Understanding changes in atmospheric carbon cycle is therefore pivotal in predicting consequences of recent global warming. Here, we report stable carbon isotopes (δ13C) of molecular land plant fossils complemented by bulk organic and inorganic carbon fractions for early Toarcian (Early Jurassic) sediments that coincided with global warming and a carbon cycle perturbation. The carbon cycle perturbation is expressed by a negative excursion in the δ13C records established for the different substrates. Based on differences in the magnitude of the carbon isotope excursion recorded in land plants and marine substrates we infer that the early Toarcian warming was paralleled by an increase in atmospheric CO2 levels from ~500 ppmv to ~1000 ppmv. Our data suggest that rising atmospheric CO2 levels resulted from the injection of  12C-enriched methane and its subsequent oxidation to CO2. Based on the cyclic nature of the CIE we concluded that methane was released from climate sensitive reservoirs, in particular permafrost areas. Moderate volcanic CO2 emissions led to a destabilization of the labile permafrost carbon pool triggering the onset of Toarcian climate change only. The main carbon cycle perturbation then subsequently was driven by a self-sustained demise of a carbon-rich cryosphere progressing from mid to high latitudes as reflected by latitudinal climate gradients recorded in land plant carbon isotopes.Throughout Earth’s history, variations in atmospheric CO As a consequence rising global temperatures can led to a reactivation of carbon stored in permafrost regions that upon its release to the atmosphere will further accelerate global warming2. Melting polar ice caps and sea level rise, climate extremes and enhanced stress for marine and continental ecosystems have been proven to be direct consequences of global warming5. Predictions on the evolution of Earth’s climate system, the carbon cycle and the response of ecosystems are, however, problematic. Thus, investigation of sediment archives that record ancient climate perturbation can serve as analogues for recent climate change and can thereby guide in predicting consequences of global warming and its cascade of consequences.Anthropogenic fossil carbon emissions steadily increase atmospheric CO2 levels and latitudinal climate gradients during the early Toarcian global warming.Here, we address changes in Earth’s climate and carbon cycle that occurred in conjunction with the early Toarcian Oceanic Anoxic Event . This study utilizes stable carbon isotopes recorded in different substrates, facilitating the reconstruction of changes in the global carbon cycle, atmospheric CO9. Rising global temperatures evolved parallel to an increase in atmospheric CO2 level inferred from stomata data10. In the marine realm global warming led to expansion of marine death zones and triggered the genesis of the Toarcian Oceanic Anoxic Event (T-OAE)11, whereas on land it caused substantial shifts in floral assemblages13.Around the globe, sediment archives that span the early Toarcian record profound environmental changes. A rapid high-amplitude sea level rise paralleled by a decline in oxygen isotope values of macrofossil calcite, has been interpreted to reflect a rise in sea water temperatures that was potentially accompanied by a reduction in the volume of land-based ice caps12C-enriched carbon into Earth’s hydro-atmosphere system18. Carbon sources are debated controversially and comprise a volcanic CO2 and/or thermogenic CH4 associated with the emplacement of the Karoo-Ferrar Large Igneous Province of southern Gondwana19, destabilization of methane hydrates16, increased rates of wetland methanogenesis17, or permafrost decay and thermokarst blowout events during global warming9. The CIE has been reported in marine and terrestrial organic matter as well as in marine carbonates15, suggesting that the carbon cycle perturbation affected the entire exchangeable carbon reservoir. A decline in δ13C documented in land plant-derived lipids indicates atmospheric 13C depletion and substantiates a perturbation of the atmospheric carbon cycle21. However, current δ13C records of land plant-derived lipids cover only a brief stratigraphic interval and provide no information on the recovery phase of the CIE and on the long-term evolution of the atmospheric carbon reservoir. Moreover, information on atmospheric CO2 concentration and its absolute change during the early Toarcian warming event are based on stomata data from a single section only and span the onset of the CIE10. Reconstruction of atmospheric CO2 concentration may further be complicated by stratigraphic gaps and methodological limitation22.A hallmark of the early Toarcian is a negative carbon isotope excursion (CIE) that is interpreted to reflect a global carbon cycle perturbation, caused by injections of 13C analysis of land plant-derived wax lipids, compounds not affected by the differential preservation of fossilized wood fragments15, provide a robust method for reconstructing changes in the isotopic composition of the atmospheric carbon reservoir. The compound-specific δ13C record is complemented by δ13C data from marine calcite that reflect changes in the oceanic carbon reservoir. The reconciliation of δ13C excursions in land plant and marine substrates allows reconstruction of changes in the entire exchangeable carbon reservoir. Moreover, parallel evaluation of marine and terrestrial carbon isotope excursions provide information not only on changes in atmospheric CO2 concentration but also on absolute atmospheric CO2 levels prior to and during the early Toarcian carbon cycle perturbation24.Here we utilize compound-specific carbon isotope data of land plant wax lipids to reconstruct changes in the atmospheric carbon reservoir across the early Toarcian carbon cycle perturbation and the associated climate event. The δEmaciatum to Serpentinum ammonite zones and the NJT5b to NJT6 nannofossil zones, cropping out at La Cerradura 25. Ammonite assemblages in combination with coccolithophore-based biostratigraphic data indicate that the sediments can be correlated with the T-OAE26, which is further supported by paleontological and geochemical data25. Sediments, mainly marlstone-limestone alternation, were deposited in a fragmented marine platform with hemipelagic sedimentation at a paleolatitude of about 26°N at the southern Iberian paleomargin. Floral assemblages suggest that during the Early Jurassic (183 Ma) the study site was located in the semi-arid climate belt27   and Hesselbo et al. (refs. 15). The −3.7‰ magnitude of the CIE at La Cerradura is similar to that reported in long chain n-alkanes from the Sichuan Basin (China)18, but is slightly lower than the −5.3‰ CIE (−4.2‰ on average) determined for terrigenic n-alkanes from the Cleveland Basin (UK)20 , molecular heterogeneity, or taxonomic impact on the isotopic signature of fossil wood15. Moreover, when preserved as jet (degraded wood), microbial reworking and impregnation by marine taxa during exposure to seawater, can alter the initial δ13C signature15.The δge) Fig.  parallell. ref. 1 and Hess13Cn-alkane records for different basins show similar trends and magnitudes of the CIE, their absolute δ13C values differ. The δ13Cn-alkane records from the Cleveland and Sichuan basins both show base values more depleted in 13C by about 4 to 5‰ when compared with base values from La Cerradura  reported pCO2 values in the range 350 to 1200 ppmv and 250 to 1800 ppmv in pre-CIE and CIE intervals, respectively. However, fragmentary deposition, stratigraphic incompleteness, and very low number of data points complicate robust stomatal pCO2 estimates. Moreover, there is also a poor calibration of the stomata proxy that can also respond towards environmental factors other than atmospheric CO234.The early Toarcian CIE was associated not only with changes in the isotopic composition of the exchangeable C-reservoir, but also with changes in atmospheric pCO2 levels is based on the observation that the isotopic fractionation of C3 land plants will vary not only with precipitation rates, but also with