas as carbon source forbacterial fermentations entails a significant reduction in the bioproductsproduction costs. Notwithstanding, the influence of transportationcosts on the final ectoine price should be taken into account in thedesign of next-generation biorefineries as it might be of relevanceif the distance to mineral salts suppliers and potential ectoine buyerswas significantly increased.In addition, transportation costs were identified as a non-negligibleoperational cost, with 8.3% of the total operational costs 3. This cFinally, the costs of energy andwater represented only 3.7% and1.5% of the total operational costs, respectively. The relevance ofthese commodities might change significantly depending on the locationof the plant and should not be neglected when studying the viabilityof future ectoine production plants. Wastewater treatment costs werenegligible compared to the total operational costs, representing ashare of only 0.3%.–1 of ectoine were obtainedfrom 67 N m3 h–1 of biogas, which wasin line with the estimated globalectoine demand ranging 10–20 t·y–1.The calculations resulted in an overall ectoine productivity of 17mg ectoine produced·m–3 biogas treated. A break-evenprice for ectoine, calculated as the selling price at which NPV20 becomes 0, of 214 €·kg–1 wasestimated. This value represented a 3-fold decrease against the lowestreported market values for ectoine production with HalomonasElongate (600–1000 €·kg–1). Interestingly, 66.6% of the break-even price was allocated tothe operational costs, while the remaining 33.4% was attributed tothe TIC and its amortization.As a result ofthe process, 10 t·y–1) and the median ectoine market value (600 €·kg–1), an outstandingly positive NPV20 of 33.6M€ was obtained. Accordingly, a IRR of 70.4% and a PP of 1.5year (2 years) were obtained. The high profitability of CH4-to-ectoine process has been previously reported by Cantera and co-workersin a preliminary techno-economic study for the coproduction of PHA,single cell protein, extracellular polysaccharides, and ectoine fromCH4 diluted streams.36 In thisregard, given the wide difference between production cost and currentectoine selling price, and the limited number of companies dedicatedto the production of ectoine, it is worth questioning if ectoine iscurrently sold following a cost-based or a market-based strategy.If ectoine commercialization follows a cost-based strategy, then theproduction of ectoine from biogas using methanotrophic bacteria couldhave the potential to displace the current industrial routes with Halomonas Elongate. On the contrary, if ectoine is followinga market-based strategy, the selling prices could drop in the future.In any scenario, the low production costs herein presented (3–6times lower than the current selling price of ectoine) guarantee acurrent and future economic feasibility of the biogas-to-ectoine process.In view of the wide differencebetween the calculated ectoine production costs . A 25% increase/decrease in the consumables purchasecosts resulted in ectoine production costs ranging from 191 to 238€·kg–1 4.–1.The ectoine production costs also showed a high sensitivity towardthe PEC estimation. A ±25% variance in the PEC resulted in a±10.1% change in the production costs of ectoine 4. Intere–1 to 18.1 €·h–1, butalso entailed a change in the Lang Factor from 3.77 to 4.42, thusaffecting the TIC , a 25% change on the unitary transportationcosts (from 60 €·t–1 to 45 and 75 €·t–1) induced negligible changes on the ectoine productioncosts from 213.8 to 214.8 €·kg–1. Interestingly,increasing transportation unitary costs by 1 order of magnitude (from60 to 600 €·t–1) increased ectoine productioncosts to only 232.5 €·kg–1. These resultsshowed a high robustness of the process toward changes on the mostinfluential parameters for other low added-value bioproducts and expandthe economic viability of ectoine production in waste treatment facilitiesto all sort of socio-economic contexts in terms of commodities (waterand energy) costs. The different cost sensitivity of low added- andhigh added-value bioproducts produced by methanotrophs in waste treatmentplants enriches the current dissertation on the scientific communityon the roadmap for future and successful CH4-biorefineries.Variations in energy and water purchase pricesresulted in negligiblechanges in the ectoine production costs of 0.6 and 0.3%, respectively.These results implied that ectoine production cost in waste treatmentfacilities might be profitable regardless of the variability of commodityprices. In contrast, recent techno-economic analyses indicated thatthe production of low added-value products such as biopolymers frombiogas exhibited an inherently high influence of these commoditieson the economic feasibility of the technology.20 and IRR in the worst-and best-case scenarios showed a high variability ranging from 28.4to 38.6 M€ and from 46.8 to 112.3%, respectively. The calculatedPP varied slightly with the 25% increase/decrease between 0.96 (1year) and 2.35 (3 years). Finally, ectoine production costs rangingfrom 158 to 275 €·kg–1 were estimatedin the best and worst case scenarios.The worst and best case scenarios for ectoine production from biogaswere calculated assuming a 25% increase/decrease in all the aforementionedparameters. The evaluation of NPVr) from 5% to10% and 15% resulted in ectoine production costs of 214, 238, and264 €·kg–1, respectively. This increasein the ectoine production costs was correlated to the increase inthe fixed and amortization costs of ectoine from 72 €·kg–1 to 95 €·kg–1 and 121€·kg–1 for r valuesof 5%, 10%, and 15%, respectively . Onthe contrary, the sensitivity analysis revealed a high robustnesstoward changes on water and energy prices, labor, and transportationcosts. In summary, this study demonstrated that large-scale productionof high added-value products from biogas represents a highly profitablealternative to the current utilization of biogas as energy source,but also a much more feasible valorization pathway than the productionof low added-value bioproducts. However, the influence of certaintechno-economic aspects such as the economy of scale or the microbialbioconversion yields of methane into ectoine on the development offuture cost-effective biogas biorefineries must be further investigated.This work constituted the first techno-economic study of the large-scaleproduction of ectoine from biogas in waste treatment plants. The resultsindicated a high profitability of the process with a payback timebelow 3 years in all the scenarios evaluated. Ectoine break-even pricesin the best and worst case scenarios considered entailed a 3- to 6-folddecrease in the ectoine production costs when compared to the currentproduction via long-time fermentation with


