Studies comparing 21% and 100% O2 during CC were conducted in postnatal models and have not shown a difference in incidence or timing of return of spontaneous circulation (ROSC). The objective of this study is to evaluate systemic oxygenation and oxygen delivery to the brain during CC in an ovine model of perinatal asphyxial arrest induced by umbilical cord occlusion. Pulseless cardiac arrest was induced by umbilical cord occlusion in 22 lambs. After 5 min of asystole, lambs were resuscitated with 21% O2 as per Neonatal Resuscitation Program (NRP) guidelines. At the onset of CC, inspired O2 was either increased to 100% O2 (n = 25) or continued at 21% (n = 9). Lambs were ventilated for 30 min post ROSC and FiO2 was gradually titrated to achieve preductal SpO2 of 85–95%. All lambs achieved ROSC. During CC, PaO2 was 21.6 ± 1.6 mm Hg with 21% and 23.9 ± 6.8 mm Hg with 100% O2 (p = 0.16). Carotid flow was significantly lower during CC (1.2 ± 1.6 mL/kg/min in 21% and 3.2 ± 3.4 mL/kg/min in 100% oxygen) compared to baseline fetal levels (27 ± 9 mL/kg/min). Oxygen delivery to the brain was 0.05 ± 0.06 mL/kg/min in the 21% group and 0.11 ± 0.09 mL/kg/min in the 100% group and was significantly lower than fetal levels (2.1 ± 0.3 mL/kg/min). Immediately after ROSC, lambs ventilated with 100% O2 had higher PaO2 and pulmonary flow. It was concluded that carotid blood flow, systemic PaO2, and oxygen delivery to the brain are very low during chest compressions for cardiac arrest irrespective of 21% or 100% inspired oxygen use during resuscitation.The current guidelines recommend the use of 100% O The infrequent use of chest compressions [2, oxygen content of arterial blood (CaO2), or oxygen delivery to the brain during chest compressions [2 during chest compressions increases oxygen delivery to the brain compared to 21% in a model of perinatal asphyxial cardiac arrest.The majority of newborn infants require limited or no assistance to undergo successful physiologic transition and stabilization at birth. Studies suggest that approximately 10% of infants require some intervention to establish regular respirations at birth with less than 1% needing extensive resuscitative measures such as chest compressions ,3. Initiressions  has imperessions . We hypo2 (EtCO2) monitor [This study was approved by the Institutional Animal Care and Use Committee at the State University of New York at Buffalo . Twenty-two time-dated term (139–141-day gestation) pregnant ewes were obtained from May Family Enterprises . Following an overnight fast, the ewes were induced for anesthesia with intravenous diazepam and ketamine. They were intubated with a 10.0 mm-cuffed endotracheal tube (ETT) and ventilated with 21% oxygen and 2–3% isoflurane at 16 breaths/min. The ewes were continuously monitored with a pulse oximeter and an end-tidal CO monitor . The fet2 administered during chest compressions: (1) 21% inspired O2 or (2) 100% inspired O2. The protocol for cardiac arrest, timing of epinephrine, and dose of epinephrine were similar between the two groups.Lambs from three different experimental protocols that followed the Neonatal Resuscitation Program (NRP) algorithm were used: two studies evaluating optimal flush volume with epinephrine and early cord clamping utilized 100% oxygen during chest compressions; one study generating preliminary data for a postnatal hypothermia trial that utilized 21% oxygen. The lambs were grouped based on the O2 by means of a T-piece resuscitator at a rate of 40 breaths/min and initial pressures of 35/5 cm H2O. Peak inspiratory pressure (PIP) was adjusted as needed to obtain adequate chest rise. Chest compressions were initiated after 30 s of effective ventilation using a two thumb technique at a 3:1 (chest compression:ventilation) ratio and a depth of one-third anterior–posterior diameter. Upon initiation of chest compressions, inspired O2 was continued at 21% or increased to 100% depending on the protocol. The first dose of epinephrine (0.03 mg/kg) was administered if return of spontaneous circulation (ROSC) had not been achieved at 1 min after coordinated chest compressions along with positive pressure ventilation, and every three minutes thereafter until ROSC or for a total of four doses. The ROSC was defined by a heart rate > 60 beats/min with a systolic blood pressure of >30 mm Hg. A baseline arterial blood sample was obtained following instrumentation of the lamb prior to cord occlusion with additional samples at the time of cardiac arrest. Thereafter, blood gases were obtained approximately every minute during resuscitation, at ROSC, and every minute until fifteen minutes following ROSC, followed by every 5 min until 30 min after ROSC. Arterial blood samples were analyzed immediately using a radiometer blood gas analyzer .A five-minute period of cardiac arrest was observed prior to initiating resuscitation to minimize chances of ROSC with positive pressure ventilation (PPV) alone. Cardiac arrest was defined by the absence of carotid blood flow, arterial blood pressure, and audible heart rate. Resuscitation began by providing PPV with 21% O2, and the fraction of inspired oxygen (FiO2) was adjusted to maintain pre-ductal saturations between 85–95%. Hemodynamic parameters were continuously monitored. Lambs were euthanized by administering 100 mg/kg pentobarbital sodium  at the completion of the study.Following ROSC, the lambs were placed on a ventilator. The PIP and rate were adjusted gradually based on tidal volumes  and PaCO2) × carotid blood flow where CaO2 = hemoglobin × 1.34 × oxygen saturation of arterial blood (SaO2)/100 + 0.0031 × PaO2 (in mm Hg).Arterial blood flow and pressures were continuously recorded using AcqKnowledge Acquisition and Analysis Software . Continuous variables were expressed as mean and standard deviation. Oxygen delivery was calculated as oxygen content in the carotid arterial blood  and XLSTAT  softwares were used. Statistical significance was defined as 2 during chest compressions and 16 lambs were ventilated with 100% inspired O2. There were no differences in baseline characteristics  between the lambs in each group, as is shown in 2 required 1.4 ± 0.8 doses of epinephrine and those ventilated with 100% O2 during CC required 1.3 ± 0.8 doses.Twenty-two lambs were asphyxiated to cardiac arrest by umbilical cord occlusion and all lambs achieved ROSC. Out of 22 lambs, 6 were ventilated with 21% inspired O2 groups (p = 0.8). Following ROSC, in the 21% group, the pH increased to 6.87 ± 0.08 at 5 min and 7.08 ± 0.11 at 30 min whereas in the 100% group the pH was 7.00 ± 0.15 (p = 0.07 compared to 21% O2 group at 5 min after ROSC) and 7.18 ± 0.15 (p = 0.16 compared to 21% O2 group at 30 min after ROSC) at 5 and 30 min respectively.Arterial pH during chest compressions was similar between the two groups—6.85 ± 0.08 in 21% and 6.86 ± 0.08 in 100% inspired O2 after asphyxia before starting ventilation was 5.5 ± 2.3 mm Hg. After initiating ventilation with 21% oxygen and chest compressions, the PaO2 increased to 21.6 ± 1.6 mm Hg. Following PPV with 100% inspired oxygen, PaO2 was 23.9 ± 6.8 mm Hg (p = 0.16 compared to 21% O2). 2 in the two groups during chest compressions  in those lambs decreased from 390 ± 70 at 5 min post ROSC to 170 ± 90 at 30 min. Similarly, in the lambs ventilated with 21% O2 the P/F ratio decreased from 240 ± 60 at 5 min to 150 ± 90 at 30 min.The preductal PaOressions A and aftressions B. Following FiO2 C. The Pa2 was 125 ± 4mm Hg. It remained similar in both the groups after initiation of PPV followed by chest compressions—113 ± 22 mm Hg in the 21% and 116 ± 21 mm Hg in 100% inspired O2 groups (p = 0.52). Following ROSC, there was a steady decline in PaCO2—86 ± 3 mm Hg and 76 ± 36 mm Hg at 5 min after ROSC (p = 0.2) and 49 ± 14 mm Hg and 42 ± 14 mm Hg at 30 min after ROSC (p = 0.08) in the 21% and 100% inspired O2 groups respectively.At the time of cardiac arrest, the arterial PaCOp = 0.07). During the recoil phase of chest compressions, carotid flow was negative. In order to capture the spurts of blood perfusing the brain during compressions, peak carotid flow was calculated and analyzed. Carotid flows increased after achieving ROSC. After ROSC, carotid flow was antegrade both during systole and diastole . After achieving ROSC, maximum systolic carotid flow increased to 43 ± 14 mL/kg/min and 49 ± 17 mL/kg/min in the 21% and 100% oxygen groups respectively as shown in p = 0.16). Systolic blood pressures achieved during chest compressions were similar in both the groups—30 ± 18 mm Hg in the 21% group and 27 ± 11 mm Hg in the 100% group (p = 0.11)  A. Similap = 0.13) (2 groups respectively and were significantly different between the two groups (p = 0.002).Similar to carotid blood flow, antegrade pulmonary blood flow was only observed during the compression phase during chest compressions A. Hence, = 0.13)  A. As shOxygen delivery to the brain during chest compressions and after ROSC are shown in p < 0.0001).Since carotid blood flow occurred in short spurts during the compression phase only, we calculated the oxygen delivery during the peak carotid flow during chest compressions. The peak oxygen delivery was not normally distributed. The oxygen delivery to the brain at the peak of chest compressions with 21% oxygen was 0.075 mL/kg/min (IQR: 0–0.2) and was significantly lower than with 100% oxygen 1.4 mL/kg/min  showed reduced mortality with 21% oxygen (8.2% vs. 12.9%) in neonates [There are minimal clinical data comparing 21% and 100% oxygen during neonatal chest compressions in the delivery room. A systematic review of 10 clinical studies published in 2008 comparing 21% and 100% oxygen for resuscitation of term newborns  compared to normal term fetal lambs (pH—7.3 ± 0.09 and SpO2—53 ± 1.4%) [2 during chest compressions after cardiac arrest was 20 mm Hg as compared to 40–60 mm Hg in a term newborn infant. We speculate that although alveolar PAO2 is increased by 100% inspired oxygen, low pulmonary blood flow results in poor gas exchange and low PaO2. An interesting observation was that oxygenation during spurts of antegrade flow in the carotid artery during chest compressions was better with 100% oxygen. The precise benefit of such intermittent perfusion with oxygenated blood and its role in preventing hypoxic injury to the brain is not known. However, after ROSC, the carotid flow increases resulting in hyperoxemia with 100% O2. We speculate this may lead to increased oxygen delivery to the brain which may potentially cause reperfusion injury to the brain. We suggest that inspired O2 concentration should be weaned to 21–30% oxygen immediately after ROSC and titrated based on preductal pulse oximetry with a target goal of 85–95%.We have developed a transitional model of prolonged asphyxiated lamb that best mimics an infant with severe perinatal asphyxia . We have ± 1.4%) . Contrar2 may be different in postnatal arrest after establishment of lungs as the site of gas exchange as compared to our perinatal asphyxia model. Despite these limitations, we provide comprehensive oxygenation and hemodynamic data during chest compressions in perinatal arrest.We acknowledge several limitations in our study. This study was not a randomized trial and the data were extracted and analyzed from lambs studied for other protocols. The oxygen concentration was not masked. However, the systolic pressures generated during chest compressions were similar between the two groups suggesting that the intensity of chest compressions were similar between the two groups. This was a model of complete cardiac arrest and response to 100% oxygen may be different with less profound asphyxia with bradycardia (heart rate < 60/min but not 0/min) necessitating chest compressions. We did not evaluate brain and lung tissue for evidence of oxidative stress or hypoxic-ischemic injury. Finally, response to 100% O2 values are low and similar to fetal baseline levels and did not differ between 21% and 100% oxygen groups, probably secondary to low pulmonary blood flow. We did not demonstrate a difference in oxygen delivery to the brain with 21% and 100% oxygen. These findings neither support nor refute current recommendations to provide supplemental 100% oxygen during chest compressions.Our results demonstrate very low pulmonary and carotid flows during chest compressions for cardiac arrest. In fact, it appears blood flow is intermittent to these essential organs. The preductal PaO2 over the first 5 min after ROSC. Further studies evaluating abrupt decrease in inspired oxygen concentration to 21–30% immediately after achieving ROSC need to be evaluated to minimize the risk of hyperoxic reperfusion injury to the brain.However, following ROSC, lambs that received 100% oxygen had post-asphyxial increased carotid blood flow and hyperoxia in spite of rapid weaning of FiOSystemic and pulmonary hypoperfusion is common during chest compressions in cardiac arrest and brain oxygen delivery is extremely low irrespective of the concentration of inspired oxygen levels. Future randomized and masked studies that compare low vs. high supplemental oxygen during newborn resuscitation with chest compressions for cardiac arrest and bradycardia are needed to assess markers of oxidative stress, as well as assess immunohistochemical changes in the brain to provide a better understanding on the optimal oxygen therapy during resuscitation of newborns.