pCO235. This CO2 effect results in a higher isotopic fractionation when pCO2 levels increase and thereby cause higher CIE magnitudes in terrigenic than in marine substrates24. Offsets in CIE magnitude of terrigenic versus marine substrates thus facilitate determination of absolute atmospheric pCO2 levels35 (for details we refer to the supplementary information). However, as pointed out by Schubert & Jahren (ref. 23) and Lomax et al. (ref. 36) under enhanced water stress the carbon isotopic signatures of C3 plants vary as a function of precipitation rates and then do not unambiguously reflect past atmospheric CO2 concentration. According to recent observations, a strong impact of precipitation rates on δ13C of land plant biomass has been documented for vegetation in areas with mean annual precipitation rates < 2200 mm/year. On the contrary, precipitation seems to have no significant impact on the land plant δ13C in areas with high mean annual precipitation rates23. The dominance of xerophytic flora in the southern Iberian paleomargin, which here is represented by the La Cerradura section, suggests low paleo-precipitation rates and eventually enhanced paleo-water stress32. When compared to localities at higher latitudes, lower paleo-precipitation rates also manifested themselves in the 13C-enrichment of the land plant biomass. We can, however, speculate only about absolute paleo-precipitation rates at the southern Iberian paleomargin, which complicate evaluating the impact of water stress on the δ13C land plant biomass.An alternative approach for determining ancient pCO2 levels and to minimize the effect of different paleo-precipitation rates, we compared data from the La Cerradura section, located in a semi-arid climate belt, with data from Yorkshire (UK)20 and from China18 that were both situated in a humid climate belt , Yorkshire20 and China18 we calculated a maximal magnitude in the CIEterrigenic of −4.2‰ (−3.1‰ on average). A higher CIEterrigenic of −5.4‰ is achieved when including δ13C data of fossil wood and phytoclasts (Table 24), we determined the magnitude of the CIE in marine substrates (CIEmarine) by using δ13Ccarb data from oxygenated marine basins only. This includes data from organic matter-lean sediments deposited at the southern part of the West Tethys Shelf. At these areas the seafloor preferentially remained oxygenated throughout the early Toarcian37. For such settings organic matter-induced carbonate diagenesis and/or CO2 recycling in stratified water bodies that may alter the δ13C signature can assumed to be minimal or can even be excluded39. Carbon isotope data from marine organic matter is not included in our calculation, as δ13Corg values can be affected by mixing of organic matter of marine phototrophic and non-phototrophic organisms or land plants24. We  calculated an average CIEmarine of −2‰ . Using the δ13Cn-alkane based CIEterrigenic and the CIEmarine we calculated a ΔCIE (ΔCIE = CIEterrigenic – CIEmarine) of −1.5 and −2.2‰, for average and maximal values of the CIEterrigenic, respectively. Including δ13C data from fossil wood yields a ΔCIE of about −3.4‰.Based on δpCO2 levels prior to the CIE (pCO2(init)) and during the climax of the CIE (pCO2(CIE)) further requires an estimation for the ΔpCO2 that here is derived from mass balance calculations in dependency of the CIEmarine and the isotopic signature of the respective carbon source. We calculated ΔpCO2 values for carbon sources with isotopic signatures characteristic for: i) biogenic methane emissions (δ13C: −70‰42) ii) gas hydrates (δ13C: −60‰43), iii) thermogenic methane (δ13C: −35‰43) and iv) a source dominated by volcanogenic CO2 (δ13C: > −10‰42) (for details see supplementary information).Calculation of pCO2(init) ∼600 ppmv and of ∼400 ppmv, respectively, whereas for pCO2(CIE) we obtained 1200 and 850 ppmv, respectively , our data attest to a doubling in pCO2 instead of a threefold increase  and a ΔCIE of −2.2‰ and −3.4‰, we calculated values for 13C-based CO2 reconstruction44, thermogenic methane release from thermal alteration of organic matter-rich sediments during the Karoo-Ferrar emplacement19 would be plausible as well. Such a scenario is, however, not supported by geochemical data46 and is further difficult to reconcile with the orbitally-forced cyclic pattern of the CIE that is only explained by carbon release from climate-sensitive reservoirs responding to changes in Earth’s solar orbit16. On the contrary, release of biogenic and thermogenic methane from glacier- and permafrost-capped reservoirs would be a plausible scenario9 that is supported by recent observations41.With respect to uncertainties in the ΔCIE value and in the δ2 emission as being the major driver of the early Toarcian climate change would require the release of enormous amounts of CO2 that would have shifted pCO2 levels from about 1000 ppmv during pre-event times to more than 4000 ppmv during the CIE  were performed on decalcified sample material9. Decalcification was achieved by treating the sample material with hydrochloric acid  to remove carbonate-bound and if present dolomite-bound carbon. Afterwards, samples were washed, neutralized with deionized water and dried in an oven at 40 °C for 48 h. Stable carbon isotope analysis was performed using a Thermo Finnigan Delta V isotope ratio mass spectrometer coupled to a Flash EA via a Conflow III interface.Stable carbon isotope analysis for bulk organic carbon  = [(Rsample − Rstandard)/Rstandard − 1] × 1000, where R is the ratio of 13C/12C of the sample and the V-PDB standard for carbon. Reproducibility and accuracy were monitored by replicate standard and sample analysis and are better than 0.1‰.The carbonate fraction was measured for its carbon isotopes using a Kiel III carbonate preparation line connected to a Thermo Fisher 252 mass spectrometer. Powered and homogenized samples were treated with 103% phosphoric acid at 70 °Cet al. (ref. 48) total bitumen extracts were separated into aliphatic, aromatic and polar hydrocarbon fractions by silica gel-column chromatography  using solvents with increasing polarity in an LCTECH automated SPE system. The aliphatic hydrocarbon fractions were treated with activated copper turnings in order to remove elemental sulfur. GC–MS measurements of the aliphatic hydrocarbon fractions were performed on an Agilent 5975B MSD interfaced to an Agilent 7890 A GC equipped with a quartz capillary . The temperature program of the GC oven used was: 70 °C  to 140 °C at 10 °C/min, then to 325 °C at 3 °C/min (held for 7 min). The quadrupole MS was operating in scan mode in the m/z 50 to 750 range. Compounds of interest were identified via characteristic mass spectra and were integrated manually using the GC/MSD Masshunter Software (Agilent Technologies)48.Total lipid extracts for selected samples were obtained from solvent extraction using a Soxhlet apparatus. As extraction solvent we used a mixture of dichloromethane (DCM) and methanol (MeOH) . Similar to the method applied by Ruebsam n-alkanes  are present as well, but occur at very low abundances  using a Thermo Scientific Trace GC Ultra interfaced to a Thermo Scientific Delta V Advantage mass spectrometer via a GC isolink and a Conflow IV. The δ13C values of the compounds were determined by integrating the ion currents of masses 44, 45 and 46, and are reported in permil (‰) relative to the VPDB standard. Reported values are the average of at least two analyses with standard deviation of <0.5‰.Gas chromatography–isotope ratio mass spectrometry (GC–irMS) was performed following the methodology described in Plet pCO2 levels follows the approach by Schubert & Jahren (ref. 24) and is based on the differences in the magnitude of a CIE reported in land plant organic matter and marine substrates. Assessment of methodical uncertainties is based on the work by Cui and Schubert (ref. 44) and varies as a function of absolute pCO2 concentration. Details on the calculations are provided in the supplementary information.Calculation of Supplementary Information.Supplementary Information2.Supplementary Information3.Supplementary Information4.