Herein, we report the synthesis of ∼15 nm LiYF4, NaYF4, and NaGdF4 nanoparticles doped with Er3+ ions and their core-shell structures. The luminescent properties are compared, showing that Er3+ ions with single-doped LiYF4 and NaYF4 nanoparticles generate stronger luminescence than Er3+ ions with doped NaGdF4, despite the difference in relative intensity at different regions. By epitaxial growth an inert homogeneous protective layer, the surface luminescence of the core-shell structure is further enhanced by about 5.1 times, 6.5 times, and 167.7 times for LiYF4, NaYF4, and NaGdF4, respectively. The excellent luminescence in both visible and NIR range of these core-shell nanoparticles makes them potential candidate for bio-applications.The availability of colloidal nano-materials with high efficiency, stability, and non-toxicity in the near infrared-II range is beneficial for biological diagnosis and therapy. Rare earth doped nanoparticles are ideal luminescent agents for bio-applications in the near infrared-II range due to the abundant energy level distribution. Among them, both excitation and emission range of Er The 4f-4f transition of rare earth elements endows them with outstanding optical properties. Specifically, the energy of photons can be converted through rational designed transition among their abundant energy levels . Further3+, which enhanced the down-shifting emission at 1550 nm under excitation at 980 nm  and 660 nm (4F9/2 → 4I15/2) in the visible range, 800 nm (4I9/2 → 4I15/2) in the NIR-I range, and 1000 nm (4I11/2 → 4I15/2) in the NIR-II range, respectively. The two-photon NIR-II to NIR-II UC process is rather unique, which have been investigated widely. However, how the matrix determines the luminescence remains unknown and deserves our efforts.The penetration depth of photons with different energies is significantly varied due to the scattering and absorption of biological tissue . Comparet 980 nm . They act 980 nm . The timightness . Er3+ ioansition . Moreove4, NaYF4, and NaGdF4, each one doped with Er3+ ions. Their luminescence properties were compared, showing different relative intensity within each matrix. With epitaxial growth an inert homogeneous protect layer, the luminescence of each kind of UCNPs achieve much improvement due to the surface passivation. A proper mechanism was proposed based on the emission intensity dependence on excitation power density.Herein, we reported the successful synthesis of three types of fluoride matrix, LiYFYttrium (III) chloride hexahydrate (99.99%), erbium (III) chloride hexahydrate (99.9%), gadolinium (III) chloride hexahydrate (99.9%), yttrium (III) oxide (99.999%), gadolinium (III) oxide (99.9%), sodium trifluoroacetate (98%), lithium trifluoroacetate (95%), trifluoroacetic acid (98%), 1-octadecene (90%), and oleic acid (90%) were purchased from Sigma-Aldrich. Sodium hydroxide (98%), lithium hydroxide (98%), and ammonium fluoride (98%) were purchased from Aladdin Co. All chemicals were used as received.The size and morphology of the resulting nanoparticles were characterized by Hitachi 7700 electron microscope. Upconversion emission spectra in visible and NIR range were recorded using Horiba Fluoromax–4. Laser excitation at 1532 nm, which was purchased from Changchun New Industries Optoelectronics Technology Co., Ltd. The emission signal from the sample in the cuvette was collected at 90° relative to the exciting laser beam. The powder X-ray diffraction (XRD) patterns were recorded by a Bruker D8 Advance diffractometer using Cu Kα radiation. The 2θ angle of the XRD spectra was recorded at a scanning rate of 5°/min.3•H2O and 0.9°mmol YCl3•H2O were added into a 100°ml flask containing 6°ml oleic acid and 15°ml octadecene. The mixture was heated to 160°C for 60°min then cooled down to room temperature. Subsequently, a solution of 4°mmol NH4F and 2.5°mmol LiOH (2.5°mmol NaOH for NaYF4:10%Er3+ and NaGdF4:10%Er3+) in 10°ml methanol was added and stirred for 30°min. The reaction mixture was then heated at 100°C for 30°min to remove the methanol, followed by heating up to 290°C (300°C for NaYF4:10%Er3+ and NaGdF4:10%Er3+) and keeping for 60°min before cooling down. A syringe needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature and precipitated by excess ethanol and collected by centrifugation. The precipitate was washed with ethanol several times, and the nanocrystals were finally dispersed in hexane.In a typical procedure, a total of 0.1°mmol ErCl4:10%Er3+@LiYF4 core-shell nanoparticles are synthesized via a thermal decomposition method. A total of 0.5°mmol of Y2O3 was dissolved in 50% trifluoroacetic acid at 95°C in a three-neck flask. Then, the solutions were evaporated to dryness under an argon gas purge. Next, 10°ml of oleic acid, 10°ml of 1-octadecene, 1.5°mmol lithium trifluoroacetate, and 1°mmol LiYF4:10%Er3+ core  were added into the flask. The resulting solution was then heated at 120°C with magnetic stirring for 45°min to remove water and oxygen. The brown solution was then heated to 310°C at a rate of about 12°C per min under argon gas protection and kept at this temperature under vigorous stirring for 45°min. A syringe needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature and precipitated by excess ethanol and collected by centrifugation. The precipitate was washed with ethanol several times, and the nanocrystals were dispersed