Dear Editor,N6-methyladenosine (m6A) RNA methylation has been found to greatly impact gene expression, leading to the field of epitranscriptomics.1 Unlike m6A that is an internal modification, a terminal modification at mRNA cap in higher eukaryotes exists, termed as N6,2′-O-dimethyladenosine (m6Am)  cap can be methylated at the 2′-hydroxyl group; and when the first nucleotide is 2′-O-methyladenosine (Am), it can be further methylated at the N6 position to become m6Am. m6Am was first identified in animal cells and virus mRNA in 19752; several years later the methyltransferase was partially purified and was proposed to be a species whose molecular weight is ~65 KD.3 Only very recently, m6Am was found to be reversible as well: the first m6A demethylase FTO also catalyzed the demethylation of m6Am, depending on its sub-cellular localizations.5 By changing FTO levels, m6Am at mRNA cap was also suggested to impair DCP2-mediated mRNA decapping.4 However, the methyltransferase of m6Am is not unambiguously identified, significantly hindering the functional and mechanistic study of m6Am.Dynamic and reversible Am) Fig. . The firN6-methylation activity   Fig. , which wPCIF1 in HEK293 cells by two independent siRNAs and confirmed the knockdown efficiency by qRT-PCR . Additionally, mouse PCIF1 protein also exhibited a robust methylation activity in vitro (Supplementary information, Fig.6Am writer, which is specific for the 5′-end capped RNA.To test whether PCIF1 possesses methyltransferase activity in vivo, we first knocked down own Fig. ; importaged Fig. , suggest Am Fig. . The abo Am Fig. . Moreove Am Fig. . Because Am Fig. , we furt6A-seq experiments for PCIF1 knockdown and control cells using an anti-m6A antibody.9 Because the antibody recognizes m6Am and m6A, both types of modifications were enriched and hence detected simultaneously.10 m6A modifications are known to be enriched around 3′-UTR, with a small portion also present internally in the 5′-UTR; while m6Am modifications localized at the 5′-end of RNA. We envisioned that the cap-specific PCIF1 should selectively alter the m6Am modification at the 5′-terminal region of transcripts. Indeed, we observed a reduction of modification peaks at the 5′-end but not the 3′-UTR regions of mRNAs upon PCIF1 knockdown (Fig. 6A-seq (Fig. 6Am peaks after PCIF1 knockdown when comparing to the m6A and m6A + m6Am categories (Fig. 6A-seq procedure that can preserve the 5′-end information of polyA+ RNA, and again found a decrease of m6Am peak intensity after PCIF1 knockdown (Supplementary information, Fig.6Am modification occurs at the transcription start sites, in accordance with the known m6Am pattern (Supplementary information, Fig.4 Thus, results from our m6A-seq experiments revealed the direct mRNA targets of PCIF1 inside of human cells (Fig. To identify the RNA targets of PCIF1, we performed mown Fig. . One exaseq Fig. ; only thies Fig. . We furtlls Fig. .6Am writer protein, characterized its biochemical property and substrate preference, and profiled its cellular targets using an epitranscriptomic sequencing approach. PCIF1 recognizes the positively charged cap structure m7GpppAm for optimal activity and is a “stand-alone” RNA methyltransferase. In comparison, the internal m6A is installed by a methyltransferase complex, the core components of which are composed of METTL3, METTL14 and WTAP. The m6A methyltransferase complex also recognizes internal adenosines, with a preference for those located within a RRACH consensus motif. Hence, while m6Am and m6A share a common eraser protein FTO, the writer proteins for the two modifications are orthogonal. Manipulating the protein levels of the writers could potentially separate the differential roles of FTO in demethylating m6Am and m6A. The functional study of m6A is greatly facilitated by the discovery and characterization of its regulation system involving the writer, reader and eraser proteins; we envision that the identification of PCIF1 as the m6Am writer will pave the path toward functional and mechanistic dissection of this dynamic and reversible epitranscriptomic mark in the future.Taken together, in this study we revealed the exact identity of the mSupplementary information, figuresSupplementary information, Table S1


Hybrid Fe3O4/SWCNT/PAMMP nanomaterial synthesis was carried out via in situ chemical oxidative polymerization of 3-amine-7-methylamine-2-methylphenazine hydrochloride in the presence of metal-carbon Fe3O4/SWCNT nanocomposites. Fe3O4/SWCNT nanocomposites were obtained by the immobilization of magnetite nanoparticles on the SWCNT surface in the course of Fe3O4 nanoparticles synthesis in alkaline medium. The developed nanocomposite materials were characterized by FTIR spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission (FE-SEM) scanning electron microscopy, atomic absorption spectrometry (AAS), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and magnetometry. The chemical structure and phase composition, magnetic and electrical properties, and thermal stability of the obtained multifunctional nanomaterials, depending on synthesis conditions, were investigated.Polymer-metal-carbon hybrid nanomaterials based on thermostable electroactive poly-3-amine-7-methylamine-2-methylphenazine (PAMMP), single walled carbon nanotubes (SWCNT), and magnetite (Fe Modern technology requires the creation of new generation materials with improved functional characteristics. Hybrid nanomaterials, described here, have a combination of organic and inorganic components providing a range of required properties ,2,3. In 3O4 nanoparticles determines the ferromagnetic behavior of the nanocomposite .,31.3O4/Snd (440) . The abs3O4/SWCNT nanocomposite, polymer-metal-carbon hybrid Fe3O4/SWCNT/PAMMP nanomaterials were synthesized via in situ oxidative polymerization of ADMPC in an aqueous solution of acetonitrile. To immobilize ADMPC on the Fe3O4/SWCNT nanocomposite surface, freshly prepared magnetite nanoparticles precipitated to SWCNT and were washed until neutral reaction without pre-drying, then were added to the reaction neutral medium of Fe3O4/SWCNT/PAMMP nanomaterial synthesis.Based on the obtained metal-carbon Fe3O4/SWCNT nanocomposite. The FTIR spectra of the Fe3O4/SWCNT/PAMMP nanomaterial .The FTIR spectroscopy data confirmed the immobilization of the monomer on the surface of the metal-carbon Fematerial  show a somposite , being a3O4/SWCNT/PAMMP nanocomposite  indicates that the polymer chains grow via the C–N bonding between 3-amine groups and the para position of the phenyl rings in relation to the nitrogen. This type of bonding is observed in the course of aniline polymerization (“head-to-tail” type) [The presence of absorption bands at 820 and 1287 cml” type) ,38,39.−1 in the ATR FTIR spectra of the Fe3O4/SWCNT/PAMMP nanocomposite  is much smaller than that of SWCNT (1067.3 m2/g) and is closer to that of polymer (15.9 m2/g) (In the process of in situ oxidative polymerization of ADMPC on the surface of Feomposite . This is.9 m2/g) . Accordi.9 m2/g) .3O4/SWCNT/PAMMP nanocomposite identify clearly the diffraction peaks of Fe3O4 at scattering angles 2θ = 46.1°, 54.2°, 66.9°, 84.8°, 91.2°, and 102.2° (CrKα-radiation) (3O4 nanoparticles determined using the EsiVision software is within the range of 2 < d < 8 nm (CFe = 17.6–45.7% (The diffraction patterns of the Fediation) . These dd < 8 nm . Accordi.6–45.7% .3O4 nanoparticles. In the Fe3O4/SWCNT/PAMMP nanocomposite, the size distribution curves of Fe3O4 crystallites are narrow. About 95–97% of Fe3O4 crystallites have sizes up to 8 nm. As it can be seen in 3O4/SWCNT nanocomposite has a wider size distribution curve. Only about 85% of Fe3O4 crystallites have sizes up to 8 nm. According to TEM data, the size of Fe3O4 nanoparticles in the Fe3O4/SWCNT nanocomposite is within the range of 6 < d < 15 nm . The residual magnetization MR of neat Fe3O4/SWCNT is 0.45 emu/g, and the coercive force HC is 6 Oe. The superparamagnetic behavior of nanocomposites at relatively high concentrations of magnetic nanoparticles is specified by the small sizes and high dispersity of the magnetic nanoparticles. Anyway, the obtained values of MR/MS for Fe3O4/SWCNT/PAMMP and Fe3O4/SWCNT nanocomposites are characteristic of uniaxial, single-domain particles. The critical size of the transition to a single-domain state for Fe3O4 is 128 nm [The magnetic properties of Fe = 45.7% , which imaterial ,40. As is 128 nm ,40.3O4/SWCNT/PAMMP nanocomposite. 