The objective of the study was to establish how patient satisfaction with surgical treatment of Parkinson's disease (PD) has been previously measured, determine whether an ideal patient satisfaction instrument exists, and to define the dimensions of care that determine patient satisfaction with the surgical treatment of PD.A systematic search of four online databases, unpublished sources, and citations was undertaken to identify 15 studies reporting patient satisfaction with the surgical treatment of PD. Manuscripts were reviewed and instruments were categorized by content and method axes. One study was found to utilize two distinct patient satisfaction instruments, which brought the total number of satisfaction instruments assessed to 16. Major factors influencing patient satisfaction were identified and served as a structure to define the dimensions of patient satisfaction in the surgical treatment of PD.Studies used predominantly multidimensional (10/16), rather than global (6/16) satisfaction instruments. Generic (12/16) rather than disease-specific (4/16) instruments were utilized more frequently. Every study reported on satisfaction with outcome and four studies reported on satisfaction with outcome and care. Six dimensions of patient status, outcome and care experience affecting patient satisfaction were identified: motor function, patient-specific health characteristics, programming/long-term care, surgical considerations, device/hardware, and functional independence. At present, no patient satisfaction instrument exists that is disease-specific and covers all dimensions of patient satisfaction in surgery for PD. For quality improvement, such a disease-specific, comprehensive patient satisfaction instrument should be designed, and, if demonstrated to be reliable and valid, widely implemented. Patient-reported outcomes are increasingly recognized as meaningful and valid measures of successful care. This is in contrast to traditional surgeon-centered outcome parameters that include morbidity, mortality, complications, and postoperative imaging findings. Patient satisfaction is a type of patient-reported outcome distinct from reports of health, disability, and quality of life. Patient satisfaction is a measurement reflecting patients’ perception of outcome of care and has been considered for use in future reimbursement schemes. Contrary to intuition, satisfaction is not necessarily concomitant with traditional measures of surgical outcomes. There is a complex interplay between preoperative factors, the patient-physician interpersonal relationship, interactions with nurses and other hospital staff, as well as the other, more traditionally measured outcomes that determine patient satisfaction. High satisfaction leads to a high likelihood that a patient will pursue further medical care, promotes patient compliance, and has been linked to decrease the incidence of malpractice suits -4. No consensus on patient satisfaction instruments exists in the surgical treatment of Parkinson’s disease (PD). A multidimensional, disease-specific patient satisfaction instrument could guide changes in surgical practice for quality improvement.The goals of this review are fourfold: (1) To assess the tools that have been used to measure patient satisfaction with the surgical treatment of PD that are currently in use; (2) To determine whether a comprehensive instrument to measure patient satisfaction exists; (3) To define the dimensions of care that consistently effect patient satisfaction in the surgical treatment of PD; (4) To describe the dimensions of an ideal instrument to measure satisfaction with the surgical treatment of PD.MethodsSearch Strategy and Study InclusionPubmed, the Cochrane Library, CINAHL and EMBASE were systematically searched for “Patient Satisfaction” AND “Deep Brain Stimulation” AND “Parkinson’s Disease,” “Patient Expectations” AND “Deep Brain Stimulation” AND “Parkinson’s Disease,” “Physician-Patient Relationship” AND “Deep Brain Stimulation” AND “Parkinson’s Disease,” “Doctor-Patient Relationship” AND “Deep Brain Stimulation” AND “Parkinson’s Disease,” “Patient Preference” AND “Deep Brain Stimulation” AND “Parkinson’s Disease,” “Patient Satisfaction” AND “Surgery” AND “Parkinson’s Disease,” “Patient Expectations” AND “Surgery” AND “Parkinson’s Disease,” “Physician-Patient Relationship” AND “Surgery” AND “Parkinson’s Disease,” “Doctor-Patient Relationship” AND “Surgery” AND “Parkinson’s Disease,” “Patient Preference” AND “Surgery” AND “Parkinson’s Disease,” yielding 33 unique publications. Abstracts of all 33 publications were read. Manuscripts were included if they were empirical, reported measurement of patient satisfaction, and referred to the surgical treatment of PD. Manuscripts were excluded if they were review papers, if there was no measurement of patient satisfaction, or if the papers did not involve the surgical treatment of PD. If the abstract and title were not clear, then full texts were read to determine inclusion or exclusion. Using these criteria, 15 manuscripts were excluded, yielding 18 manuscripts. Then, all full-length manuscripts were read, and an additional eight manuscripts were excluded because exclusion criteria were met, yielding 10 manuscripts. Citations in all papers were reviewed, and an additional five manuscripts meeting inclusion criteria were discovered. In total this yielded 15 empirical studies measuring patient satisfaction in the surgical treatment of PD  for deep brain stimulation (DBS) arrays. Patient satisfaction was defined as any attempt of a study to gather evaluations or “affective responses” regarding a patient’s health care experience -5.All manuscripts were critically read, and data were categorically extracted. Categories included: study type , procedures performed , number of surgeons, length of follow-up, country of participants, number of participants, outcome measurements, patient satisfaction instrument used, intake format (self-administered or interviewer administered), validity of the instrument, and findings of the study. Siderowf et al.  utilizedFrom these data the characteristics of patient satisfaction instruments used in each study were classified using the criteria described by Hudak et al . These aThrough review of the findings of each study, a taxonomy of patient satisfaction with PD surgery was developed identifying the major characteristics of providers and medical care influencing patient satisfaction. In the pioneering work in elucidating the dimensions of patient satisfaction with medical care, Ware et al. formed such a taxonomy from reviewed studies to act as the basis for the development of the dimensions of care that affected patient satisfaction . In a siResultsCurrent Measurement InstrumentsEach patient satisfaction measurement instrument described in the 15 manuscripts were categorized using the model utilized by Hudak et al.  for patiThe methods of the instruments used exclusively affective rather than factual questions. Close-ended questions rather than open-ended questions were used exclusively. Most instruments were self-administered (87.5%) rather than interviewer-administered (12.5%). The most common response format was a Likert scale (93.75%) with a varying number of gradations.The most commonly used multidimensional patient satisfaction instrument was the Short From-36 (SF-36) instrument, which was administered in four studies . The QueDimensions of Patient SatisfactionOf the 15 papers included in this study, three , 14-15 dMotor FunctionThe interaction between the improvement in motor function and patient satisfaction was explored in five studies , 13, 16.Patient-Specific CharacteristicsPatient-specific characteristics are intrinsic traits that cannot be modified in the acute phase prior to an operation. This includes co-morbid conditions that are not directly treated by the surgical intervention. These characteristics were found to be related to patient satisfaction in five studies -18, 20. Decreased rates of satisfaction were found to be more prevalent as patients were farther out from surgery in one study . This fiAge and gender differences were also found to affect satisfaction. Multiple studies found age to be negatively associated with patient satisfaction and one specifically found younger people to be more satisfied with a battery that could be percutaneously recharged , 20. HarProgramming/Long-Term CareThe link between satisfaction and long-term care is clear due to the current practice of DBS being the primary surgical intervention for PD and the resultant requirement of a long-term relationship with a movement disorder specialist for programming and maintenance of the device. Farris and Giroux found that suboptimal programming of the device led to dissatisfaction early in the post-operative period . KuehlerSurgical ConsiderationsDetails of surgical planning and procedure, both with the intracranial portion of the process and with implantation of a pulse generator if required, were found to be linked to satisfaction in three studies , 17, 19.Farris and Giroux found that intracranial leads placed in suboptimal positions led to early dissatisfaction with the surgical result . The choSon et al. found that patients were more satisfied with a trans-axillary, sub-pectoral implantation of a pulse generator as opposed to the standard sub-clavicular location. Patients were found to be pleased with the decreased visibility both of the pulse generator and surgical scar . KuehlerHardwareWith the predominant use of DBS as surgical treatment for PD and the necessity for expensive hardware that has a finite lifespan, the presence of a linkage between hardware and patient satisfaction is not surprising. Farris and Giroux noted that hardware erosion and dysfunction are associated with dissatisfaction with surgical treatment . MultiplIndependenceThe importance of functional independence as a determinant of satisfaction was explored in three studies -13. HashFerrara et al. and Kuehler et al. all found that functional independence and the perception of increased energy post-operatively were linked to increased patient satisfaction , 13.DiscussionPatient-reported outcomes such as patient satisfaction are increasingly recognized as valid markers for therapeutic efficacy and there are discussions concerning the use of patient satisfaction in the determination of reimbursement schedules. Beyond the financial implications lie patient care issues. It is well established that patients who are more satisfied with their medical care have better long-term outcomes. Satisfied patients are more compliant with treatment regimens, develop lasting relationships with their medical providers and are more apt to present promptly with new signs and symptoms -2, 4, 28Currently the field of surgical treatment for PD utilizes patient-reported outcome measures such as the UPDRS and Parkinson’s Disease Questionnaire (PDQ-39) routinely. Both of these patient-reported outcome measures fail to take into account the patients affective response to the disease process or treatment and thus do not meet the standard to be considered a patient satisfaction instrument . Both of An ideal instrument would be a disease-specific instrument that uses a multidimensional approach to measuring satisfaction with both care and outcome. Questions should be structured in a direct manner as this is more appropriate to assess satisfaction with a specific procedure or treatment -31. QuesThe only instrument that we identified that approximates an ideal instrument is the QLS that was initially described by Kuehler et al.  and lateThe absence of consideration of patient expectations is a major limitation of the QLS specifically and the current state of the measurement of patient satisfaction in the surgical treatment of PD in general. A patient’s expectations are increasingly identified as a major influence on outcomes and patient satisfaction -39. ExpeLimitations and Future DirectionsThese dimensions of care have been defined using a literature review technique. While this approach elucidates dimensions of care, there is no means of applying value to each dimension appropriately and understanding whether there are collinear dimensions that will cancel each other out. It is highly unlikely that each of these dimensions of patient satisfaction is of equal value in determining overall satisfaction. Only through design of an instrument using these dimensions and psychometric testing can appropriate values and collinear variables be established to provide utility to such a model.Patient satisfaction is increasingly understood as a distinct and important patient reported outcome measure. Despite consistent measurement of other outcome measures, including those reported by patients and observed by physicians, the current state of measurement of patient satisfaction in the surgical treatment of PD is haphazard and inconsistent. Design of an ideal instrument to measure patient satisfaction with the surgical therapy of PD would be beneficial in determining or proving the efficacy of and necessity for surgical treatment of PD.