in hexane.The LiYF3+ was doped into three matrix LiYF4, NaYF4 and NaGdF4 according to a previous synthesis procedure (–1), which are beneficial for efficient UC luminescence. As shown in 4 was shaped with spindle morphology  and (200) plane of the cubic phase of NaGdF4. Previous reports have concluded doping ions with larger size will efficiently reduce the energy and time for transformation from cubic to hexagonal phase  and Y3+ (1.159 Å). The differential between Gd3+ and Er3+ is more remarkable than that of Y3+ and Er3+. As a result, the NaGdF4 requires more energy to complete the phase transfer. Then we further grew a homogeneous shell on each type of core UCNP as protect layer since the inert epitaxial shell growth could enhance the UC luminescence by overcoming the concentration quenching effects. The Transmission electron microscopy (TEM) imaging shows the sizes of three types of NPs grow larger, and the morphologies are much more uniform and monodisperse after coating the shell layer , 4F9/2 → 4I15/2 (660 nm), 4I9/2 → 4I15/2 (800 nm), and 4I11/2 → 4I15/2 (1000 nm), respectively. The optimized concentration of Er3+ ions is determined to be 10% according to integration of the total emission intensity  of NaYF4 is about 1.7 times stronger than LiYF4, while on the contrary, the 4I11/2 → 4I15/2 transition (1000 nm) of LiYF4 is about twice of the NaYF4. As we all know, the luminescence of rare earth can be ascribed to the local field splitting, which is highly depended on the crystal symmetry. Then it is understandable the tetragonal and hexagonal phases with different symmetry structures will favor variable energy transition, corresponding to the diverse dominant luminescence character. This explains the intensities of different peaks are not identical for the NaYF4 and LiYF4 matrix, and so the NIR-II emission bands of LiYF4:Er3+ are more suitable than NaYF4:Er3+ NPs. As for the NaGdF4, it contains both cubic and hexagonal phase. For the parity forbidden essence, the phase with lower symmetry exhibits stronger emission intensity than the phase with higher symmetry  and red (4F9/2) emission bands as well as the two-photon process of 800 nm (4I9/2) and 980 nm (4I11/2) emission peaks.Then, we measured the pump power dependences of four bands for the colloidal LiYFechanism . Briefly4:Er3+@LiYF4, NaYF4:Er3+@NaYF4, and NaGdF4:Er3+@NaGdF4 core-shell UCNPs under excitation at 1532 nm. Homogeneous epitaxial growth of the shell layer promoted the luminescence intensity efficiently. Notably, NaGdF4 completed the phase transfer during the shell coating, which enhanced the core luminescence intensity about 167.7 folds. Taking LiYF4:Er3+@LiYF4 as example, the intensities of four UC peaks showed linear dependence on the excitation power, which can be explained by a saturation effect of 4I13/2 energy level. These core-shell UCNPs are pronounced as ideal NIR-II agent for biological related applications.In conclusion, we have observed four intensive emission bands for three colloidal and uniform LiYF


The differentiation of cardiac fibroblast to myofibroblast is the key process of cardiac fibrosis. In the study, we aimed to determine the function of E2F Transcription Factor 1 (E2F1) in human cardiac fibroblasts (HCFs) differentiation, search for its downstream genes and elucidate the function of them in HCFs differentiation. As a result, we found that E2F1 was up-regulated in TGF-β1-induced HCFs differentiation. Silencing the expression of E2F1 by siRNA in HCFs, we found that the expression of differentiation-related genes  was significantly suppressed, combining with proliferation and migration assay, we determined that HCFs differentiation was decreased. Luciferase report assay and immunoprecipitation proved that the oncogene CCNE2 was a direct target gene of E2F1, overexpression of CCNE2 was found in differentiated HCFs, silencing the expression of CCNE2 by siRNA decreased HCFs differentiation. Our research suggested that E2F1 and its downstream target gene CCNE2 play a vital role in TGF-β1-induced HCFs differentiation, thus E2F1 and CCNE2 may be a potential therapeutic target for cardiac fibrosis. Myocardial infarction (MI) remains to be the leading cause of mortality and morbidity in humans, after MI, cardiac fibroblasts are activated to avoid cardiac rapture, but over-activation of cardiac fibroblasts will lead to cardiac fibrosis. Cardiac fibrosis is characterized by cardiac fibroblast-to-myofibroblast differentiation and extracellular matrix (ECM) deposition , is wildE2F transcription factor 1 (E2F1) is one of the E2F transcription factors family. Recent research shows that E2F family of transcription factors are the key regulators of numerous genes involved in DNA damage repair, cell apoptosis, and cell cycle progression ,11. The In this research, we aimed to determine the function of E2F1 in HCFs differentiation, search for its downstream genes and elucidate the function of them in HCFs differentiation. In our hypothesis, we assumed that E2F1 participated in TGF-β1-induced HCFs differentiation by promoting the transcription activity of its downstream gene CCNE2. Our data showed that in HCFs, E2F1 promoted the fibroblast-to-myofibroblast differentiation. What’s more, we found that E2F1 directly promoted the transcriptional activity of its downstream target gene CCNE2. Suppression of CCNE2 attenuated the differentiation of myofibroblast after TGF-β1 treatment. To our knowledge, this finding was also the first time to illustrate E2F1 and CCNE2 genes in regulating HCFs differentiation.2. To avoid the impact of biomechanical input, the culture plates used were pre-coated with gelatin. The 3 to 6 passages of cultured cells were used in our experiments. Before transfection, HCFs were starved in DMEM/F12 without serum for 6 h and incubated with 10 ng/ml TGF-β1 . In our research, E2F1 siRNA (100 pmol/2 mL) and pcDNA3.1 (4 μg/2 mL), and CCNE2 siRNA (100 pmol/2 mL) were transiently transfected by Lipofectamine 3000 for 8 h. The E2F1 siRNA (100 nM), E2F1 pcDNA3.1 (100 nM), NC siRNA (100 nM), NC pcDNA3.1 (100 