3O4/SWCNT/PAMMP nanocomposite compared to PAMMP when heated up to 1000 °C in the argon flow and in air. The nanocomposite content of carbon nanotubes CSWCNT = 10 wt % relative to the monomer weight. According to AAS data, the content of C3O4Fe = 23.5%. In TGA and DSC methods were used to study the thermal stability of the Fe3O4/SWCNT/PAMMP nanocomposite is slightly higher than that of PAMMP. In an inert medium above 320 °C, the weight loss of the samples occurs gradually. PAMMP loses half of its initial weight in an inert atmosphere at 865 °C. For the nanocomposite at this temperature, the weight loss is only 44% and at 1000 °C the residue is 51%. The processes of thermo-oxidative degradation begin at 315 °C. The 50% loss of weight is observed at 475 °C for the polymer, and at 485 °C for the nanocomposite.The thermal stability of the Feac) in the Fe3O4/SWCNT/PAMMP nanocomposite has been studied. The Fe3O4/SWCNT/PAMMP nanocomposite demonstrates a weak frequency dependence on σac conductivity in the range of 0.1–5.0 × 104 Hz. As the frequency of the alternating current grows in the range of 7.7 × 103–1.15 × 106 Hz, the conductivity increases from 8.3 × 10−6 S/cm to 2.4 × 10−5 S/cm (The frequency dependence on the ac conductivity (σ0−5 S/cm .ac) in the SWCNT/PAMMP nanocomposite, containing no magnetite nanoparticles, demonstrates a gradual increase in electrical conductivity in the whole of the investigated frequency range  and SWCNT/PAMMP (σdc = 4.4 × 10−12 S/cm). Hence, the magnetite nanoparticles make a significant contribution to the conductivity σdc of the nanocomposite. An ionic component of the conductivity also plays an important role in the low-frequency region. Thus, for the Fe3O4/SWCNT/PAMMP nanocomposite, weak conductivity changes in the low-frequency range are determined by a growth of percolation degree provided by the high content of magnetite and SWCNT presence, which increases the probability of indirect electron tunneling.At low frequencies, the main contribution to the conductivity is made by the component σncreases . The meaac of the Fe3O4/SWCNT/PAMMP nanocomposite starts to rise.As the frequency increases, the influence of the ionic component on electrical conductivity is leveled and the hopping conduction mechanism begins to play a crucial role. Therefore, in the high-frequency region, the conductivity σ3O4) nanoparticles were synthesized for the first time. The obtained thermostable ternary nanomaterials are multifunctional and exhibit certain electrical and magnetic properties. Fe3O4/SWCNT/PAMMP nanocomposites are superparamagnetic (nκ = MR/MS ~ 0). The size of the Fe3O4 nanoparticles is within the range of 2 < d < 8 nm. The saturation magnetization MS depends on the iron concentration and is 17.65–47.24 emu/g at CFe = 17.6–45.7%. The Fe3O4/SWCNT/PAMMP nanocomposite demonstrates a weak frequency dependence on the σac conductivity. As the ac frequency rises in the frequency range of 7.7 × 103 Hz–1.15 × 106 Hz, the conductivity increases from 8.3 × 10−6 to 2.4 × 10−5 S/cm. The nanocomposite loses half of its initial weight in air at 485 °C. In an inert atmosphere at 1000 °C the residue is 51%. The prepared multifunctional hybrid nanomaterials are very promising for modern technologies and can be employed for information storage systems and for creating sensors, electromagnetic shields, materials absorbing electromagnetic radiation in different wavelength ranges, and contrast agents for magnetic resonance tomography. High thermal stability of the obtained hybrid nanomaterials in the air and in an inert atmosphere provides a possibility to use them in high temperature processes.Polymer-metal-carbon hybrid nanomaterials based on thermostable electroactive poly-3-amine-7-methylamine-2-methylphenazine (PAMMP), single walled carbon nanotubes (SWCNT), and magnetite (Fe


Stencil kernels have been implemented using a naïve OpenMP   Specifications TableValue of the data•The data are output of reproducible executions of the source codes on different architectures, from CPU to GPU.•The data allow to compare the performance obtained using different approaches to parallelize stencil computations: naïve OpenMP  •Several stencils have been implemented and their execution performance registered, thus broadening the impact of these data to several communities.•The configuration data available, allow to reproduce the experiments eve n without using PROVA!  1Information like version of the compiler, compilation flags, configurations, experiment parameters and raw results are fundamental contextual information for the reproducibility of an experiment. All these information are automatically stored by PROVA! when creating a project, a method, or an experiment. Each of them holds a descriptor that stores the relevant information and makes them available to the tool and the users, when needed.For each used software PROVA! stores the building and installing recipes, in the form of easyconfig files (used by EasyBuild), the compilation and execution commands, together with their environment , self-documenting the whole research from the creation of a project until the run of an experiment.•○EASYCONFIGS○▪●OUT (raw output per thread used)●○Makefile○Sources○INPUT_PARAMETERSSRC●METHOD DESCRIPTORMETHOD (implementation)▪●PROLOGUE infos●EPILOGUE infos●RESULTS (ordered in a json)●PLOTTING SCRIPT●FIGUREEXPERIMENTAL RESULTS▪●COMPILATION SCRIPT●RUN SCRIPT●METHOD_DESCRIPTOR (containing the environment module used)METHOD_TYPECOMPUTE SYSTEMPROBLEMThus the general structure of the data in the repository is:22.1•a naïve C + OpenMP•a C source with PLUTO directives•a source in a Domain Specific Language used as input for PATUS.In this experiment has been solved a classical wave equation with a fourth order-in-space and second order in time finite difference method. After the discretization, 3 implementations have been produced:•Mint cluster at university of Basel: 4 compute nodes of dual socket AMD Opteron 6274 “Bulldozer” with a nominal clock speed of 2.2 GHz and 16 cores per chip. Each CPU has a 2 × 6 MiB L3 cache shared among a NUMA domain (8 cores), every 2 cores are sharing a 2 MiB L2 cache , and a 16 KiB core private L1 cache. The peak performance of one core is 8 flops per cycle in DP or 16 flops per cycle in SP. Each node is equipped with 128 GiB of RAM (DDR3–1600) per socket and has a maximum memory bandwidth of circa 20 GB/s per socket (measured via the STREAM COPY benchmark). The machine is running a 64-bit Ubuntu 14.04.4 LTS (kernel 3.13.0-93-generic).•CentOS release 6.7(kernel 3.10.0-327.22.2.el7.x86_64).LiMa cluster at Regionales RechenZentrum Erlangen: 500 compute nodes of dual socket Xeon 5650 “Westmere” with a nominal clock of 2.66 GHz and 6 cores + SMT per chip. Each CPU has 12 MiB L3 cache shared among all cores, and core-private L2 and L1 caches of 256 KiB and 32 KiB respectively. The peak performance of one core is 4 flops per cycle in DP or 8 flops per cycle in SP. Each node is equipped with 24 GiB of RAM (DDR3-1333) per socket and has a maximum memory bandwidth of circa 21 GB/s per socket (measured via the STREAM COPY benchmark). The machine is running a 64-bit Two different compute systems have been used:•GCC/4.9.3-2.25•GCC/4.9.3-2.25, PLUTO-pet/0.11.0 (pet branch)•ecl/16.0.0)GCC/4.9.3-2.25, PATUS/0.1.4, Java/1.7.0_79, Maxima/5.37.2 , over 100 timesteps.The experiment has been conducted using three dimensional grids of size 2002.2σ denotes the width (i.e. standard deviation) of the bell-shaped function. Gaussian filters are isotropic if the filter matrix is large enough  to provide a sufficient approximation.The second problem we chose to solve is a blur filtering. The filter matrix of the smoothing used, corresponds to a discrete two dimensional Gaussian function: 2 points and we calculate 50 timesteps in IEEE single precision arithmetic.The size of our grids is 1024The same systems and methods of Experiment 1 have been used.2.32 and we calculate 100 timesteps in IEEE single precision arithmetic.A classical heat equation, describing the temperature change over time, given initial temperature distribution and boundary conditions. A finite differencing scheme is employed to solve the heat equation numerically on a square region. The size of the grids is 512The implementation used makes use of MPI for the parallelization.The compute system used is Mint (details presented above). To complement that description, it is equipped with Mellanox Infiniband MT26428 [ConnectX VPI PCIe 2.0 5GT/s - IB QDR / 10GigE].The module used is OpenMPI/1.10.1-GCC-4.9.3-2.25.2.4•miniHPC cluster: 22 compute nodes of dual socket Xeon E5-2640 v4 “Broadwell” with a nominal clock of 2.4 GHz and 10 cores+SMT per chip. Each CPU has 25 MiB L3 cache shared among all cores, and core-private L2 and L1 caches of 256 KiB and 32 KiB respectively. The peak performance of one core is 16 flops per cycle in DP or 32 flops per cycle in SP. Each node is equipped with 32 GiB of RAM (DDR4-2133 ECC) per socket and has a maximum memory bandwidth of circa 35 GB/s per socket . Additionally, 4 KNL nodes are available, each being a 64 Core Intel Xeon Phi 7210 1.50 GHz Processor (16GB on-Die Memory), equipped with 96GB 2133 MHz DDR4 ECC memory and Omni-Path 100 GBits Host Fabric Interface, with a maximum memory bandwidth of 102 GB/s (as displayed on the Intel website). The machine is running a 64-bit CentOS release 7.2.1511(kernel 3.10.0-327.el7.x86_64).The problem solved is the same presented in the Experiment 1, using a naïve implementation, parallelized with OpenMP, using NUMA-aware initialization. The compute system where it was run is the KNL partition of theThe environment module used is GCC/ 4.9.3-2.25.2.52, 10242, 20482 and 40962), calculating 10,000 time-steps. The implementation uses CUDA to parallelize the kernel. The experiments ran on a machine hosting a•NVIDIA GeForce GTX 1060: 10 Multiprocessors, 128 CUDA Cores/MP, warp size of 32, 1.85 GHz max clock rate, 1.5 MiB L2 cache and 6 GiB of 4 GHz RAM. The device to device memory bandwidth is circa 14 GB/s, while the host to device and device to host peaks at 12GB/s .In this experiment a 2D Jacobi calculation is carried out, computing a new value for each element of the grid, such a value being an average of the element׳s current value and the values of its neighbors. Several grid sizes were used (512The environment module used is CUDA-8.0.44-GCC-5.4.0-2.26.


Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922 and Proteus hauseri ATCC 13315. The results were compared with those of silver nitrate, a silver sulfadiazine drug and appropriate ligands. The most significant antibacterial properties were exerted by silver(I) complexes containing benzimidazole derivatives. The cytotoxic activity of the complexes was examined against B16 (murine melanoma) and 10T1/2 (murine fibroblasts) cells. All of the tested silver(I) compounds were not toxic to fibroblast cells in concentration inhibited cancer cell (B16) viability by 50%, which ranged between 2.44–28.65 µM. The molecular and crystal structure of silver(I) complex of 2,6-di(hydroxymethyl)pyridine was determined by single-crystal X-ray diffraction analysis. The most important features of the crystal packing and intermolecular non-covalent interactions in the Ag(I) complex were quantified via Hirshfeld surface analysis.Selected aspects of the biological activity of a series of six nitrate silver(I) complexes with pyridine and (benz)imidazole derivatives were investigated. The present study evaluated the antibacterial activities of the complexes against three Gram-negative strains:  Due to the rapidly increasing phenomenon of drug resistance, the search for new and effective antimicrobial agents is a worldwide concern. Benzimidazole and pyridine derivatives are known to possess desirable antibacterial, antifungal, antiviral and antiproliferative properties ,3,4,5,6.Many silver compounds have been shown to have antimicrobial activity. Silver sulfadiazine (SSD or AgSD) is the most popular silver drug used in wound therapy and medicinal devices ,16,17. AAlthough silver complexes also demonstrate significant antitumor activity, the body of research in this area is still very poor compared with that devoted to other metals’ complexes. Nevertheless, a lot of silver(I) complexes have been found to exhibit greater cytotoxic activity than that of cisplatin, a drug used in cancer treatment, with relatively low toxicity to normal human cells. Ag(I) complexes show selectivity toward various types of tumor cells, and this is dependent on the type of ligand linked to the silver(I) ions. This dependency is probably connected with the stability of the complexes and the hydrophilicity-lipophilicity of the complexes formed by the type of the ligand ,25,26,27The present study describes the antibacterial activity and cytotoxic activity of a series of silver(I) complexes  13,28,2,228,29. Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922, and Proteus hauseri ATCC 13315. These microorganisms can cause urinary tract infections, diarrhea, pneumonia, and other life-threatening diseases. The bacteria from this group are also responsible for diverse nosocomial infections   .2OH)py ligand tends to be tridentate donor in transition metal  (where sac = saccharinate) structure the relatively short Ag–N distance of 2.1490(15) Å was found. In the latter structure, the silver-donor bond lengths and related angle  correspond well with those determined for 3 . In theiii/Cg2···Cg1iii distance of 3.914(1) Å and corresponding interplanar distances of 3.489(1) Å/3.597(1) Å; Cg1 and Cg2 are the centroids of the N1/C1–C5 and N2/C8–C12 rings, respectively ) encompassing the distances of any surface point to the nearest atom outside (ed) and inside (id) the surface and the van der Waals radii of the atoms ddnorm function mapped onto it: red color highlights short contacts, white color is used for weak contacts around the van der Waals separation, and blue color corresponds for a region to be free of significant contacts.A red–white–blue color scheme is characteristic for the Hirshfeld surface projection with i vs. ded, referred to as “fingerprint plots”, are useful tool in identifying and comparing different kinds of intermolecular interactions. Fingerprint plots are unique for a given crystal structure [The associated two-dimensional histograms being a combination of tructure .2OH)py)2]+ and corresponding two-dimensional fingerprint plots showing all types of contacts and these reduced to selected contact types: Ag···O/O···Ag, O···H/H···O, C···H/H···C.3 are typical without extremely short ones.As expected, the H···H contacts comprise 39% of the surface area due to the small van der Waals radius of hydrogen and packing effects in molecular structures , likewis2OH arms of the 2,6-di(CH2OH)py ligands are mostly involved in quite strong O-H···O hydrogen bonds (the shortest interactions are shown as long spikes) and weaker C-H···O interactions. Another type of weak C···H/H···C contacts give only 12.4% of the surface. The aromatic π-π stacking interactions mainly represented by C···C and C···N contacts  as well as N···H/H···N contacts (3.5%) make a small contribution to the Hirshfeld surface comparable to the all interactions with metal Ag1 atom (4.2%). Among short contacts of Ag···X  dominate the Ag···O ones (2.6%).Furthermore, the O···H/H···O contacts play a dominate role with a significant contribution of 33.7%. It was found that CH1–6, were compared with those of two commonly-used silver antimicrobial agents (AgNO3 and AgSD) andthe appropriate free ligands  and MBC  values. The activity of silver compounds  ligands . The stuactivity . CompounCC 27853 .5, which inhibited the growth of E. coli and P. aeruginosa at a concentration 2.6-fold lower than that determined for AgSD. Its MIC value against P. hauseri was almost four-fold lower than that of reference AgSD. In the case of P. aeruginosa, complex 5 demonstrated good antimicrobial activity and inhibited the growth of bacteria at a concentration almost three-fold lower than that of AgSD. The MBC values . To summarise, complexes 2 and 5 containing a benzimidazole ring in their structure demonstrated much higher activity than that containing pyridine. Moreover, mentioned compounds were more effective than AgSD and AgNO3 toward the selected tested Gram-negative pathogens.Satisfactory antibacterial properties were also displayed by complex C values  obtained6, containing metronidazole and nitrate counter-ion, exhibited satisfactory antimicrobial properties against tested Gram-negative bacteria. Antimicrobial activity of 6 was lower or comparable with those presented by AgNO3 [Our previous studies also indicated that silver(I) complex by AgNO3 .N-heterocyclic carbene complexes are widely described in the literature as effective antibacterial, antifungal, and antiviral agents [et al. synthesized dinuclear silver complexes with N-heterocyclic carbene, which possessed good antibacterial activity. The authors also demonstrated that compounds containing benzimidazole moiety exhibited significantly higher activity than complexes with imidazole [Staphylococcus aureus strains or silver-resistant Escherichia coli [Silver l agents . Haque emidazole . The 4-vmidazole . Moreovehia coli .in vitro cytotoxic activity of silver complexes 1–6 and free ligands towards murine melanoma (B16) cell lines was determined and compared with that of AgNO3, AgSD (silver sulfadiazine) and cis-DDP (cisplatin). The IC50 values (µM), i.e., the concentrations of compounds that cause 50% reduction of cellular viability, are given in Firstly, the 50 values of complexes 1–6 range from 244 to 2865 µM. Complexes 4 and 5 demonstrate the highest cytotoxicity against B16 cells, with respective IC50 values of 2.44 and 7.05 µM. The activity of complex 4 is four times greater than that of AgNO3 and eight times greater than AgSD. Complex 5 demonstrates slightly greater cytotoxicity than AgNO3 and about twice as much as AgSD. Moreover, complex 5 exhibits IC50 value in the same range as cisplatin, whose IC50 value is 7.87 µM, whereas complex 4 is three-fold higher in IC50 value compared to cisplatin. Though complexes 4 and 5 contain different heterocyclic rings, i.e., pyridine and benzimidazole, they both include the hydroxymethylene groups, the presence of which may determine the activity of the complexes.The ICHowever, the cytotoxic activity of the free ligands is very low (>200 µM), their presence is crucial for activity of the complex as a whole. It is hard to conclude how the ligands influence the cytotoxic activity of the complexes, the barrier is the lack of a defined mechanism of action of Ag(I) complexes. It has been previously proposed that the activity of the silver complexes is not only dependent on the metal ions but is connected with the presence of the metal together with the ligands ,55.1–6 and referenced silver compounds (AgNO3 and AgSD) on non-cancerous cells, we also examined their cytotoxicity on murine fibroblasts cells, 10T1/2. The percentage of survival 10T1/2 cells was measured just for concentrations, in which 50% of murine melanoma cells (B16) remain viable . The results  complexes  results  are very50 0.9 µM) [50 values ranged from 1.3 to 5.0 µM [The limited amount of research concerning the cytotoxicity of silver(I) complexes towards mentioned cell lines (B16 and 10T1/2) exists. Cytotoxicity studies of silver(I) complexes of substituted (2-aminophenyl)phosphines have shown them to be active against B16 cells in the range of comparable to cisplatin (IC 0.9 µM) . Similaro 5.0 µM .1–6 were performed according to procedures reported previously [3 , silver sulfadiazine AgSD , and cisplatin (Sigma-Aldrich Sp. z.o.o); the ligands: 2,6-di(CH2OH)py, 4-CH2OHpy, 2-CH2OHbim (Sigma-Aldrich Sp. z.o.o), MTZ (Sigma-Aldrich Sp. z.o.o) were all used as supplied. The ligands: 4-pmOpe and 2-bimOpe was synthesized according to previously described procedures [The syntheses of complexes eviously ,28,29. Tocedures ,58.1 was performed on an Agilent SuperNova diffractometer  with an Atlas detector using MoKα radiation and ω scan at 100 K. Multi-scan absorption [Uiso(H) = 1.2 Ueq(C) or 1.5 Ueq(methyl C).The X-ray measurement of sorption  correctisorption . During Ak:Bk = 0.69(3):0.31(3). Moreover, geometrical similarity constraints were applied to all N–O bonds within the disordered anion molecule, using the SADI instruction.Three oxygen atoms  in nitrate group were found to be disordered and refined with the final site-occupation factors for the two alternative positions to http://www.ccdc.cam.ac.uk/conts/retrieving.html .CCDC1405058 contains the 3 and AgSD) was performed against three Gram-negative strains: Eschericha coli ATCC 25922, Proteus hauseri ATCC 13315, and Pseudomanos aeruginosa ATCC 15442. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by a modified broth microdilution method according to the recommendation of Clinical and Laboratory Standards Institute (CLSI M07-A8). In the experiments the Mueller-Hinton broth was used, the final organism density was about 5 × 105 CFU/mL and the tests were performed 20 h at 37 °C. The studied compounds were dissolved in water  or in DMSO , and the appropriate amounts were added to samples (to biotic controls was added pure solvent). The compounds were tested in concentration ranging from 0 mg/L to 100 mg/L, except free ligands which were tested up to 500 mg/L.The antimicrobial susceptibility testing of synthesized complexes, free ligands, and references compounds (AgNO3 (B16) and 3 × 104 (10T1/2) per well/24 well plate into a medium containing DMEM GlutaMAX , 10% bovine serum  added, and Antibiotic Antimicotic Solution 1:100 (Sigma-Aldrich). The plates were stored in a humidified incubator at 37 °C, 5% CO2. After 24 h, the cells were treated with the following compounds: compounds 1–6, free ligands, AgNO3, and AgSD (except control cells—wells without tested compound). Silver complexes 1, 3, 4, 6, and AgNO3 were dissolved in water. Complexes 2, 5, and AgSD are DMSO-soluble. Next, all chemicals were diluted into water to make a concentration 1, 0.1, 0.01 mg/mL and as a working solution were added to the medium. After 72 h of incubation, the cells were washed and incubated with 0.25 mg/mL MTT (Sigma-Aldrich) solution and were incubated for another 3 h. Isopropanol was added to dissolve the formazan crystals. Absorbance at 540 and 690 nm was read using Epoch Software version 2.01.14 .The B16 (murine melanoma) cells and C3H10T1/2 (mouse fibroblast) cells  were see3) was completed herein.Antibacterial and cytotoxic activity of a series of silver(I) complexes of phosphate and hydroxymethylene derivatives of pyridine and (benz)imidazole was determined. Crystal and molecular structure of silver(I) complex with 2,6-di(hydroxymethyl)pyridine  and AgNO3. Moreover the complex 2 exhibited antibacterial activity at concentrations that were not toxic to the eukaryotic cells.The complexes containing benzimidazole derivatives, in vitro cytotoxicity has been assessed against the cancerous B16 (murine melanoma) and non-cancerous 10T1/2 (murine fibroblasts) cell lines. Two of the tested silver(I) complexes containing hydroxymethylene groups, i.e., 4 and 5, displayed greater cytotoxic activity against B16 cells (murine melanoma) than that of AgNO3, AgSD, and cisplatin. All the complexes 1–6 were found to have relatively low toxicity to non-cancerous 10T1/2 cells (murine fibroblasts). The percentage of survival of 10T1/2 cells treated with the mentioned compounds in concentrations equal to IC50 values for B16 cells, was in the range of 90%–100%. Results are favourable. Any potential anticancer agents should be non-toxic for healthy cells and toxic towards cancer. The


Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156. Enzymes are nature's biological catalysts. They perform chemical reactions with remarkable speed and specificity, making them critical for the biochemical processes that sustain life. Most enzymes have evolved to perform their function under moderate environmental conditions that support the majority of life on our planet. However, many organisms thrive in physically and chemically extreme environments such as hot springs, hydrothermal vents, hypersaline ponds, evaporation salterns, and acidic or soda lakes. These extremophiles often produce enzymes with biochemical properties that have been uniquely shaped by their extreme surroundings. As such, the study of enzymes from microbial communities that populate extreme environments is of interest to both basic science and biotechnology.en masse from microbes that populate an environmental niche. This environmental DNA is cloned into a fosmid vector and introduced into a host cell, often Escherichia coli . In the present study, we conducted a functional metagenomic screen of an environmental DNA library from a freshwater thermal spring  for enzymes capable of hydrolysis of sialic acids .Functional metagenomics has been used to identify a variety of carbohydrate hydrolases (glycosidases) from environmental libraries –4. Sever–5–9–N-glycans, O-glycans, glycosphingolipids, and glycosylphosphatidylinositol anchor side branches. Additionally, many bacteria that closely associate with animals incorporate sialic acids into their cell walls  via an α2–3 or α2–6 linkage. However, other linkages are also possible, including polysialic acid chains, in which many Neu5Ac residues are each connected via an α2–8 linkage 16–24–Sialidases are enzymes that catalyze hydrolysis of the glycosidic bond that links a sialic acid to a subterminal sugar in an oligosaccharide. They are widely distributed in biology and have been found in animals, fungi, protozoa, bacteria, and many viruses. Based on their amino acid sequences, sialidases have been classified into four CAZy . Additionally, we show that this newly found enzyme catalyzes sialic acid hydrolysis via an inverting reaction mechanism, the first example of a WT exosialidase with this ability.E. coli, and 616 individual clones were arrayed into two 384-well plates to facilitate enzyme screening. Restriction fragment analysis of 12 randomly selected library clones indicated an average cloned insert size of ∼30–40 kb (A). Additionally, Sanger sequencing of the ends of each insert revealed that cloned DNA originated from both known and unknown bacterial species. Among the known species were the thermophiles Caldilinea aerophila and Thermomicrobium roseum d-N-acetylneuraminic acid (4MU-α-Neu5Ac) substrates in agar plate and cell lysate assays, respectively. In the agar plate screen, a single colony (designated G7) hydrolyzed X-Neu5Ac and turned blue after an overnight incubation at 37 °C and several days of incubation at 4°C (B). In the cell lysate screen, the same clone, G7, was identified by measuring an increase in fluorescence caused by the hydrolysis of 4MU-α-Neu5Ac (C). No additional clones gave rise to detectable sialidase activity.To identify active sialidases, the arrayed clones were screened with 5-bromo-4-chloro-3-indolyl α-n at 4°C B. In theα-Neu5Ac C. No addde novo assembly of circular sequences), a single contig corresponding to the full insert nucleotide sequence  was obtained (GenBankTM accession number MH016668). The sequence coverage was ∼8,000×. The fosmid insert did not match any DNA sequence in GenBankTM, indicating that it was from a previously unsequenced organism.To identify the gene responsible for the observed sialidase activity, fosmid G7 was sequenced using the PacBio RS II platform. A total of 44,770 reads were generated with an average insert size of 7,523 bp and an average polymerase read length of 15,285 bp. The reads were assembled with the RS_HGAP_Assembly.3 algorithm . After rTM protein sequence database was performed using each predicted ORF protein sequence as a query. Based on the observed similarity of each ORF to homologous proteins in GenBankTM, the 40 ORFs were classified into three categories: (i) proteins of known function, (ii) proteins involved in saccharide utilization, and (iii) hypothetical proteins of unknown function (A and B). Of the 40 ORFs, four showed similarity to proteins involved in saccharide utilization, and none showed similarity to proteins from the known CAZy sialidase families GH33, GH34, GH58, and GH83.The fosmid G7 DNA sequence encoded 40 putative ORFs that were predicted by MetaGeneMark . BLASTP function , A and BFig. S1). Seventeen mutants with abolished sialidase activity were identified. Each of these mutants was bidirectionally Sanger-sequenced with transposon-specific primers to identify the element's insertion site. Multiple transposon insertion events were observed for seven mutants, and they were not further investigated. A map of the G7 insertion sites for the remaining ten mutants is shown in Fig. 3A. All loss-of-function insertions clustered to a ∼5-kb region of fosmid G7 containing several ORFs with similarity to enzymes that hydrolyze or metabolize sugars (A and 3A). The majority of insertions occurred in ORF9 (annotated as encoding a putative α-l-fucosidase) or ORF12 (annotated as encoding a “protein of unknown function”).Because sequence analysis did not identify an obvious sialidase candidate among the 40 sequenced ORFs, transposon mutagenesis was performed to disrupt expression of the sialidase activity. Tn5 mutagenesis was used to randomly insert a kanamycin cassette into fosmid G7 under conditions that minimized multiple insertion events. A total of 192 mutants were assayed for sialidase activity using the substrate 4MU-α-Neu5Ac (e sugars A and 3A.in vitro protein synthesis system (B) and tested for its ability to cleave 4MU-α-Neu5Ac (C). The expressed product from ORF12 (ORF12p) generated a fluorescence signal ∼27 times higher than the no DNA control reaction . Expression of ORF9 yielded no sialidase activity. Because the deduced protein sequence of ORF9 (ORF9p) had sequence homology to α-l-fucosidases, the ability of in vitro expressed ORF9p to cleave the chromogenic fucosidase substrate 2-chloro-4-nitrophenyl α-l-fucopyranoside was also tested. Light absorbance at 405 nm was approximately six times higher for ORF9p than the negative control, confirming that the ORF9 encoded an α-fucosidase, not an exosialidase. Together, these data support the conclusion that the gene product from ORF12  is solely responsible for the hydrolysis of 4MU-α-Neu5Ac observed during primary screening. Furthermore, because ORFs 8–12 are co-directionally oriented and are each related in some way to saccharide metabolism, it is plausible that these genes are co-transcribed in the organism from which the locus originates and in E. coli. This could account for the observation of reduced sialidase activity upon insertion of Tn5 into multiple positions in this region.To determine whether either ORF9 or ORF12 encoded a protein with sialidase activity, each was expressed using the PURExpress s system B and tesα-Neu5Ac C. The exE. coli and purified using nickel-affinity chromatography (A).To facilitate biochemical characterization of ORF12p, an expression DNA construct encoding ORF12p with a C-terminal hexahistidine tag (ORF12p–His) was assembled. ORF12p–His was expressed in tography A.B). The enzyme showed optimal activity at 37 °C and retained at least 50% activity at 60 °C (C). No metal ions were required for activity (D). Reaction kinetics were determined using the substrate 4MU-α-Neu5Ac. The initial reaction rates were determined at several substrate concentrations (E). The mK was calculated to be 62 μm, and the kcat was calculated to be 2.3 s−1.The substrate 3′-SLN-2AB was used to determine the optimum pH and temperature of the ORF12 sialidase as described under “Experimental procedures.” A pH optimum of 5.0–5.5 was observed B. The enat 60 °C C. No metactivity D. Reactitrations E. The KmN-acetyllactosamine substrates , a GD3 ganglioside headgroup glycan (Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc-2AB) and G2S2 biantennary N-glycans with two terminal Neu5Gc or Neu5Ac residues (G2S2-Ac or G2S2-Gc). Digestion products were analyzed by ultra-HPLC with fluorescence detection (UPLC–HILIC–FLR) . A positive control reaction using commercial α2–3,6,8,9-neuraminidase A  resulted in a shift of each substrate, illustrating complete removal of α2–3–, α2–6–, and α2–8–terminal sialic acid . This same peak shift was observed for 3′-SNL-2AB and 6′-SNL-25AB substrates when incubated with purified ORF12p–His, indicating that this enzyme was able to hydrolyze both terminal α2–3– and α2–6–Neu5Ac  and confirming the ability of ORF12p–His to function as an exosialidase. The peak corresponding to the released glycan from GD3 ganglioside did not shift upon incubation with ORF12p–His, confirming the enzyme's inability to hydrolyze α2–8–linked sialic acid . N-Glycan substrates G2S2-Ac and G2S2-Gc were both hydrolyzed by ORF12p–His, indicating that this enzyme can hydrolyze both terminal Neu5Ac and Neu5Gc from complex N-glycan structures . However, the enzyme showed a preference for Neu5Ac. In addition, the enzyme was assayed on a 2AB-labeled O-glycan library  and showed the ability to hydrolyze terminal sialic acid linked to GalNAc or galactose residues.The specificity of purified ORF12p–His was assayed using 2-aminobenzamide–labeled 3′- and 6′-sialyl-LIC–FLR) . NegativGlycoside hydrolases are grouped into two mechanistic classes: inverting or retaining enzymes, depending on whether the stereochemistry of the substrate's anomeric carbon is inverted or retained upon hydrolysis. To date, known exosialidases function as retaining glycoside hydrolases, because they hydrolyze terminal Neu5Ac with net retention of its overall anomeric configuration –27.