The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activation, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features.The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue characterised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized contractile structures. The non-receptor protein tyrosine kinase Arg  has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular signals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in ArgThis article has an associated First Person interview with the first author of the paper. Summary: The non-receptor tyrosine kinase Arg and its isoforms modulate the extra cellular matrix production that is relevant in fibrosis and tumour growth, this may open future novel therapeutic approaches. The fibrotic process, after a chronic injury, results in an excessive scar tissue deposition and development of fibrosis, and has in the activated fibroblasts (myofibroblasts) the main players . In factThe non-receptor tyrosine kinase Arg   shares w−/− murine embryonal fibroblasts (MEF) cell line, in particular focusing on their capacity to activate fibroblasts, to modulate their functionality and to remodel ECM.The aim of this study was to analyse more deeply the difference of two 1ALCTL and 1BLCTL Arg isoforms transfected in Arg−/− MEF  and the EGFP empty vector has been transfected in wt and Arg−/− MEF . After 2–4 steps of purification by cell sorting, the percentage of EGFP positive cells was from 90 to 95% for all types of transfection . Western blot confirmed that 1ALCTL MEF and 1BLCTL MEF expressed the Flag-Arg proteins at the expected molecular weight and at the same level of wt MEF  . We performed an in vitro kinase assay on Hek cells transfected with the two Arg isoforms and we showed that both immunoprecipitated isoforms were able to phosphorylate the enolase protein . In addition, the transfected Hek cells treated with Imatinib, an inhibitor of Arg tyrosine kinase activity, evidenced that the two isoforms were sensitive to the drug, in particular to Imatinib concentration of 10 µM  A. The tyof 10 µM D.Fig. 1−/− MEF. The 1ALCTL isoform maintained the MEF proliferation at the level of Arg−/− MEF, while 1BLCTL induced a significantly higher proliferation activity than Arg−/− and only slightly lower, in a non-significative manner, with respect to wt MEF . It is of note that the different MEF studied have a different capacity to produce TGFß1. In particular, the absence of Arg determined the increase of TGFß1 expression . Arg−/− MEF were characterised by less evident focal adhesions and by thinner stress fibres that had a prevalent cortical distribution. 1ALCTL MEF had stress fibres crossing the cytoplasm and focal adhesions more evident in the retraction tail and in the opposite side of retraction tail (lamellipodia). 1BLCTL MEF maintained a cortical F-actin and a delocalisation of focal adhesions to filopodial structures , we analysed phospho-Y118 paxillin. As shown in Fig. S2D). In −/− MEF had a more spheroid shape without a typical F-actin organisation. 1ALCTL and wt MEF inside the collagen were associated in groups of cells, 1BLCTL MEF had filopodia structures. 1ALCTL and 1BLCTL showed diffuse paxillin staining, but at 4 h of migration they did not show the focal adhesion organisation. Finally, the different MEF types were analysed for their ability to shrink the collagen plug in which they had been embedded for 72 h. The Arg−/− MEF lost the ability to contract the collagen matrix. Both 1ALCTL and 1BLCTL restored the contraction to wt values . This finding suggests that Arg is necessary for fibroblast activation and that the fibrotic or the primary tumour stroma could be characterised by 1BLCTL Arg upregulation. Also an intriguing finding, in Arg−/− MEF, showed us that TGFß1 expression was inversely related to Arg expression  may justify their adhesion capacity  may counteract the lack of functional focal adhesion preserving cell contractility.The ECM-contraction ability, another feature of myofibroblasts , demonst−/− MEF failed to deposit the collagen I, which is currently deposited by myofibroblasts during fibrosis and tumour progression  according to the manufacturer's instructions. Transfected cells, resuspended in growth medium without antibiotics were plated and after 24 h transferred in growth medium containing 1 mg/ml of G418 (Sigma-Aldrich) for a 10 day selection. Transfected cells were stained by Propidium Iodide solution (Biolegend) to exclude dead cells and sorted on the basis of EGFP expression using MOFLO Astrios Cell Sorter and analysed by Kaluza software (Beckman Coulter). All the transfected cells were sorted to reach a EGFP purification level from 90 to 95%.Wild-type (wt) or Arg−/− MEF  were gro2. These cell types (75×104 cells) have been transfected using the vectors pCX-1ALCTL-EGFP, pCX-1BLCTL-EGFP in the Hek cells, pCX-C1-EGFP in 786-O cells using Lipofectamine 3000 Reagent (Invitrogen) following the manufacturer's instructions. The transfected Hek cells, when requested, were cultured for 3 h in presence of 1 µM or 10 µM Imatinib mesylate as Arg tyrosine kinase inhibitor .The human embryonic kidney Hek cell line, and the 786-O renal cell carcinoma cell line (ATCC) have been cultured in DMEM supplemented with 10% FBS (Euroclone), at 37°C and 5% COCell lysates were prepared, separated by SDS Nupage 4–12% and blotted on nitrocellulose membrane  as described . The pro−/− MEF. The ability to tyrosine phosphorylate the enolase substrate (Sigma-Aldrich) has been evaluated by an in vitro kinase assay on the immunoprecipitated Arg isoforms transfected in Hek cells (0.5 mg of cell lysates). The reaction has been performed in 20 µl of the following solution: 16 µl of 50 mM MgCl2, 2 µl of 1 mM ATP (Sigma-Aldrich) and 2 µg of enolase. After 30 min of incubation at 30°C, the reaction was stopped by adding SDS sample buffer. The reaction product was blotted and probed with antibodies against anti-PhosphoY and anti-enolase (Santa Cruz Biotechnology Heidelberg) and visualised by the ECL system.The tyrosine kinase activity of 1ALCTL and 1BLCTL isoforms has been assessed as described  on the i5) were seeded for 4 h on glass coverslips coated with collagen I Rat tail , fixed and incubated with antibody against paxillin  to evidence focal adhesion or against α-sma . Stress fibres have been labelled by Alexa 594-phalloidin  and nuclei were counterstained with Mounting DAPI (Molecular Probes Invitrogen). The cell surface area was evaluated as described  with a solution of 0.3% collagen, composed by type I (85%) and type III (15%) collagen from chicken. The manufacturer's instructions were followed and 60×103 fibroblasts were plated on the upper chamber. After 3 h at 37°C, we stained and microphotographed the porous membrane. The migrated cells were counted by two blinded operators at 400× in 10 different fields randomly chosen for each sample in all independent experiments with ImageJ software.Wound healing has been performed as described . Monolay3 fibroblasts embedded in 100 µl of collagen I rat tail. The mixture of collagen and cells was seeded on a 35-mm glass-bottom MetTek dish. Once the gels were set, photographs have been taken (t0) and gels containing fibroblasts were maintained in culture medium for 72 h (t72) when other photographs have been taken. To obtain the gel contraction value, the specific areas of the gel at t0 and t72 were measured using ImageJ software. The percentage of gel contraction of different independent experiments was calculated using the formula 100×[(gel area t0−gel area t72)/gel area t0].Adhesion assay has been performed as described . ECM con5 MEF of each type were respectively plated on coverslip. When the cells were confluent, ascorbic acid  was added and the medium changed every 2 days for 10 days. Cells were then removed by extraction buffer . Solubilized cellular material was then gently washed two to three times for 5 min each on ice with a washing buffer composed of 2 mM Tris-HCl, pH 8.0, supplemented with protease inhibitors. A subsequent treatment at 37°C with DNAse I (Sigma-Aldrich) for 20 min was performed to assure removal of DNA associated with nuclear debris . Cell pictures have been taken with an Eclipse E800 microscope (Nikon) and LuciaG 5.0 software (Nikon) supported by a digital camera . In three different independent experiments 20 to 40 cells have been analysed for each conditions.Cell-derived matrices were obtained on coverslips prepared as described . 1.5×105r debris . These Mr debris . 15×104 P<0.05 were considered statistically significant. In the box/dot graphs showed, representing at least three independent experiments, the individual dot represents the single independent experiment, the boxes indicate the 25°–75° percentile, the continuous horizontal line into the box represents the median (—), while the dotted horizontal line represent the mean (- - -). Max and min (⊤ and ⊥) are indicated.All molecular and functional effects of different cellular types were evaluated and/or quantified by two different operators blinded to experimental treatment. Differences between multiple groups were analysed using one-way ANOVA followed by post-hoc Tukey's test using OriginPro 2016 64BIT software. Values of


Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP).In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells.ab initio design of cells’ microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the  Indeed, in addition to the delivery and retention of cells, hydrogels allow the mechanical properties and highly hydrated nature of the NP to be recapitulated with the aim to preserve and/or restore its mechanical functionality. One particular family of hydrogels that has attracted significant attention in the last decade due to their biocompatible and non-immunogenic nature are peptide-based hydrogels In this work we explore the use of GO as a nanofiller for the design of hybrid peptide hydrogels for the delivery of NP cells. For this purpose, we first investigated the effect of adding GO has on the physico-chemical properties of the hydrogels. Subsequently NP cells were encapsulated and cultured over 7 days to demonstrate the hybrid hydrogels potential suitability as scaffold for their delivery.22.1FEFKFEFK peptide  was purchased from Biomatik Corporation . The peptide purity (94%) was confirmed in-house by mass spectroscopy (MS) and reverse phase high performance liquid chromatography (HPLC). All solvents and reagents were purchased from Sigma-Aldrich and used as received.2.23 (9 g) and concentrated H2SO4 (338 mL) at room temperature (RT) for 3 h to obtain intercalated graphite. The mixture was then cooled in an ice bath and 45 g of KMnO4 gradually added. After addition of the oxidizing agent, the reaction mixture was stirred at room temperature for one week to complete the oxidation. The oxidised graphitic slurry was then diluted with a solution of 5% H2SO4, followed by slow addition of 5 g of H2O2 as solution. The resulting GO was purified by repeated centrifugation and re-dispersion in deionised (DI) water until the pH of the supernatant was neutral. The size distribution of GO flakes was characterised using scanning electron microscopy (Zeiss Ultra FEG SEM). GO flakes (50 µg/mL−1) were spin-coated onto a plasma-treated Si/SiO2 wafer at 3500 rpm and acceleration of 350 rpm/s for 5 mins to get monolayer coverage with minimal flake overlap and left to dry overnight before imaging. For each dispersion the size of ∼300 flakes from three SEM images was measured manually using ImageJ® software. In order to avoid shape anisotropies to skew the distribution results, measurements were performed in the horizontal direction through the centre of the flake assuming that the drying process did not introduce any orientation anisotropy.GO used in this study was prepared by a modified Hummers method 2.3−1 aqueous solution after NaOH addition to prevent GO precipitation, as observed previously −3 per sample) using a SteriBeam sterilization system. The name and formulation of the samples used in this study are listed in Depending on final concentration targeted, the desired amount of peptide powder was dissolved in HPLC grade water. The hydrogels were then prepared by adjusting the pH to 4 by of a 0.5 M NaOH solution. When hybrid hydrogels were prepared GO was added as a 3 mg mL2.42-scrubbed air. Absorbance spectra were obtained using the average of 128 scan with a 4 cm−1 resolution. HPLC grade water background was subtracted from each spectra using the built-in OMNIC software provided with the instrument. All measurements were done in triplicate. The FTIR spectra were smoothed using a Savitzsky-Golay filter with a 9-points window. A baseline was subtracted in the Amide I region (1500–1700 cm−1) from the original spectra using OriginPro v8.5.1 software.Measurements were performed on a Thermo-Fisher Nicolet 5700 FTIR spectrophotometer equipped with an attenuated total reflectance (ATR) diamond accessory. 20 µL of hydrogel was pipetted onto the crystal surface of the spectrophotometer and the beam path purged with dry CO2.52O. 50 µL of diluted hydrogel were then drop-cast on freshly-cleaved mica and left for 2 min. Excess solution was then removed from the surface of the specimens and the mica stubs rinsed 10-times by pipetting 100 µL of ddH2O directly onto the surface. Excess liquid was finally removed using Whatman No. 1 filter paper. Samples were left to air-dry overnight before imaging. AFM imaging was performed on a Bruker Multimode 8 instrument . Samples were scanned in air at room temperature in ScanAsyst mode, using a ScanAsyst-Air probe with a resonant frequency, f0, of 70 kHz and a nominal spring constant, k, of 0.4 N m−1. AFM images were acquired at a 512 × 512 pixels resolution over scanning areas ranging from 2 × 2 to 10 × 10 μm at a scan frequency of 1 Hz and analysed with Nanoscope Analysis 1.40 software.Hydrogels were first diluted 10-fold using ddH2.620. A carbon-coated copper grid  was then placed sequentially on a: 10 µL sample droplet for 1 min, 10 µL ddH2O droplet for 10 s, 10 μL 1% uranyl acetate (negative staining) solution droplet for 30 s, 10 µL droplet of ddH2O for 10 s. Excess liquid was removed following each step using a lint-free tissue. Samples were then left to air-dry before imaging. TEM images were obtained using a FEI Tecnai12 BioTwin at 100 KeV with Gatan Orius SC1000A CCD camera.Hydrogel samples were first diluted 10-fold using ddH2.72O or bovine nucleus pulposus cell culture media. After media exposure, samples were removed from the inserts by peeling-off the bottom membrane of the insert and transferred onto the rheometer plate. The upper rheometer head was then lowered to the desired gap size and samples were left to equilibrate for 3 min at 37 °C. Frequency sweep experiments were performed at 0.2% strain, within the linear viscoelastic region in the frequency range: 0.01–10 Hz. For shear-thinning recovery experiments, samples were tested in time-sweep mode by alternating cycles of recovery (5 min at 0.2% strain and 1 Hz frequency) with cycles of high-shear (5 min at 100% strain and 1 Hz frequency). For all experiments a solvent trap was used to minimise sample evaporation. All measurements were repeated at least three times to ensure reproducibility.Oscillatory shear rheometry was performed on a Discovery Hybrid 2 (DHR-2) rheometer  using a 20 mm parallel plate geometry with a gap size of 500 µm. Samples were prepared by pipetting 300 μL of hydrogel into ThinCert well inserts . The inserts were then placed into 12-well plates and incubated at 37 °C overnight in either 1 mL of ddH2.8−1 penicillin, 100 μg ml−1 streptomycin, 0.25 μg ml−1 amphotericin, 10 μM ascorbic acid 2-phosphate, 100 mM of sodium pyruvate) containing 10% (v/v) foetal calf serum. The cells were passaged at 70–80% confluency using a 1× trypsin/EDTA solution and the cell suspension collected in a 50 mL falcon tube. A cell pellet was then recovered from the cell suspension by centrifugation (350 g for 5 min) and re-suspended in fresh culture media to the desired cell density. Hydrogels were pre-warmed at 37 °C and bovine NP cells encapsulated by gentle pipetting and mixing 100 μL of cell suspension into 1 mL of hydrogel to a final cell density of 4 × 106 cells mL−1 of hydrogel. After encapsulation, 100 μL aliquots of cell-laden hydrogels were dispensed in ThinCert well inserts and conditioned by adding 1 mL of cell culture media in each culture well. The cell culture plates were incubated at 37 °C and 5% CO2. Media was changed every 20 min during the first hour and every two days thereafter.NP cells were isolated from bovine tails obtained from a local abattoir and expanded in monolayer culture using DMEM culture media . Media was removed from the cell culture wells and the hydrogels incubated for 1 h with a membrane integrity assay as a surrogate measure of viability LIVE/DEAD assay solution , 20 μL ethidium homodimer-1 and 5 μL of calcein AM). After incubation the LIVE/DEAD assay solution was gently removed and the cell-laden hydrogels transferred onto microscope glass slides for imaging. Cell images were collected using a Nikon Eclipse 50i fluorescence microscope  at days 1, 4 and 7. Three samples per time point were used. The fluorescence composite images in red, green and blue (RGB) were split in their individual channels using ImageJ® (v.1.51) to produce three 8-bit greyscale images. Greyscale images were thresholded using Huang’s approach 2.102. After incubation the AlamarBlue solution was gently removed and transferred to a 96-well plate (100 μL per well) and fluorescence measured using a BioTek™ FLx800™ microplate fluorescence reader . The fluorescence intensity measured was normalised using a blank hydrogels (without cells) fluorescence.To assess cell metabolic activity, media was removed from the cell culture wells and the hydrogels incubated for 2.5  h with a 10% AlamarBlue DMEM solution at 37 °C and 5% CO2.11To assess cell clustering the area distribution of the green fluorescent images obtained during LIVE/DEAD staining were used. The images were processed as described above and the greyscale images corresponding to the green channel used to create frequency plots of cell and cell clusters area. Cell areas were then obtained using the ‘analysing particles’ plugin on the greyscale image relative to the green channel and results were plotted as area-size-frequency plots. The cell area value below which 50% of the cell area distribution is contained (D50) was also obtained.2.12All quantitative values were presented as mean ± standard deviation. All experiments were performed using at least three replicates. Data were compared using one-way analysis of variance (ANOVA) implemented in Microsoft Excel 2016. Two level of significance: 0.05 (**) and 0.001 (***) were used.3We recently have investigated the interactions between a range of graphene derivatives, including GO, and this family of β-sheet forming peptides In −1 and a weaker one at ∼1690 cm−1 are observed. These absorption bands are characteristics of the adoption by these peptides of a cross-β-sheet conformation and the formation of extended β-sheet