nM), CCNE2 siRNA (50 nM), and siRNA negative control (50 nM) were purchased from Sangon . Following the guidance of manufacturer’s instruction, all these procedures were performed.Following our previous routines , human cTotal RNAs were extracted by EZ-press RNA Purification Kit (EZ-bioscience) and reverse transcribed by HiScript III RT SuperMix for qPCR (+gDNA wiper) cDNA Synthesis Kit  by following manufacturer’s instruction. Quantitative realtime RT-PCR was performed by ChamQ SYBR GREEN qPCR Master Mix . The sequences of primers in this experiment are listed below: α-SMA , COL-1A1 , FN1 , E2F1 , E2F2 , E2F3 , CCNE2 , GAPDH .RIPA  was used for harvesting cell lysates, and BCA protein assay kit  was used for determining the concentration of protein samples. Same amounts of proteins were separated by SDS-PAGE gels (10%) and transferred to PVDF membranes. After 2 h of blocking in 5% Bovine Serum Albumin , PVDF membranes were incubated in primary antibodies under the temperature of room (24°C) for 2 h. After that, PVDF membranes were washed 3 times in 15 min with TBST and incubated in horseradish peroxidase-conjugated secondary antibody  for 1 hunder 24°C. Finally, an enhanced chemiluminescence  was used for imaging western blots. Image data were analyzed by ImageJ software . The antibodies used in this experiment are listed as below Anti-FN-1 , anti-Col-1a1 , anti-α-SMA , anti-E2F1 , anti-CCNE2 , anti-GAPDH  for western-blot.12-well plates with microscope slides were used for culturing HCFs, after 24 h of incubating, EDU Alexa Flour 555 imaging kit  was used for detecting the proliferation of HCFs. All procedures were performed by following the manufacturer’s instructions. HCFs were incubated for 2 h after adding 1 μL EDU solution into each well. After incubating, HCFs were fixed in 75% ethyl alcohol on condition of −20°C overnight, then, fixed cells were penetrated by 0.3% PBST for 30 min, after washing 3 times in 0.1% TBST for 15 min. HCFs were incubated in EDU click solution (with Alexa Flour 564) away from light for 30 min. Images were captured by fluorescence microscope . The proliferation of HCFs were calculated by counting the percentage of EDU positive cells (red fluorescence) to DAPI stained cells (blue fluorescence).6-well plates were used for culturing HCFs to 90% confluence and starved the HCFs into serum-free medium for 6 h to avoid the effect of serum on cell migration. After starving, 6-well plates were scratched with a 200 μl pipette tip by following the guidance of scratch ruler. PBS was used for washing plates, after three times of washing, the cells were incubated in different treatment groups (listed in legends of figures). Images were captured at 0 h and 24 h after scratching by an inverted light microscope  and the wound closure (%) was accessed.5 cells were resuspended in 500 μl of DMEM/F12 serum-free medium in the upper chamber after different treatments (listed in legends of figures), 500 μl of DMEM/F12 medium supplemented with 25% FBS was added in the lower chamber. After 24 h of incubating, the migrated cells were fixed in 4% formaldehyde and penetrated by 0.3% PBST for 30 min, after washing 3 times in 0.1% TBST for 15 min, HCFs were stained with crystal violet . Under a light microscope , five fields were randomly selected, and the average number of migrated HCFs was calculated to evaluate the ability of migration.Transwell chamber  was used for culturing HCFs. To evaluate the migration ability of HCFs in transwell migration assay, 1 × 10Two pmirGLO dual luciferase vectors  were designed to carry CCNE2 5ʹUTR sequences of wild type and mutant E2F1 binding sites, respectively. Then, vectors and pcDNA3.1s (to overexpress E2F1) were transfected into HCFs by Lipofectamine 3000 (invitrogen). After 24 h, luciferase activity was detected by a dual-luciferase assay kit  under the guidance of manufacturer’s instructions.6-well plates were used for culturing HCFs. HCFs were collected after treatments. BCA protein assay kit was used for determining the concentrations of cell lysates. 1 mg of total proteins were used for immunoprecipitation. The samples were incubated in rabbit-controlled Ig-G antibody and anti-E2F1  antibody under condition of 4°C overnight. Then the samples were suspended with 25 μL of protein A/G PLUS-Agarose. After 2 h of incubation and washing, the samples were western-blotted, and anti-CCNE2  antibody was incubated to evaluate the conjunction amount of E2F1 and CCNE2.Datasets in bar charts are shown in the form of means ± SD. The samples were compared by non-paired student’s t-test (for 2 different groups) and One-way-ANOVA (for over 3 different groups). P value <0.05 was considered a statistically significant difference, different P values were marked over the bar chart, and all analyses and graphic drawings were performed in GraphPad Prism 7.In our results, we found that expressions of both differentiation associated proteins and E2F1 were upregulated in TGF-β1, stimulated HCFs, silenced the expression of E2F1, demoted the expressions of differentiation associated proteins, cell proliferation, and migration abilities. We also found that CCNE2 was the downstream gene of E2F1 by luciferase assay and immunoprecipitation. E2F1 promoted the TGF-β1-induced HCFs differentiation through promoting the transcription of CCNE2.Western blot assay and qRT-PCR were performed to assess the fibrosis level. As a result, in TGF-β1-treated HCFs, expression of fibrosis-related genes  was significantly increased in 12, 24 h, and reached the peak in 48 h ). The plE2F1 siRNA were transfected into HCFs to further validate the effect of E2F1 on cardiac fibroblasts differentiation. Our findings showed that the mRNA and protein levels of fibrotic markers were attenuated in TGF-β1-treated HCFs with E2F1 siRNA while negative control (NC) siRNA was ineffective in non-treated HCFs ). MoreovCellular signal pathway database KEGG  predicteA siRNA was designed to silence the expression of CCNE2. We found that silenced CCNE2 significantly demoted the HCFs differentiation. When E2F1 pcDNA3.1 and