–1H NMR) spectroscopy was used to determine whether ORF12p functions via a retaining or inverting mechanism. In this experiment, purified ORF12p–His was incubated with the 4MU-α-Neu5Ac substrate. Data analysis focused on the up-field region of the NMR spectra, where signals from the axial and equatorial protons in the 3-position of Neu5Ac appear. The initial NMR spectrum (A) showed only the axial (α-MU-H3a) and equatorial (α-MU-H3e) protons from the substrate 4MU-α-Neu5Ac at 1.99 and 2.86 ppm, respectively. The first additional set of signals (H3a and H3e) appeared after only a few minutes at 1.81 and 2.20 ppm. These signals corresponded to β-Neu5Ac that had been released from the substrate by the enzyme. The signal set of H3a and H3e corresponding to α-Neu5Ac (at 1.61 and 2.72 ppm) appeared after ∼1 h and remained very small throughout the remaining reaction. This reflects spontaneous mutarotation of the β-Neu5Ac product to its α conformer and is not a result of the action of the sialidase. These data unambiguously show that the ORF12 sialidase initially produces β-NeuAc from 4MU-α-Neu5Ac and supports the conclusion that the enzyme functions via an inverting mechanism.Proton NMR (spectrum A showed B) showed the axial (H3a) and equatorial (H3e) protons corresponding to 4MU-α-Neu5Ac at 2.03 and 2.89 ppm, respectively. Upon initiation of the reaction, the first additional signals appeared after ∼30 min and corresponded to the α-anomer (1.64 and 2.76 ppm). These peaks were initially larger than the H3a and H3e β-Neu5Ac peaks . However, after 130 min, the peaks of the β-anomer became more prominent than those of the α-anomer, consistent with the spontaneous mutarotation of the α-Neu5Ac product to its β-Neu5Ac anomer. This was previously reported to reach an equilibrium mixture of 92.1% of β-anomer and 7.5% of α-anomer and three open-chain species at pH 8.0 (A control experiment was run with purified NeuA (New England Biolabs), a known retaining exosialidase from the GH33 family B showed t pH 8.0 . This stTM sequence repository using the BLASTP algorithm. The 38 nearest homologs of ORF12 were retrieved and used for study of ORF12p phylogeny. Signal peptides that were present in many sequences were trimmed along with C-terminal extensions present for two homologs (OGV64360 Lentisphaerae and KXK35835 Omnitrophica). Trimmed sequences ranging from 477 to 578 amino acids were then aligned with Muscle, and a phylogenetic tree was made using BLOSSUM62 substitution matrix and the neighbor-joining method (A). It is noteworthy that all 38 ORF12p homologs showed no similarity to known CAZy glycoside hydrolases or other proteins of known function, and all were annotated as hypothetical proteins.ORF12p homologs were identified in the GenBankg method A. It is All sequences comprising the ORF12p family originated from bacteria and were distributed within eight prokaryotic phyla. The majority of sequences originated from organisms in the phylum Bacteroidetes or the superphylum Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) that contains the phyla Planctomycetes, Verrumicrobia, Omnitrophica, and Lentisphaerae. Other sequences were from organisms from the phyla Firmicutes, Armatimonadetes, and Acidobacteria. Based on our phylogenetic analysis, ORF12p was most closely related to sequences from organisms of the PVC superphylum. Notably, no homolog was found within the phylum Proteobacteria, which contains most of the well-studied bacteria, or in the phyla Actinobacteria or Cyanobacteria, which contain many species widely represented in soil and aquatic environments. Similarly, no eukaryotic or archaeal proteins had similarity to ORF12p.Armatimonadetes was expressed in E. coli, and crude lysate was assayed with 4MU-α-Neu5Ac. Lysate from E. coli expressing ORF12p was also assayed. A high fluorescence signal was detected for both ORF12p and OIO94155 lysates, indicating that OIO94155 is also an exosialidase (B). A multiple sequence alignment of 10 proteins (including ORF12p and OIO94155) showed the conserved regions within this family . Strictly conserved aspartate, glutamate, and histidine residues are indicated . These types of residues are commonly involved in catalysis in most inverting glycoside hydrolases .The cloned fosmid DNA sequence encoding ORF12p was unique and showed no significant match to DNA sequences from known organisms, thus providing no clue to its precise origin. The gene encoding ORF12p resided in a gene cluster that also encoded a GH29 family α-fucosidase and a GH20 family minidase . Althougminidase , 41, theE. coli. The DNA fragment originated from an unknown microorganism and encoded no ORFs with homology to known sialidase families. Tn5 mutagenesis was used to identify a 505-amino acid ORF responsible for the observed sialidase activity (ORF12). We undertook the phylogenetic analysis of the ORF12 protein (ORF12p) family and biochemical characterization of this new sialidase.In the present study, we utilized functional metagenomic screening to search for environmental enzymes with the ability to hydrolyze the fluorogenic sialidase substrate 4MU-α-Neu5Ac. Our screen identified a 41-kb environmental DNA fragment from a hot spring metagenomic library that produced sialidase activity in i.e. Proteobacteria), archaea, or eukaryotes. Thus, this new sialidase family appears to be highly niche-specific in bacterial biology. The ORF12 protein sequence family defines a novel CAZy glycoside hydrolase family that has been designated GH156.Glycoside hydrolases and other enzymes that assemble or modify oligo- and polysaccharides are classified into families based on their amino acid sequence similarity . These emK = 62 μm; kcat = 2.3 s−1) were similar to reported values for other enzymes from the GH33 family of bacterial exosialidases - and α-Neu5Ac from the terminal position of oligosaccharides, indicating that it functions as an exosialidase. Despite lacking protein sequence similarity with known exosialidases, ORF12p shares similar basic biochemical properties with them –44. For –alidases  45–47).–47.Km = 1H NMR data unambiguously showed that ORF12p liberated β-Neu5Ac as the primary product of hydrolysis of the substrate (4MU-α-Neu5Ac)  , indicat-Neu5Ac) –27. Howe were from Prozyme . The 2AB-labeled di-Neu5Gc-terminated biantennary complex N-glycan (termed G2S2-Gc) was from Tokyo Chemical Industry . All DNA enzymes and enzymes for SMRTbell complex library preparation were from New England Biolabs. MilliQTM purified water (Millipore Sigma) was used in all experiments.Chemicals and solvents were from Sigma–Aldrich. The substrate X-Neu5Ac was from Sigma–Aldrich. The substrate 4MU-α-Neu5Ac was from Toronto Research Chemicals . The 2AB fluorescently labeled substrates 3′ and 6′-sialyl-d-galactopyranoside , 0.4 mm isopropyl β-d-1-thiogalactopyranoside (IPTG), and 5% (w/v) sucrose) and incubated overnight at 37 °C. A total of 616 colonies were harvested and archived in two 384-well plates in 20% sterile glycerol (v/v).Samples from a hot spring located in Dixie Valley, Nevada, were collected in 2013. Environmental DNA was subsequently extracted and frozen until use. Isolated DNA was concentrated to 548 ng/μl using a 0.45× volume of AMPure PB magnetic beads  and used to produce a DNA library using the CopyRight v2.0 Fosmid cloning kit  following the manufacturer's instructions. Briefly, DNA was end-repaired and size-selected using a 1% low melting point agarose gel run overnight at 35 V. DNA fragments from 30–70 kb were extracted from the gel using 1 unit of β-agarase I (New England Biolabs) for each 100 μl of melted agarose. The end-repaired and size-selected DNA was ligated to the pSMART FOS cloning vector. Phage packaging of the resulting clones was performed using Gigapack III XL packaging extracts . Replicator FOS cells were transfected with the packaging reaction and plated on YT-CXIS agar medium . Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector. Digestion products were separated by electrophoresis on a 1% agarose gel to estimate the average insert size of cloned DNA.Restriction digestion was used to assess the average insert size in the fosmid DNA library. Fosmids were individually isolated from 12 randomly selected library clones using the FosmidMAXTable S1). These primers allowed sequencing of ∼500–800 bp from the insert ends. BLASTN was used to probe the GenBankTM sequence repository for known identical sequences to assist in identification of the species of origin for the cloned DNA fragments.To assess the cloned DNA insert species diversity, the 12 randomly selected clones were subjected to Sanger sequencing using the T7 universal primer or pCC1 forward and reverse sequencing primers   supplemented with 60 μg/ml X-Neu5Ac dissolved in dimethylformamide using a 384 Slot Pin Multi-BlotTM replicator . After overnight incubation at 37 °C, the plates were stored at 4 °C until blue color developed . For sialidase activity screening in E. coli cell lysates, the library clones were grown in 50-μl LB liquid cultures ) in 384-well plates and incubated overnight at 37 °C. 50 μl of lysis buffer (Thermo Fisher Scientific) containing 40 μg/ml of the sialidase substrate 4MU-α-Neu5Ac were added to the liquid cultures. The mixtures were incubated overnight at 37 °C in a static incubator. Fluorescence at λex = 365 nm and λem = 445 nm was read with a SpectraMax Plus 384 microplate reader .The metagenomic library was screened for sialidase activity using two different approaches. In an agar plate assay, library clones were spotted onto agar plates  following the manufacturer's instructions. DNA was isolated from 40-ml LB cultures supplemented with 12.5 μg/ml chloramphenicol and 1× inducing solution (Lucigen Corporation). A 10-kb SMRTbell library was prepared as follows. 