fibres −1 samples imaging was more challenging due to the density of the fibrillar network and the difficulty when preparing the specimens, to dilute/disentangle the peptide fibres. Nevertheless in all cases long semi-flexibles fibres can be observed. For the sample containing GO the flakes where found in all cases to be fully embedded within the fibrillar network.First we investigated the effect that incorporating GO in the hydrogels had on the ability of the peptide to form β-sheet fibres. In In order to gain further insight into the molecular interactions between the peptide and GO high resolution AFM images were obtained of GO flakes embedded in the GO-F820 sample A. As forThese results confirm that the presence of GO does not affect the ability of this peptide to form β-sheet fibres nor hydrogels. They though highlight the presence of strong interactions between the GO flakes and the peptides that are expected to have a significant effect on the bulk mechanical properties of the hydrogels.Next we investigated the effect that adding GO flakes had on the shear moduli of the hydrogels after formulation (i.e.: pH = 4) and after conditioning the gel with cell culture media (i.e.: pH 7.4). The latter condition was used as these will be the mechanical properties actually experienced by the cells when encapsulated in the hydrogels. Changing the hydrogels’ pH from 4 to 7.4 will significantly alter the nature of the interactions between peptide fibres themselves as well as between peptide fibres and GO flakes, as the fibres will go from carrying a positive net-charge to becoming neutral . It shou−1 samples but to half for the 20 mg mL−1 sample. The detrimental effect on G′ at high peptide concentrations (>20 mg mL−1) of incorporating GO in these hydrogels was discussed in our previous work, where we showed that the adhesion of the peptides on the graphene derivatives was mainly driven by hydrophobic interactions −1) is tough puzzling. We hypothesise that this effect is linked to a change in the nature of the electrostatic interactions between the peptide fibrillar network and the peptide-coated GO flakes. Indeed, at pH 7 when electrostatic interactions are removed , no such effect is observed  were found to have significantly higher G′. The overall mechanical properties of these hydrogels is linked to the nature of the interactions between the peptide fibres which control the level of network crosslinking. At pH 4 there is a competition between hydrophobic interaction that promotes crosslinking and electrostatic repulsion that prevents crosslinking. At pH 7 the fibre do not carry a charge resulting in the elimination of the electrostatic repulsion leading to an increase in the level of hydrophobic crosslinking and a stiffening hydrogel i.e. and increase in G′. As stated above, conditioning the samples with media in addition to a change in pH also introduces significant amount of ions in the system resulting in charge screening effects being also present, once again promoting higher levels of crosslinking −1 samples in both cases, with and without GO, the hydrogels had the same rheological signature. When sheared at high shear strain (strain = 100%) the hydrogels shear-thin becoming liquid-like (G″ > G′) and when the shear strain is decreased (strain = 0.2%) within the LVR the hydrogels recover their solid-like behaviour (G″ < G′). This effect was found to be highly reproducible over the 3 cycles tested. In One of the key features of these hydrogels is their injectability or in other words their shear thinning and recovery properties. In order to confirm that he incorporation of the GO did not affect the hydrogels injectability a series of experiments where performed in-situ in the rheometer during which high (hydrogel breaking) and low (hydrogel recovery) shear strains where applied successively. As can been seen from Finally, we investigated the potential of these hydrogels as delivery platform of IVD repair by culturing in 3D bovine NP cells. The cells where initially encapsulated in the hydrogel as formulated (i.e.: pH 4). The cell containing hydrogel were then plated in cell culture inserts and conditioned by surrounding the inserts with cell culture media . Cell culture was performed over a period of 7 days. In In 2 corresponding roughly to clusters of 15–20 cells.Another characteristic of NP cells in their native environment is the tendency to cluster into small groups of cells, which have been referred as the “functional units” for disc repair, within the NP The results obtained, in particular for the GO-F820 sample, clearly highlight the potential of these hybrid hydrogels as injectable scaffold for the culture and delivery of NP cells.4We have designed and characterised a series of FEFKFEFK-GO hybrid hydrogels. Our results clearly confirm the presence of strong interactions between this peptide and the GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions affect the bulk properties of the final hybrid hydrogels, which are a function of the peptide concentration and hydrogel pH. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels. At low peptide concentration, where attractive electrostatic interactions are present, the incorporation of GO result in an increase in the hydrogel G′ while at high peptide concentration, where electrostatic iterations are absent or repulsive, it results in a decrease in G′. At pH 7, after conditioning with cell culture media, electrostatic interactions are removed and the main interactions controlling the final properties of the hydrogels are thought to be hydrophobic. At this pH, G′ was found to increase at all concentrations upon incorporation of GO. Our results also show that the incorporation of GO does not affect their injectability.In order to explore the potential of these new hybrid hydrogels as cell delivery platform for IVD regeneration, NP cells were culture in 3D. Our results clearly show that the incorporation of GO improve cell viability and metabolic activity, in particular the GO-F820 hybrid hydrogel promotes high cell viability and maintain cell metabolic activity over the 7 days of culture. This hydrogel has mechanical properties similar to the NP and was shown to be injectable making it a good candidate as injectable scaffold for the delivery of NP cells in the IVD.


Holes confined in quantum dots have gained considerable interest in the past few years due to their potential as spin qubits. Here we demonstrate two-axis control of a spin 3/2 qubit in natural Ge. The qubit is formed in a hut wire double quantum dot device. The Pauli spin blockade principle allowed us to demonstrate electric dipole spin resonance by applying a radio frequency electric field to one of the electrodes defining the double quantum dot. Coherent hole spin oscillations with Rabi frequencies reaching 140 MHz are demonstrated and dephasing times of 130 ns are measured. The reported results emphasize the potential of Ge as a platform for fast and electrically tunable hole spin qubit devices. Germanium’s electronic structure and large, tunable spin-orbit coupling makes it a good material for constructing hole-based quantum devices. Here the authors demonstrate the fabrication and two-axis control of a hole spin qubit in a germanium double quantum dot. One of the key ingredients for realizing such circuits will be a strong coupling of spins to superconducting resonators4. This has been recently achieved for Si by dressing electrons with synthetic spin-orbit coupling6. Ge, on the other hand, with its strong and tunable spin-orbit coupling10 could be an alternative material for the realization of scalable qubits.Spins in isotopically purified Si have shown record coherence times13. Here, we study hut wires (HWs), Ge nanowires monolithically grown on Si. They have a triangular cross section with a width of about 20 nm and a height of about 2 nm18. As has been very recently reported16, holes localized in Ge HWs are of almost pure heavy-hole (HH) character making them thus an appealing system for hosting hole qubits with long dephasing times19.In the past few years several studies have addressed the properties of Ge/Si core/shell nanowires and Ge self-assembled nanocrystals20 mechanism and the electric dipole spin resonance (EDSR) technique in order to demonstrate the addressability of single holes. By varying the duration of the radio frequency (RF) burst, Rabi oscillations with frequencies higher than 100 MHz are observed. Finally, Ramsey fringes-like measurements reveal dephasing times of 130 ns, twice the dephasing time reported for holes in Si21.In this work we demonstrate the ability to capture holes in double quantum dots (DQDs) fabricated from Ge HWs. We make use of the Pauli spin blockade (PSB)SD and measuring the resulting current I to test our DQD device. Thereby, we vary the voltages VG1 and VG2 tuning the electrochemical potentials of our dots. The low temperature measurements reveal the formation of quantum dots (QDs) below the deposited top gates, presumably due to strain22. Therefore, the two gates (with voltages VG1 and VG2) are already sufficient to fully define and operate the DQD. The stability diagram of the DQD device A showing characteristic bias triangles23 is depicted in Fig. 24 of about 1 aF the triangles are merged already at relatively low bias voltages. The base of the triangle marks current flowing through the ground states. The parallel lines within the triangles denote transport through excited states. Energy level separations of up to ~1 meV and a relative lever arm ΔVG1/ΔVG2 = 0.7 are observed23. Since