CCNE2 siRNA transfected together into HCFs, CCNE2 siRNA significantly rescued the fibroblast-to-myofibroblast differentiation promoting effect of E2F1 pcDNA3.1, differentiation-associated markers were decreased in HCFs transfected with CCNE2 siRNA and E2F1 pcDNA3.1 compared with those who transfected E2F1 pcDNA3.1 alone ). BesideExcessive activation of HCFs caused by fibroblast-to-myofibroblast differentiation will lead to myocardial dysfunction and heart failure. The activation of the differentiation induced by TGF-β1 is now regarded as a critical stimulus of the process. In our research, we firstly demonstrated that E2F1 was up-regulated in TGF-β1-induced HCFs differentiation. Secondly, we silenced the expression of E2F1 and suggested that HCFs differentiation was inhibited. Then, we validated that E2F1 promoted the transcription of CCNE2. Finally, we demonstrated that the differentiation effects of overexpressed E2F1 can be rescued by CCNE2 knockdown in TGF-β1-induced HCFs. Our data proved that E2F1 promoted the TGF-β1-induced HCFs differentiation through promoting the transcription of CCNE2.Cardiac fibrosis is a remarkable pathological process after myocardial infarction that was characterized by fibroblast-to-myofibroblast differentiation and ECM deposition ,20, and Classic cellular signal pathway of cardiac fibrosis induced by TGF-β1 is TGF-β–Smad2/3 signaling pathway. After TGF-β1 activating, it binds with its receptor TβRI and TβRII. TβRI phosphorylates its downstream transcription factor SMAD2/3. TβRI binds with SMAD4 and enters together with TβRI into cell nucleus to promote transcription of fibrosis-related genes . Direct Cyclin E2 (CCNE2) is a less known member of Cyclin E family. Those highly conserved proteins regulate cell cycle by activating CDK2 to promote cell cycle progression. Cyclin E2 is less known than cyclin E1, but it is also related to proliferation. Genomic instability and high expression correlate to poor outcome in malignancies . In prevOur study showed that E2F1 is up-regulated after TGF-β1 stimulation and E2F1 promoted TGF-β1 induced fibroblast-to-myofibroblast differentiation of HCFs by promoting the transcription of CCNE2. These findings suggested that E2F1 and CCNE2 could be a potential therapeutic target for cardiac fibrosis.


The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron–CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron–CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions. Cardiac function is modulated by the sympathetic and parasympathetic branches of the cardiac autonomic nervous system (cANS), which can stimulate or inhibit, e.g., the heart rate and contraction force. In vivo, neuronal axons innervate cardiac tissue ,2,3, whiPreviously reported coculture devices used for neurons and CMs were compartmentalized polydimethylsiloxane (PDMS)-based chips that allowed the isolation of neuronal somas from the CMs and restricted cell-to-cell interactions to axons extending through microtunnels between the compartments ,11,12. CPrimary human neurons and CMs are difficult to harvest, while hiPSCs can be easily differentiated into neurons and CMs in large quantities . As manyThe 3D3C chip consists of three serial compartments connected via microtunnels, allowing the culture of up to three different cell or tissue types in a single chip. Here, it was used in the coculture of hiPSC-neurons and -CMs, as the chip isolated neuronal somas in their own compartment, allowing axonal growth to the cardiac compartment through the microtunnels , whereas their expression in the neuronal samples was almost undetectable, with significant differences when compared to the cardiac samples. TUBB3 was robustly expressed in neuronal samples, with significantly lower expression in cardiac samples. However, 4-week cardiac coculture samples had higher TUBB3 expression than other cardiac samples suggesting an increase in the axonal expression of TUBB3 in the coculture. It was shown earlier that TUBB3 is detectable axon-specifically in neurons ..33].Plastic and MatTek 48-well plate control samples were prepared following the same coating protocol except using 0.1 mg/mL PLO for 1 h and 15 µg/mL LN521. Wells for neurons and neuron–CM cocultures were coated with LN521, whereas wells for CMs were coated with gelatin. Neuron–CM cocultures were cultured in 1:1 neuronal and EB medium, while neuronal and cardiac monocultures were cultured in their respective media.2O provided in the kit, after which the RNA quality and concentration were measured using a NanoDrop (Thermo Scientific).Samples were collected, and RNA was extracted with a NucleoSpin RNA kit  following the manufacturer’s instructions immediately after video microscopy recording of CMs and chemical stimulation of neurons. Samples were lysed in buffer RA1, containing 3.5 µL of b-mercaptoethanol separately from the neuronal and cardiac compartments, and the content of three cell-specific compartments of the 3D3C chips were pooled to make one sample. Control samples in plastic well plates were lysed similarly, with one well containing one sample. RNA was extracted from the cell lysate and eluted in 20 µL of RNase-free HReverse transcription was performed with a High-Capacity cDNA Reverse Transcription kit  following the manufacturer’s instructions. Briefly, 10 µL of prepared 2× RT Master Mix and 10 µL of RNA sample were mixed. In some cases, RNA samples were diluted in nuclease-free water to even the RNA concentration in the reactions. Samples were then run in Mastercycler EP Gradient S  using the following protocol: 10 min at +25 °C, 120 min at +37 °C, 5 min at +85 °C, and +4 °C until storage at −20 °C.ADRB2, ADRB3, CHRM2, MYBPC3, TNNT2, CHAT, TH, DBH, TUBB3, GAPDH, EEF1A1; EE+, and GUSB. Reactions were prepared by mixing 25 µL of TaqMan PreAmp Master Mix, 12.5 µL of 0.2× TaqMan Assay Pool and 12.5 µL of the cDNA sample. Samples were run in Mastercycler EP Gradient S using the following protocol: 10 min at +95 °C, 14 cycles of 15 s at +95 °C, and 4 min at +60 °C, 10 min at +99 °C, and at +4 °C until storage at −20 °C.cDNA preamplification was performed due to the low RNA concentration of the 3D3C chip samples by using 2× TaqMan PreAmp Master Mix (Applied Biosystem) following the manufacturer’s instructions. The TaqMan 20× assays used are listed in −ΔΔCt method [GAPDH, EEF1A1; EE+ and GUSB were used as endogenous controls. For ADRB2, ADRB3, CHRM2, MYBPC3, and TNNT2, a 2-week cardiac control was used for normalization, whereas for CHAT, TH, DB, and TUBB3, a 2-week neuron control was used for normalization.qPCR for preamplified cDNA samples was performed by using TaqMan Fast Advanced Master Mix (Applied Biosystem) following the manufacturer’s instructions. The same TaqMan 20× assays were used as in the preamplification step. Briefly, master mixes for each gene were prepared as instructed in the kit. Preamplified cDNA samples were diluted to nuclease-free water 1:5 (20 µL of sample in 80 µL of water). Two µL of the diluted sample was added to 6 µL of the master mix, and three technical replicates were used for each sample for each gene. The plates were run in a CFX384 Touch Real-Time PCR System  with the following protocol: 2 min at +50 °C, 20 s at +95 °C, and 40 cycles of 3 s at +95 °C, and 30 s at +60 °C. The 2t method  was usedImmunocytochemistry (ICC) was performed at 2 and 4 weeks of coculture immediately after video microscopy recording of cardiomyocytes and chemical stimulation of neurons. The ICC protocol was used for 3D3C chip samples as described earlier , with thForty-eight MatTek control samples were stained as previously described . The dil+ solution to the neuronal compartment. The extracellular solution contained 93 mM of NaCl, 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM of D-glucose, 50 mM of KCl, 1.25 mM of NaH2PO4, 2 mM of CaCl2, and 1 mM of MgCl2 (pH adjusted to 7.4 with NaOH). CM activity was recorded before neuronal stimulation for baseline measurement (1. Rec), during medium change in the neuronal compartment to evaluate the effect of the liquid change on CM beating (2. Rec), during neuronal high K+ exposure to capture the immediate CM response (3. Rec) and approximately 2 min after neuronal stimulation as a follow-up (4. Rec). All videos of CM activity were recorded for 40 s at 44 fps using an Eclipse TS100 microscope  and IGV-B1620M-KC000 camera . MUSCLEMOTION (version 1.0) software [+ solution was added directly to CMs, which caused the CMs to cease beating (Neurons were stimulated through the addition of a high-Ksoftware  using in beating .rstatix (version 0.7.0) package [p < 0.05 was considered statistically significant. The data are presented as the mean ± standard deviation in the text and as boxplots and boxplots with individual data points in the figures.Statistical analysis was performed using R and the  package . A nonpa


SOX2, SOX9, and CD44 in detached cells. KDM6B occupies the promoter region of both SOX2 and CD44 to regulate their expression epigenetically. We also noticed an increased occupancy of the HIF1α promoter by KDM6B, suggesting its regulatory role in maintaining hypoxia in detached cancer cells. This observation was further strengthened as we found a significant positive association in the expression of both KDM6B and HIF1α in various cancer types. Overall, our results reveal a novel transcriptional program that regulates resistance against anoikis and maintains stemness-like properties.Epithelial cancer cells that lose attachment from the extracellular matrix (ECM) to seed in a distant organ often undergo anoikis’s specialized form of apoptosis. Recently, KDM3A (H3K9 demethylase) has been identified as a critical effector of anoikis in cancer cells. However, whether other histone demethylases are involved in promoting or resisting anoikis remains elusive. We screened the major histone demethylases and found that both H3K27 histone demethylases, namely, KDM6A/B were highly expressed during ECM detachment. Inhibition of the KDM6A/B activity by using a specific inhibitor results in reduced sphere formation capacity and increased apoptosis. Knockout of KDM6B leads to the loss of stem cell properties in solitary cells. Furthermore, we found that KDM6B maintains stemness by transcriptionally regulating the expression of stemness genes  Normal epithelial cells are non-tumorigenic and are anchorage-dependent, i.e., attach well with the matrix for obtaining nutrition and physiological cues. However, tumorigenic epithelial cells require detachment from the matrix and move to different body sites to initiate metastasis . Severalα prompts the expression of several JmjC- Jumonji-domain histone demethylases (KDMs) and histone methylases under hypoxic conditions  . Circulanditions .The HIF alters the chromatin in different ways by changing the expression level of different KDMs and DNA methyltransferase and increases transcription factors to promote EMTs .In addition to this, the transcriptomic adjustment of the circulating, matrix-detached tumorigenic cells is also documented due to diverging epigenetic changes at DNA and histone levels .Therefore, in the current study, we aimed to explore the impact of matrix detachment on the epigenome, mainly KDM(s), in various cancer types. Furthermore, we aimed to assess the role of identified KDM(s) in maintaining the stemness and survival of matrix-detached cancer cells.2. All the cell lines, namely, HCT116, HeLa, and 22Rʋ1 used in the current study, were obtained from ATCC (United States). The cells were grown to 70–90% confluence, and the media was changed every two days. In addition, the cell lines were routinely checked for any Mycoplasma contamination.Various cancer cell lines were maintained in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin–streptomycin (Invitrogen) at 37°C in 5% CO2 incubator at 37°C. The assessments of matrix detachment were performed in a cell suspension culture. First, cells were dislodged by simple agitation in the presence of trypsin, followed with washing with PBS, resuspended at 0.5×106 cells/ml in serum-free culture media containing BSA, and finally were cultured in an ultra-low attachment