10 μg of DNA in 150 μl of TE buffer  were sheared using g-tubes . The g-tubes were centrifuged at 4500 rpm (Eppendorf 5424 centrifuge) for 30 s with the cap up, followed by 30 s with the cap down, three times each for a total of six spins. Sheared DNA was concentrated using 0.45× volume of AMPure PB magnetic beads (Pacific Biosciences). The beads were incubated for 10 min in a ThermoMixer C  at 1,400 rpm and then washed twice on a magnetic rack with 500 μl of freshly prepared 70% ethanol. DNA was eluted from the beads by addition of 20 μl of TE buffer and incubation for 2 min in a ThermoMixer C at 1400 rpm 2 μl of PreCR Repair Mix (New England Biolabs) and 5 μl of NEBNext End Repair Reaction Buffer (New England Biolabs) in a 50 μl of total volume reaction was added to repair the damaged DNA template. The reaction was incubated at 37 °C for 20 min. DNA was end-repaired by addition of 2.5 μl of NEBNext End repair mix (New England Biolabs) (incubation at 25 °C for 5 min) and then purified with 0.45× volume of AMPure PB magnetic beads and eluted with 20 μl of TE. DNA was blunt-ligated with 2.5 μl of 80 μm SMRTbell adaptors  using 17.5 μl of blunt/TA ligase master mix (New England Biolabs). The ligation reaction was incubated at 25 °C for 15 min. Ligase was removed using the Monarch PCR and DNA cleanup kit (New England Biolabs). Unligated DNA fragments were removed by digestion with exonuclease III and VII (100 units each) in the presence of 4 μl of buffer for T4 DNA ligase with 10 mm ATP. Finally, two AMPure PB magnetic bead purifications were performed. The SMRTbell library was ultimately eluted with 15 μl of TE buffer. MagBead complexes were prepared on the day of sequencing using the binding calculator tool and enzymes from Pacific Biosciences. The final library was sequenced on a PacBio RS II platform using the P6 chemistry and one single-molecule real-time cell for a 360-min movie.Fosmid DNA was extracted using the FosmidMAXde novo using the RS_HGAP_Assembly.3 protocol . ORFs encoded by the assembled contig were predicted using MetaGeneMark  following the manufacturer's instructions. Briefly, an equimolar amount (0.007 pmol) of the EZ-Tn5TM <KAN-2> transposon and target DNA (G7 fosmid) were used to maximize the insertion efficiency while minimizing multiple insertion events. The 10-μl reaction was incubated at 37 °C for 2 h and stopped upon addition of 1 μl of EZ-Tn5 10× stop solution and incubation at 70 °C for 10 min. 1 μl of the reaction was added to 50 μl of thawed electrocompetent cells EC300110 (Lucigen Corporation) in a prechilled tube. Electroporation was performed with a Gene Pulser XcellTM electroporation system (Bio-Rad) following the manufacturer's instructions . SOC medium (New England Biolabs) was added immediately after electroporation (950 μl), and the cells were transferred to a 15-ml tube for incubation at 37 °C with shaking for 1 h. Transformed cells were plated on LB + 12.5 μg/ml chloramphenicol and 50 μg/ml kanamycin. 192 random mutants were arrayed in two 96-well plates. Sialidase activity in both plates was investigated using the lysis-based assay with 4MU-α-Neu5Ac substrate as described above.A library of random mutants of fosmid G7 was generated using the EZ-Tn5in vitro using the PURExpress in vitro protein synthesis kit (New England Biolabs) following the manufacturer's instruction. PURExpress DNA templates were generated by PCR using primers specific to ORF9 or ORF12 (Table S1). PCR was performed using 250 ng of fosmid G7 as template, 0.75 μl of each primer (20 μm), 15 μl of Q5 hot start high-fidelity 2× Master Mix (New England Biolabs) in a 30-μl total reaction volume. Thermocycling consisted of 25 cycles . The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific). Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.Sialidase candidates ORF9 and ORF12 were each expressed Table S1). Linearization of the vector was performed by PCR with Q5 hot start high-fidelity 2× Master Mix (New England Biolabs) for 25 cycles . An insert was also prepared by PCR using 25 cycles of 98 °C for 10 s, 60.5 °C for 30 s, and 72 °C for 2 min with Q5 hot start high-fidelity 2× Master Mix. The construct was assembled using the NEBuilder HiFi DNA assembly cloning kit (New England Biolabs) and transfected into New England Biolabs 5-α competent E. coli cells following the manufacturer's instructions. The clone was verified by Sanger sequencing and used to transform New England Biolabs Express competent cells. Expression under the Ptac promoter was performed at 18 °C overnight upon addition of IPTG to 0.4 mm. Cells from 1 liter of LB culture were harvested by centrifugation at 15,000 × g for 10 min at 4 °C. The cell paste was resuspended in 30 ml of 20 mm sodium phosphate, pH 7.4, 500 mm NaCl, and 20 mm imidazole buffer and lysed using a TS Benchtop Series cell disruptor  at 32 kilo pounds per square inch (kPsi). Expressed protein was purified on a 5-ml His-TrapTM FF column . The bound ORF12–His6 protein was eluted with 20 mm sodium phosphate, pH 7.4, 500 mm NaCl, and 500 mm imidazole by gradient elution over 20 column volumes (from 0 to 100% of elution buffer). Fractions containing pure protein were pooled and dialyzed against 20 mm sodium phosphate, pH 7.4, containing 500 mm NaCl, 1 mm EDTA.ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end  and cloned into the pJS119K vector . Primersm sodium phosphate, pH 7.4. The effect of pH and temperature on enzyme activity was investigated by incubating 0.5 unit of enzyme with 1.7 pmol of 3′-sialyl-N-acetyllactosamine-2AB for 3 h at 37 °C. These conditions led to incomplete cleavage so that both substrate and product could be monitored. A range of pH from 4.5 to 9.5  at 37 °C and temperature ranges from 15 to 70 °C in 50 mm sodium acetate, pH 5.0, were tested. The effect of metal ions was tested by incubating 0.5 unit of enzyme with 1.7 pmol of 3′-sialyl-N-acetyllactosamine-2AB in 50 mm sodium acetate, pH 5.0, with 5 mm of NiSO4, CaCl2, MnSO4, MgCl2, FeSO4, ZnCl2, or CuSO4, respectively, for 3 h at 37 °C. After incubation, the reactions were dried by vacuum evaporation and resuspended in 1.8 μl of water and 13.2 μl of acetonitrile for a 12:88 ratio. 12 μl were injected into a Waters Acquity BEH glycan amide column  on a Waters Acquity UPLC H-Class instrument  equipped with a quaternary solvent manager and a fluorescence detector. 50 mm ammonium formate buffer, pH 4.4, and 100% acetonitrile were used, respectively, as solvent A and B. The gradient used was 0–1.5 min, 12% solvent A; 1.5–35 min, 47% solvent A; 35–36 min, 70% solvent A; and 36.5–42 min, 12% solvent A with a flow rate of 0.561 ml/min. The samples were kept at 5 °C prior to injection, and separation was performed at 30 °C. The fluorescence detection wavelengths were λex = 330 nm and λem = 420 nm with a data collection rate of 20 Hz. The quantity of uncleaved substrate and released product were analyzed with Empower 3 chromatography work station software (Waters Corporation). Peak areas were quantitated by integration, and relative activity was calculated.One unit of the ORF12 sialidase was defined as the amount of enzyme required to cleave Neu5Ac from 1 nmol of 4MU-α-Neu5Ac in 1 h at 37 °C in 20 mN-acetyllactosamine substrates , G2S2-Ac, G2S2-Gc, and the GD3 ganglioside-released glycan (Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc-2AB). The released glycan headgroup substrate from GD3 ganglioside was prepared as follows: glycans from 10 nmol of GD3 ganglioside dissolved in methanol were released with 20 milliunits of endoglycoceramidase I (New England Biolabs) in a 10-μl reaction and incubated for 24h at 37 °C. The reaction mix was passed through a Nanosep 10K Omega centrifugal device  and centrifuged at 12,000 rpm for 4 min to remove the enzyme. The filtrate was dried in a vacuum evaporator prior to 2AB labeling for 2 h at 65 °C with 10 μl of 2AB labeling mix . Labeled glycans were cleaned-up with a HILIC detergent removal Microspin cartridge .Sialidase specificity was assessed by testing its efficacy on 2AB-labeled 3′ and 6′-sialyl-m sodium acetate, pH 5.0, and incubated at 37 °C overnight. Negative and positive control experiments were performed in the same conditions with no enzyme and 1 μl of NeuA (New England Biolabs), respectively. Reactions were dried in a vacuum evaporator and analyzed by UPLC–HILIC–FLR as described above. ORF12p–His activity was also tested on a fetuin O-glycan library purchased from Ludger  and 2AB-labeled as described above.Enzyme (1 or 10 units) was mixed with each of the substrates in 20-μl reactions in 50 mm) in 50 mm sodium acetate, pH 5.0. Each substrate concentration was tested in triplicate. Aliquots (50 μl) of each reaction were removed at different time points over a 5-min incubation. They were immediately mixed with 50 μl of 1 m sodium carbonate (pH 10.9) to stop the reaction. Fluorescence was read at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.To determine the kinetic parameters of ORF12p–His, 20 units of ORF12p–His was incubated at 37 °C with different concentrations of 4MU-α-Neu5Ac , 60 μl of 200 mm sodium phosphate in D2O, pH 7.4 , and 190 μl of D2O. The solution was mixed, and the NMR tube placed into a 600-MHz Inova NMR spectrometer  at 25 °C. A one-dimensional proton NMR experiment with water presaturation (using the two-step purge option) was recorded as the t = 0 measurement. Then 30 units of ORF12p–His sialidase or 240 units of NeuA (New England Biolabs) was added, the NMR tube was inverted several times to ensure good mixing, and the tube was replaced into the NMR spectrometer. One-dimensional proton spectra with water presaturation were recorded at time intervals with 32 transients each. Chemical shifts were referenced relative to the residual HDO peak, set at 4.78 ppm.NMR was performed by the Complex Carbohydrate Research Centre . A solution was prepared in a 5-mm NMR tube with 320 μl of 1.78 mg/ml 4MU-α-NeuAc in DArmatimonadetes was synthesized by GenScript  in pUC57. The gene was subcloned into pJS119K using the HiFi DNA assembly cloning kit (New England Biolabs) with primers designed with NEBuilder assembly online tool (Table S1). The clone was verified by Sanger sequencing and used to transform New England Biolabs Express competent cells. Expression under the Ptac promoter was performed at 30 °C for 4 h upon addition of IPTG to 0.4 mm. Crude lysate was prepared by sonication and assayed with 4MU-α-Neu5Ac by mixing 5 μl of crude lysate with 4 μl of 100 μg/ml 4MU-α-Neu5Ac and 15 μl of 20 mm MES buffer, pH 6.5. After 1 h of incubation at 37 °C, the fluorescence of the reaction was read at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer. Lysate from New England Biolabs Express cells containing the empty pJS119K vector and lysate from the pJS119K-ORF12p construct were prepared and assayed in the same conditions to serve as negative and positive controls, respectively.The gene encoding the hypothetical protein OIO94155 from L. C., M. B. G., and B. H. data curation; L. C., M. B. G., and B. H. formal analysis; L. C. and M. B. G. methodology; L. C. and C. H. T. writing-original draft; L. C., M. B. G., E.R., B. H., and C. H. T. writing-review and editing; M. B. G. validation; E. R. and C. H. T. supervision; C. H. T. conceptualization; C. H. T. funding acquisition; C. H. T. investigation; C. H. T. project administration.


Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of β-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat.Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene  •Rex1-EGFP transgene is a neutral reporter of pluripotency and self-renewal in the rat•Rex1-EGFP transgene responds appropriately to self-renewal and differentiation signaling•Rex1-EGFP transgene allows the discrimination between rat ESC pluripotent states Rex1-EGFP stem cell reporter as a sensitive tool to monitor and interrogate the regulation of rat ESCs, and contribute to understanding stem cells and fate determination in mammals.Rat embryonic stem cells (ESCs) differentiate into all adult cells, including functional gametes, and uniquely qualify as a comparator with mouse ESCs to study self-renewal and pluripotency. In this article, Burdon and colleagues generated a  Embryonic stem cells (ESCs) are pluripotent, immortal, embryo-derived cell lines that can differentiate into all cells of a developing embryo, including the germ cells, and can be used to introduce genetic modifications through the germline . Althougde novo from blastocysts using this 2i+LIF culture medium, but rESC lines are typically less stable than their mouse counterparts under conditions of clonal expansion and continuous culture (Standard mouse ESC culture conditions containing serum and leukemia inhibitory factor (LIF) do not support rESC culture . However culture . This in culture . Understin vitro in 2i+LIF cultures that most closely represents pluripotent stem cells in the preimplantation blastocyst embryo  promoter, replacing the entire coding region of the Rex1 gene  and blastocyst (day E4.5) stage embryos , Rex1-EGFP heterozygous (60%), and Rex1-EGFP homozygous (20%) pups  but could not detect any obvious difference in average litter numbers at E4.5 or mid-gestation between homozygous and wild-type rats, suggesting that the fertility of Rex1 mutant male rats at the ages we tested was normal  pups . Moreove-fertile . We thers normal .Rex1 affected the derivation and maintenance of rESCs in vitro. The culture of E4.5 embryos generated by cross-breeding heterozygous Rex1-EGFP rats showed that the derivation efficiency of Rex1-EGFP homozygous ESCs under 2i+LIF conditions (83%) was close to that obtained for wild-type (100%) and heterozygous (91%) lines  lines . Furtherpe cells C. Notabl of REX1 , thus co of REX1 D. Import of REX1 E. In sumRex1-EGFP reporter, we monitored the correspondence between expression of Rex1 mRNA and Rex1-EGFP mRNA under culture conditions that promote ESC differentiation. Withdrawal of LIF and 2i inhibitors from rESCs in suspension cultures to promote embryoid body differentiation reduced the level of EGFP expression as assessed by fluorescence microscopy  >3 μM efficiently suppressed Rex1-EGFP-dependent fluorescence  activity in association with the transcription factor LEF1 drives rat ESC differentiation, causing a loss of ESC colony morphology, induction of mesendoderm-associated differentiation markers Brachyury and Cdx2, and increased instability and collapse of rESC cultures . We therrescence A and 4B rescence C. Depletrescence A and S3B-renewal C and 4D.ectively C. TransfRex1-reporter could also be reactivated upon dedifferentiation into a pluripotent stem cell state through induced pluripotent stem cell (iPSC) reprogramming, we transfected rat embryonic fibroblasts (REFs) derived from a Rex1-EGFP heterozygous embryo with a piggyBac vector co-expressing human cDNAs encoding OCT4, SOX2, KLF4, and cMYC , which typically represented the majority (>90%) of cells in the culture; cells expressing an intermediate or low level of EGFP (Rex1-EGFPlow), which constituted a variable and less distinct population (1–10%); and cells that did not express EGFP (Rex1-EGFPneg) and contributed to 1–3% of the overall population , replating the cells in 2i+LIF ESC self-renewal conditions, and monitoring the growth of the resulting cultures for 4 days . A significant proportion of the EGFPhigh cells (55%) also produced colonies that expressed uniformly low levels of EGFP. Rex1-EGFPlow sorted cells gave rise to fully differentiated Rex1-EGFPneg colonies (31%), but also produced morphologically undifferentiated colonies that maintained a low level of EGFP expression (65%), indicating that the Rex1-EGFPlow state could be maintained through multiple cell divisions. By comparison, Rex1-EGFPneg cells exhibited reduced cloning efficiency, gave rise to some Rex1-EGFPlow colonies (22%) and produced the greatest proportion of morphologically differentiated Rex1-EGFP-negative colonies (67%). The clonal analysis demonstrated that expression of the Rex1-EGFP reporter could discriminate between different rat ESC states. It also identified a loss of pluripotent potential among the Rex1-EGFPlow population and indicated that this state could persist for a period of time under standard 2i+LIF culture conditions. To assess how this loss of potential affected developmental potency in vivo, we purified Rex1-EGFPlow cells by FACS, allowed them to recover in culture for 4 days, and tested their contribution to embryonic development by blastocyst injection. The cells contributed to live-born chimaeras with an efficiency close to the parental ESCs (6 chimaeras out of 26 pups: 23%), demonstrating that the Rex1-EGFPlow cells either fully retained the capacity to functionally reintegrate into the developing embryo or were in a state where this potential was readily reinstated within the blastocyst environment.We next examined the stem cell self-renewal potential of cells within the r 4 days D. Most Red cells D. To stred cells E and 5F.Rex1-EGFPlow intermediate cell type, we assessed the transcriptional activity of cells within the three populations of Rex1-EGFP cells in 2i+LIF culture by single-cell RNA sequencing. We sorted cells into replicate 96-well plates according to their levels of EGFP expression and followed the Smart-seq2 protocol, with minor modifications as we have described previously  and Rex1-EGFPneg (Rex1mRNA−/EGFP−) cells, both Rex1 alleles were active or inactive, respectively (Rex1-EGFPlow population expressed high levels of Rex1 mRNA (Rex1mRNA+/EGFP−). To assess the differentiation potential of Rex1-EGFPlow cells in vitro, we purified the cells by FACS, expanded the population for 4 days after plating, and then analyzed the cells by flow cytometry and single-cell sequencing. In line with previous results, a significant proportion of the cells retained a Rex1-EGFPlow state  gene expression is a benchmark marker of pluripotency in cultures of ESCs and reprogrammed iPSCs . This pointed to selective loss of transcriptional activity from the targeted Rex1-EGFP allele and might arise from a loss of positive regulatory elements from the targeted Rex1-EGFP allele or interference induced by insertion of the targeting cassette, thus sensitizing expression of the EGFP knockin allele to a weakening of control of pluripotency. An alternative possibility, however, is that asynchronous expression of Rex1 alleles might occur normally in this peri-naive ESC transition state. Indeed, the detection of Rex1-EGFPhigh cells expressing low levels of endogenous Rex1 mRNA (Rex1mRNA−/EGFP+), thus mirroring the Rex1-EGFPlow (Rex1mRNA+/EGFP−) population, supports the notion that the EGFP transgene might simply reflect asynchrony in the response of Rex1 alleles to relaxation of the control of pluripotency in the earliest stages of differentiation. Indeed, the pluripotency factor Nanog has been reported to exhibit mono-allelic expression prior to attaining full biallelic expression in the ICM of the blastocyst  also tended to express lower levels of naive pluripotency regulators (Rex1-EGFPhigh (Rex1mRNA+/EGFP+) cells. Pathway analysis of genes differentially regulated between these two populations also pointed to the involvement of chromosome reorganization during this transition. Interestingly, we did not observe significant levels of reversion of the Rex1-EGFPlow cells to the Rex1-EGFPhigh state in single-cell clonal assays, in line with the results reported for mouse Rex1-EGFPlow ESCs cultured in 2i+LIF conditions  and GSK3 (CH) inhibitors were supplied by Axon Medchem (http://www.axonmedchem.com).ESCs were derived from E4.5 rat blastocysts following removal of the zona pellucida with acidic Tyrode solution. The intact blastocyst was then cultured in the well of a 96-well plate, in 2iL  on γ-irradiated (5 Gy) OF1 mouse fibroblasts for 7 days prior to passaging. ESCs were maintained on irradiated OF1 or DR4 mouse fibroblasts in 2iL. Colonies were passaged every 2–3 days using TVP  and plated at a density of (0.5–1) × 10Rex1 coding sequence was replaced with a Kozak-EGFP-loxP-IRESneo-loxP-bGHpA reporter/selection cassette. Approximately 1 × 107 DAK31 rat ESCs . Electroporated cells were plated into 10 cm2 wells containing 2iL medium. The aminoglycoside G418 (80 μg/mL) was added 48 h after electroporation and G418-resistant colonies picked 9 days later onto DR4 fibroblasts in 2iL in wells of a 96-well plate and expanded.A targeting vector was constructed using BAC recombineering which consisted of approximately 3 kb and 2.7 kb, 5′ and 3′ homology arms respectively, in which the entire rat ESCs  in 0.6 m5/cm2 in 2iL. The cells were cultured for 2 days then transferred into fibroblast medium  for a further 2 days. Rat ESCs differentiated by culturing at a density of 5 × 103/cm2 in either 2iL with concentrations of PD lower than 1 μM for 3–4 days or in 2i (no LIF) with concentrations of CH >3 μM for 3–4 days.For embryoid body differentiation, a single-cell suspension of rat ESCs was plated into non-coated, low-adherence plastic wells of a 6-well plate at a density of 2 × 105Rex1-EGFP REFs were resuspended in Resuspension Buffer R (Invitrogen) and transfected with 1.5 μg of pPBTRE-hOSMK + 0.5 μg of PBase + 0.5μg of PB-rTTA (3/cm2 on γ-irradiated (100 Gy) STO mouse fibroblasts in M15G+SB medium  and kept at 37°C in 5% CO2. The medium was changed the day after transfection and every 2 days thereafter. Colonies emerged by day 10 and M15G+SB media changed to t2i+Lif . The colonies were fixed for immunocytochemistry at day 14.Approximately 5 × 10 PB-rTTA , using tCells were fixed in cold 100% methanol (5 min at −20°C), washed with PBS, then blocked in PBS + 10% FCS for 1 h at room temperature. GATA4 primary antibody  diluted in PBS/2%FCS was applied at room temperature for 2 h, followed by washes with PBS. Secondary antibody  was diluted in PBS/2%FCS and applied for 1 h at room temperature in the dark. The cells were washed with PBS, then 10 μg/mL DAPI applied for 5 min at room temperature in the dark followed by washes with PBS. NANOG protein was detected in cells fixed with 4% paraformaldehyde/PBS (15 min at room temperature) and washed with PBS. The cells were permeabilized using ice-cold methanol (−20°C for 10 min), washed with PBS, and incubated with blocking solution (PBS/0.3% Triton X-100/10% goat serum) for 1 h at room temperature. NANOG primary antibody  was diluted in blocking solution and applied overnight at 4°C followed by washes with PBST (PBS + 0.3% TritonX100). Secondary antibody  was diluted in blocking solution and applied for 1 h at room temperature in the dark. The cells were stained with DAPI as described above.The parameters for gating cell populations by flow cytometry were established using the EGFP-negative parental DAK31 cells. EGFP fluorescence was measured in single-cell suspensions of ESCs in N2B27 medium on a BD FACSCalibur or BD LSR Fortessa. Cell populations or single cells were sorted in 2iL using a BD FACSAria III cell sorter. Single cells were sorted into wells of a 96-well plate coated with mouse fibroblasts and containing 2iL medium.RNA (1 μg), purified using an RNeasy Mini Kit (QIAGEN) was used to synthesize cDNA using SuperScript First-Strand Synthesis System (Invitrogen). Approximately 1/60 of the cDNA was amplified using a Platinum SYBR Green QPCR kit (Invitrogen) under the following conditions: 50°C for 2 min, then 95°C for 2 min followed by 40 cycles of 95°C for 15 s, then 60°C for 30 s, with a final cycle consisting of 95°C for 1 min, 60°C for 30 s, and 95°C for 15 s. Primer sequences are in Rex1-EGFP cells maintained in 2iL were sorted into single cells using a BD FACSJazz cell sorter. Wild-type DAK31 parental cells .al cells  were useprotocol . IlluminUnless specified, all experiments were performed on triplicate biological or experimental samples. The data presented represented means ± SD or SEM. Single-cell sequencing data were analyzed using R to generate principal-component analysis (PCA) and box plots.S.M., J.W., A.J., and T.B. conceived and designed the study. S.M., J.W., T.O., J.O., L.S., and T.W. performed the study. T.C. and A.S. planned and performed the single-cell RNA sequencing experiments. D.F.C. constructed the β-catenin TALENs. S.M., J.W., A.J., T.C., and T.B. analyzed the data. S.M. and T.B. wrote the manuscript. All authors reviewed the manuscript.