the two top gates G1 and G2 are very close to the HW, a relatively strong coupling is obtained, leading to large gate couplings of α1 = 0.62 eVV−1 and α2 = 0.43 eVV−124.A schematic and a scanning electron micrograph of a typical DQD device are shown in Fig. 26. PSB occurs in a  →  or an equivalent  →  charge configuration  can cause oscillations in the position of the confined hole wave function  of the resonance peak at a certain RF power30. For HH states it has been predicted that the direction of the applied magnetic field has a strong influence on the dephasing times19. Indeed, optical measurements of hole spins confined in GaAs self-assembled QDs have shown very long dephasing times31. In order to obtain such longer dephasing times, the external magnetic field needs to be aligned perpendicular to the direction of the Overhauser field, which for HH states is perpendicular to the growth plane19. By repeating the EDSR measurement for an in-plane magnetic field and an RF power of −14 dBm  on 4-inch intrinsic Si001) wafers in two different systems. Two different wafers were used for the realization of the three devices A, B and C, which differ in several growth parameters, as can be seen in Table  wafers iDevice A and C (device B) were fabricated using a 100 kV (20 kV) e-beam lithography system. For the source and drain contacts 25 nm Pt (5/25 nm Pd/Al) were deposited. The gates (3/25 nm Ti/Pt) were evaporated onto an about 6–8 nm hafnium oxide layer. The oxide was created by atomic layer deposition of 80 cycles of Tetrakis(dimethylamido)hafnium (Tetrakis(ethylmethylamido)hafnium)/80 cycles of water at 130 °C (150 °C).9 was used for the current measurements. All low-frequency lines are filtered at three stages. Pi filters are used at room temperature, LC filters at the mixing chamber stage and a single stage RC filters on the printed circuit board (PCB) on which the sample was mounted. High-frequency signals were applied to the gate G1 through a 20 GHz bandwidth coaxial line and attenuated by 44 dB from attenuators distributed at the different stages of the dilution refrigerator. To apply periodic square voltage pulses for fast switching between the CB and the PSB regime, one channel of an arbitrary wave generator Tektronix AWG5014C was used. Two other channels were connected to the I and Q inputs of the R&S SMW200A vector signal generator for the RF burst creation. Both the RF and the voltage square pulses were merged by a diplexer before entering the dilution refrigerator. Such a signal was further merged with a DC signal via a bias tee positioned on the PCB.All the measurements were done with low-noise electronics and in a He-3/He-4 dilution refrigerator at a base temperature of ~40 mK. A current to voltage amplifier with a gain of 10Supplementary Information


Adding cyclic short sustained inflations (sigh) to assisted ventilation yields optimizes lung recruitment, decreases heterogeneity and reduces inspiratory effort in patients with acute hypoxemic respiratory failure (AHRF). These findings suggest that adding sigh to pressure support ventilation (PSV) might decrease the risk of lung injury, shorten weaning and improve clinical outcomes. Thus, we conceived a pilot trial to test the feasibility of adding sigh to PSV (the PROTECTION study).2O. Standardized protocols will guide ventilation settings, switch back to controlled ventilation, use of rescue treatments, performance of spontaneous breathing trial, extubation and reintubation. The primary endpoint of the study will be to verify the feasibility of PSV + sigh evaluated through reduction of failure to remain on assisted ventilation during the first 28 days in the PSV + sigh group versus standard PSV (15 vs. 22%). Failure will be defined by switch back to controlled ventilation for more than 24 h or use of rescue treatments or reintubation within 48 h from elective extubation. Setting the power to 80% and first-risk order to 5%, the computed size of the trial is 129 patients per arm.PROTECTION is an international randomized controlled trial that will be conducted in 23 intensive care units (ICUs). Patients with AHRF who have been intubated from 24 h to 7 days and undergoing PSV from 4 to 24 h will be enrolled. All patients will first undergo a 30-min sigh test by adding sigh to clinical PSV for 30 min to identify early oxygenation responders. Then, patients will be randomized to PSV or PSV + sigh until extubation, ICU discharge, death or day 28. Sigh will be delivered as a 3-s pressure control breath delivered once per minute at 30 cmHPROTECTION is a pilot randomized controlled trial testing the feasibility of adding sigh to PSV. If positive, it will provide physicians with an effective addition to standard PSV for lung protection, able to reduce failure of assisted ventilation. PROTECTION will provide the basis for a future larger trial aimed at verifying the impact of PSV + sigh on 28-day survival and ventilator-free days.NCT03201263. Registered on 28 June 2017.ClinicalTrials.gov, The online version of this article (10.1186/s13063-018-2828-8) contains supplementary material, which is available to authorized users. Ever si lacking . Around  lacking  and earlFrom a pathophysiological standpoint, during assisted ventilation, derecruitment and lung heterogenity may facilitate the generation of high regional barotrauma and volutrauma , thus inAs prospective clinical trials including long-term use of sigh during assisted ventilation have not been performed yet, we conceived a pilot randomized controlled trial (RCT) to verify the clinical feasibility of the addition of sigh to PSV (PSV + sigh) in comparison to standard PSV. Therefore, the objective of the present trial is to determine whether ventilation by PSV + sigh can be applied early and safely in intubated patients with AHRF and successfully maintained until extubation. Specifically, the primary endpoint is to evaluate whether PSV + sigh, as compared to PSV, is associated with reduced failure of assisted ventilation, with failure defined as (1) prolonged switch to controlled ventilation; (2) use of rescue therapies for refractory hypoxia or (3) early need for reintubation after elective extubation.www.clinicaltrials.gov with code NCT03201263 in June 2017. The Standard protocol items: recommendation for interventional trials (SPIRIT) checklist can be found in Additional file The PROTECTION (Pressure support ventilation + sigh in acute hypoxemic respiratory failure patients) trial is an investigator-initiated, international, multicenter, parallel randomized controlled two-arm trial that will be performed in intubated and ventilated patients with AHRF, who are admitted to the intensive care unit (ICU). The study will be conducted in adherence to the principles of the World Medical Association’s Declaration of Helsinki and in accordance with the Medical Research Involving Human Subjects Act (WMO). Ethics approval will be sought from each participating institution before starting enrollment and consent will be obtained for each patient following local regulations. The Institutional Review Board of the coordinating center approved the protcol on 12 June 2017 under reference number 318_2017bis. The trial was registered at The PROTECTION trial will be performed in the ICUs of 23 centers around the world . The coordinating center will be Policlinico Maggiore Hospital in Milan, Italy.2)/fraction of inspired oxygen (FiO2) ratio ≤ 300 mmHg  and FiO2 values); clinical PEEP ≥ 5 cmH2O and stable Richmond Agitation-Sedation Scale (RASS) value of − 2 to 0. Exclusion criteria will be clinical PEEP ≥ 15 cmH2O; partial pressure of arterial carbon dioxide (PaCO2) > 60 mmHg; arterial pH < 7.30; PaO2/FiO2 ratio ≤ 100 mmHg ; age < 18 years; presence of central nervous system or neuromuscular disorders; history of severe chronic obstructive pulmonary disease or fibrosis; AHRF fully explained by cardiac failure or fluid overload; evidence of active air leak from the lung ; cardiovascular instability  < 90 mmHg despite vasopressors); clinical suspicion of elevated intracranial pressure; extracorporeal support; moribund status and refusal by the attending physician.Consecutive patients admitted with AHRF to each participating ICU, who have been intubated and switched to PSV between 1 and 7 days after intubation will be screened between 4 and 24 h from the start of PSV. Further inclusion criteria will be partial arterial pressure of oxygen  for exclusion will be registered.The Consolidated Standards of Reporting Trials (CONSORT) diagram of PROTECTION trial is presented in Fig. 2 will be titrated to obtain pulse arterial oxygen saturation (SpO2) of 90–96% maintaining clinical PSV settings including PEEP; then, all patients will undergo a clinical test of PSV + sigh to assess the prevalence of improved oxygenation in sigh responders vs. non-responders. To this end, we will first record the SpO2/FiO2 ratio at the start of the test; then, 30 min after the introduction of sigh as the cyclic pressure control phase set at 30 cmH2O for 3 s once per minute [2/FiO2 will be collected again to quantify the number of patients in whom this ratio increased  [2O with a 3-s inspiratory time and then a 57-s expiratory time [To explore the possibility of implementing predictive enrichment in a larger future RCT , we planr minute , SpO2/Fionders”) . To deliory time .At the end of the sigh test, patients will be randomized through an online automatic centralized and computerized system to one of the two study groups (1:1 ratio): PSV or PSV + sigh. Due to the nature of the intervention, blinding