plate at 37°C for various time points, which resulted in the formation of spheroids. The spheroids were treated with either a vehicle control (0.1% DMSO) or with different concentrations of GSK J4  for five days. The images were captured by using a Nikon (United States) inverted light microscope. Images were analyzed for size measurement by ImageJ software (https://imagej.net/Invasion_assay).We have followed the well-established method of the cell detachment model in our experiments . BrieflyCT) values obtained from the instrument’s software were used to calculate the respective mRNAs’ fold change. ΔCT was calculated by subtracting the CT value of the housekeeping gene from that of the mRNA of interest. ΔΔCT for each mRNA was then calculated by subtracting the control’s CT value from the experimental value. The fold change was calculated by formula 2CT−ΔΔ.Briefly, RNA was extracted from all the cell lines at the end of different experimental conditions using the RNAeasy kit (Qiagen) and reverse-transcribed with a high-capacity cDNA reverse transcription kit (applied biosystems). cDNA (1–100 ng) was amplified in triplicates using gene-specific primers . Thresho6 were obtained. They were washed with ice-cold PBS three times in 500 g for 10 min in 500 µL of lysis buffer 10 mM HEPES, pH 7.9, with 1.5 mM MgCl2 and 10 mM KCl (add 5 µL of 0.1 M DTT and 5 µL of protease inhibitor cocktail) and incubated for 15 min in ice. The supernatant was removed after spinning, and the pellets were resuspended in 200 µL of the lysis buffer, and five gentle strokes were performed using a glass homogenizer. It was allowed to spin for 20 min at 12,000 g. The cytosolic fraction (supernatant) was removed, and 150 µL of nuclear extraction buffer 20 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25% (v/v) glycerol was added with the addition of 1.5 µL of 0.1 M DTT and 1.5 µL of protease inhibitor cocktail. It was incubated in ice for 30 min with a gentle shake every 2 min and allowed to spin at 21,000 g for 10 min to obtain the supernatant (Nuclear protein).Histone H3K27 demethylase KDM6A/B activity was measured by using the Abcam- KDM6A/B activity quantification kit (ab156910). The protocol was performed based on the Kit guidelines. 15 µg of the nuclear extract was used. The nuclear protein was extracted without using detergent. The ECM-detached cells with and without treatment of GSK J4 and ECM-attached cells 2 × 10The H3K27-specific methyltransferase activity was performed using a commercially available kit from Abcam [H3K27 methylation Assay Kit (Colorimetric) # ab156910]. The assay kit could measure the activity or inhibition of H3K27 mono/di/tri subtypes and required cellular nuclear extracts. Briefly, equal amounts of protein samples were added in each independent experiment; however, the overall protein concentration ranged from 100 to 300 ng H3K27 modifications were calculated according to the manufacturer’s instructions, which also accounts for protein amounts, and the final values for each modification were presented as the percentage over untreated control.75 flask (1 × 106/flask). After 24 h, plating cells in ultra-low attachment plates were treated with GSK J4 for the indicated dose for five consecutive days. The new treatment was added every 48 h; following completion of treatment, media was aspirated, and cells were washed with cold PBS (pH 7.4) and pelleted in 15-ml falcon tubes. Ice-cold lysis buffer was added to the pellet. The composition of the lysis buffer was 50 mM Tris–HCl, 150 mM NaCl, 1 mM ethylene glycol-bis(aminoethyl ether)-tetraacetic acid, 1 mM ethylenediaminetetraacetic acid, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4 with freshly added protease inhibitor cocktail . Then, cells were passed through the needle of the syringe to break up the cell aggregates. The lysate was cleared by centrifugation at 14,000 g for 30 min at 4 °C, and the supernatant (nuclear lysate) was used or immediately stored at −80°C. For western blotting, 4–12% polyacrylamide gels were used to resolve 30 μg of protein, transferred onto a nitrocellulose membrane, probed with appropriate monoclonal primary antibodies, and detected by chemiluminescence after incubation with specific secondary antibodies  were cultured in a Ttibodies .HeLa, HCT116, and 22Rʋ1 cells were grown in an ultra-low attachment plate for six days with and without treatment of GSK J4. Cells were washed two times in ice-cold PBS and resuspended in 100 µL of staining solution (5 µL PE-conjugated anti-CD 70 (BD Bioscience), CD 133 (Miltenyibiotec-130-090-853), and FITC-conjugated anti-CD 105(BD Bioscience- 56143) and anti-CD44(Miltenyibiotec -130-098-210), 1% FBS, in 1X PBS and incubated at room temperature under the dark condition for 2 h. Then, cells were washed three times in a wash buffer (1%FBS in 1X PBS) and then analyzed by using a Guava Easy-Cyte flow cytometer. For all assays, 10,000 cells were taken for measurement and also for analysis. For each plot, 1000 cells were displayed.For immunofluorescence assay, cells were grown in ultra-low attachment plates for six days with and without treatment of GSK J4. Then, cells were collected and washed two times with ice-cold PBS, and cells were stained with a staining solution containing  1% FBS in 1X PBS and incubated at room temperature in the dark for 2 h. Then, cells were washed two times in ice-cold PBS, and cells were analyzed using a Amins image stream flow cytometer.pI. After incubation for 20 min in RT (dark condition), the cells were analyzed by using The Guava® easy, yet 5 flow cytometer, and the percentage of apoptosis cells was calculated  three times. The spheroids were broken down by multiple pipetting and resuspended in 100 μL 1X binding buffer, 10 μL Annexin V-FITC, and 5 μL lculated .The CHIP experiments were performed by using the Abcam (ab185913) chip kit. The protocol was followed as per standard guidelines of the manual with minor modifications. The spheroids (ECM detached), ECM-attached cells, and ECM-detached cells were harvested and washed with ice with 3 ml PBS. Cells were resuspended in 1% formaldehyde in the DMEM and incubated at RT for 10 min with the minor rocking platform, and 300 μL of 1.25 M glycine was added for crosslinking washed with ice-cold PBS. The pellet was resuspended in 300 μL lysis buffer, and the chromatin was sheared using a water bath sonicator (15 cycles on and 15 cycles off for 20 min). Three micrograms of the sheared chromatin were taken to the well coated with KDM6B antibodies, input (negative control) along with nonimmune IgG and incubated overnight at 4°C. The unbounded chromatins were removed and washed with the wash buffer. DNA was released using the DNA release buffer and incubated at 60°C for 45 min in a water bath; subsequently, the solution was transferred to PCR tubes heated at 95°C for 15 min at a thermocycler. The DNA was purified by using the column provided in the kit. The PCR was performed by using the targeted primers  of speciKDM6B, SOX2, CD44, and HIF1α, a gene expression profile was taken from two different studies. 1) A metastatic melanoma sample was taken from Hugo et al., 2016 (PMID: 26997480). The study provided the normalized gene expression data in FPKM values from 27 samples. 2) Metastatic breast cancer data of the Metastatic Breast Cancer Project (www.mbcproject.org). The study provided the normalized gene expression data in RSEM values from 146 samples. The normalized expression values were transformed into log2 (x+0.1), and then, the Pearson correlation coefficient was measured with the ggpubr package.For the correlation analysis of gene expressions among t-test was used. Data points in graphs represent mean ± SD, and p values <0.05 were considered significant.Data were analyzed using GraphPad Prism . The two-tailed, unpaired 5 per HeLa cells were plated at a 6-well plate. First, 1 μg plasmid was transfected by using Lipofectamine 3000. After transfection, at 48 h, the cells were collected for extraction of total protein. The knockout efficiency was verified by Western blotting.CRISPR case-based gene knockout experiments, the plasmid-containing CRISPR double nickase was constructed by SANTA CRUZ biotechnology sc-401883-NIC for KDM6B and non-targeted + ve control (sc-437281). For plasmid transfection, 1 × 10To investigate key histone demethylase, promote or regulate anoikis resistance. We measured the expression of various histone demethylases in detachment conditions of different cancer cell types . Quantitative gene transcript analysis at early time points (from 30 min to 24 h) showed statistically significant induction of various histone demethylases such as JARID1A, JARID2, JMJD1A, and KDM6A/B in all the three cancer cell types grown in detached conditions . We deciSince both KDM6A/B are H3K27 me3 demethylases and were most consistently upregulated at all time points in all cell types, we decided to focus our further work on KDM6A/B. We performed quantitative activity assay for KDM6A/B demethylases and found a statistically significant increase in the KDM6A/B demethylase activity in HeLa, 22Rv1, and HCT116 cell lines in detached conditions when compared with the attached. . Based oMatrix detachment of cancer cells tends to form spheroids. Therefore, to investigate the effect of KDM6A/B inhibition in spheroid formation, we treated spheroids of HeLa, HCT116, and 22Rv1 that formed during matrix detachment with GSK J4 , a highlThe reduction in the spheroid size is associated with cell death. So we measure the percentage of cell death that might have occurred in ECM-detached cancer cells during GSK J4 treatment. As expected, GSK J4 significantly induces apoptosis in all the ECM-detached cancer cells. Among them, 22Rv1 showed the maximum percentage of apoptotic cells (57.7%) when compared with HeLa (34.4%) and HCT116 (22.8%) . OverallWe observed that GSK J4 treatment significantly reduces the KDM6A/B activity in all cell lines and KDM6B expression , along wSOX2, SOX9, and CD44 and found significantly increased levels of these stemness-related genes in all matrix-detached cancer cell lines when compared to their attached counterparts  for a total period of 5 days after the initial 24 h of anoikis (detachment). Our data showed that GSK J4 treatment significantly reduces the transcript levels of  control . Next, wntreated .SOX2 and CD44 but not on SOX9 in detached cancer cells. Furthermore, GSK J4 treatment significantly reduces the occupancy of KDM6B on promoters of both SOX2 and CD44, suggesting the KDM6B-driven regulation of these stemness genes in detached cancer cells   along with CD44 and SOX2  in melanoma cancers. We also found that metastatic breast samples had a significantly high positive correlation between CD44 and HIF1α   . Similar 0.0031) .SOX2, CD44, and HIF1α to regulate survival and stemness of cancer cells.Anoikis (matrix detachment) can be regulated by histone demethylases such as KDM3B . HoweverKDM6B was shown to regulate epithelial to mesenchymal (EMT) conversion of cancer cells by regulating the expression of the EMT process’s essential transcription factors . Our resSOX2, SOX9, and CD44, suggesting their epigenetic regulation in matrix-detached conditions. The post-translational modifications of SOX2 are shown to regulate its protein stability and transcriptional activity. Akt-mediated phosphorylation at Thr118 promotes the transcriptional activity of SOX2 in ESCs , MBD1, MBD2, and MeCP2 is well-reported . Our worHypoxia has been recently associated with the matrix detachment of cancer cells and can regulate the matrix-detached cancer cells . Our resSOX2 and CD44-mediated stemness to enhance anchorage-independent survival of various cancer patients’ cells. Additional consequences of the context-specific increase and regulation of HIF1α by KDM6B might be likely to further aid in survival in response to changing nutrient microenvironments. Our study highlights an essential role of KDM6B in cancer and has important implications for targeting this protein for anticancer therapies.Our data suggest that the rapid and sustained upregulation of KDM6B following matrix detachment is necessary for