will not be feasible.2 will be left as selected before the pre-randomization sigh test, while PEEP will be left as clinically set.After randomization, clinicians will set PSV to meet the following targets: tidal volume (Vt) of 6–8 mL/kg of predicted body weight (PBW), with respiratory rate (RR) 20–35 bpm. FiO2O for 3 s delivered once per minute  [2 will be left as selected before the pre-randomization sigh test with clinical PEEP.PSV will be set with the same protective targets of the PSV group (see above) but cyclic pressure control phase at 30 cmH., sigh)  will be 2O if Vt > 8 mL/kg PBW and/or RR < 20PSV support will be decreased by 2 cmH2O if Vt < 6 mL/kg PBW and/or RR > 35 and/or in the presence of respiratory distress PSV support will be increased by 2 cmH2 will be increased by 2 cmH2O and 0.1 if SpO2 is < 90%PEEP and then FiO2 and then PEEP will be decreased by 0.1 and 2 cmH2O if SpO2 is > 96%.FiOIn both groups, PSV will be adjusted, at least every 8 h, as follows:The PSV group will be treated by protective PSV settings until day 28 or death or performance of a spontaneous breathing trial (SBT); the PSV + sigh group will be treated by protective PSV settings with the addition of sigh until day 28 or death or performance of a spontaneous breathing trial (SBT). Sigh settings will be left unchanged until day 28, death or the SBT.2O; PEEP ≥ 15 cmH2O; unstable hemodynamic status (SBP < 90 mmHg with vasoactive drug); active cardiac ischemia (dynamic ST changes on cardiac monitor or electrocardiogram); unstable arrhythmias; uncontrolled hypertension (SBP > 180 mmHg); abrupt decrease in the level of consciousness (RASS <− 3); dangerous agitation (RASS > + 2); pH < 7.30; PaO2/FiO2 ratio ≤ 100 mmHg; necessity to perform diagnostic test  or bronchoscopy).In both groups, switch to protective controlled ventilation will be indicated if patient develops at least one of the following conditions: PSV support > 20 cmH2. Controlled ventilation will be thereafter adjusted according to clinical evolution. Patients switched to controlled ventilation will be reassessed at least every 8 h and they will be switched back to PSV or PSV + sigh (to maintain study group assignment) targeting the aforementioned settings and adjustments as soon as all the following conditions are met: patient is able to trigger ventilator breaths; PaO2/FiO2 > 100 mmHg; PEEP < 15 cmH2O; pH ≥ 7.3; stable hemodynamic status with stable or decreasing doses of vasopressors for ≥ 6 h.Controlled ventilation will be set on volume mode with Vt 6–8 mL/kg PBW, RR to control pH, unchanged PEEP and FiO2 ≤ 90%) it will be crucial to exclude hemodynamic impairment as a possible cause. Also, airway obstruction and ventilator malfunction must be ruled out. Provided those factors are excluded, a rescue step-up strategy will be allowed: institution of protective controlled mechanical ventilation (see above for settings) and performance of recruitment maneuvers at 40–50 cmH2O, PEEP ≥ 15 cmH2O, prone positioning and inhaled nitric oxide, extracorporeal membrane oxygenation. Patients undergoing controlled ventilation and rescue treatments will be reassessed at least every 8 h and switched back to PSV or PSV + sigh (to maintain study group assignment) with the aforementioned settings and adjustments as soon as all the following conditions are met: patient can trigger ventilator breaths; PaO2/FiO2 > 100 mmHg; PEEP < 15 cmH2O; pH ≥ 7.3 and stable hemodynamic status with stable or decreasing doses of vasopressors for ≥ 6 h.In the case of desaturation  respiratory rate > 35 bpm; heart rate > 140 bpm; SBP > 180 or < 80 mmHg; marked complaint of dyspnea; increased somnolence with elevated PaCO2 and/or pH < 7.3; active cardiac ischemia (dynamic ST changes on cardiac monitor or electrocardiogram); abrupt decrease in the level of consciousness with RASS <− 3 or if cough is not strong enough to clear secretions.The SBT will last at least 60 min with a combination of PEEP 0–5 cm HPatients who fail the SBT will be switched back to PSV or PSV + sigh (to maintain study group assignment) and clinical criteria will be checked again to repeat the procedure after at least 6 h.Patients who pass the SBT will be extubated or, in the presence of tracheostomy, mechanical ventilation will be discontinued. If a patient is reintubated or mechanically ventilated through a tracheostomy again within 48 h, PSV or PSV + sigh (to maintain study group assignment) will be restored. If a patient remains extubated or separated from the ventilator for > 48 h data collection only will continue.2 < 90% and/or RR > 35 bpm despite non-invasive ventilation; decreased level of consciousness impairing ability to protect airway; hemoptysis or hematemesis impairing ability to protect airway; abundant secretions that cannot be effectively cleared or are associated with lobar collapse, acidosis, hypoxemia or change in mental status; surgical/invasive procedure requiring sedation/anesthesia +/− neuromuscular blockade such that patient is no longer be able to sustain unassisted breathing or hemodynamic instability with SBP < 80 mmHg despite vasoactive drugs.After elective extubation, reintubation should be promptly performed if at least one of the following criteria is present: cardiac arrest; respiratory arrest (respiratory pauses with loss of consciousness or gasping for air); respiratory failure with SpOIn all patients, standard of care for intubated patients with hypoxemic acute respiratory failure  will be granted throughout the whole ICU stay in accordance to local protocols.Enrolled patients will be observed until day 28, ICU discharge or death, whichever comes first.2O, prone positioning, inhaled nitric oxide, extracorporeal membrane oxygenation or reintubation within 48 h from extubation following one of the aforementioned reasons.The primary endpoint will be to verify the clinical feasibility of PSV + sigh vs. PSV. Feasibility will be assessed by comparing the number of patients in each group experiencing at least one of the following failure criteria : switch The study will also provide a preliminary evaluation of the safety of PSV + sigh by comparing the incidence of the following adverse events in the two study groups: hemodynamic instability with hypotension  despite vasoactive drugs; arrhythmias with heart rate < 40 or > 140 bpm; radiographic evidence of barotrauma  and long-term  sigh responders defined by improved oxygenation. Finally, it will assess the following clinical outcomes: 28-day mortality; ventilator-free days; PSV level, PEEP value and oxygenation index on day 1–3; number of days on assisted ventilation until day 28; use of rescue treatments; number of quadrants involved on chest x-ray on day 1–5; ICU and hospital length of stay and tracheostomies. The listed variables will be analyzed by comparing the two study groups considering, first, the whole study population and, then, only patients who improved oxygenation during the pre-randomization sigh test (sigh responders).2, PSV level).At enrollment, before the sigh test we will anonymously collect patients’ demographic information , past  and recent  medical history, severity of lung injury ) and of systemic involvement  and finally, ventilation data , heart rate, arterial blood pressure, central venous pressure, cumulative fluid balance, patient’s comfort assessed using a visual analog scale (VAS), RASS value, SOFA score and dosage of sedative agents and vaso-active drugs.From day 1  to day 28 or death or discharge from the ICU, the following data will be collected every day between 6:00 and 10:00 in the morning: arterial SpO2O, prone positioning, inhaled nitric oxide, extracorporeal membrane oxygenation), tracheostomy, switch from the allocated treatment to the other study arm for ≥ 24 h, switch to controlled ventilation for ≥ 24 h, reason for switch to controlled ventilation, adverse events , SBT performance and reason for SBT failure, extubation or separation from mechanical ventilation, reintubation and reason for reintubation with time. We will collect data on day 28 for all enrolled patients on mortality, extubation that occurred outside the ICU and readmission to ICU.Moreover, each day we will collect information on use of rescue treatments . To assess the feasibility of PSV + sigh as compared to PSV , we will compute the proportion of patients experiencing at least one of the following events in each arm: switch to controlled ventilation for ≥ 24 h consecutively; use of PEEP ≥ 15 cmH2O, prone positioning, inhaled nitric oxide, extracorporeal membrane oxygenation or reintubation within 48 h from extubation.t test or Wilcoxon signed rank test for continuous variables. Time to death or extubation will be analyzed using Kaplan-Meier survival curves. Adjustement for potential confounding factors will be evaluated using appropriated regression models.We will compare the two proportions using the one-tailed non-inferiority test for proportions with a 5% tolerance. We will use 0.05 as the significance level. The same method will be adopted to evaluate the safety of PSV + sigh, which is a secondary endpoint. Prevalence of sigh responders in the two arms will be compared using the chi-squared or Fisher’s exact test. Ventilator-free days will be calculated as the difference between the duration of follow up (up to 28 days) and the number of days of intubation before successful extubation or separation from mechanical ventilation for tracheostomized patients. For all the secondary endpoints, we will use the chi-squared or Fisher’s exact test in the case of binary variables or the The steering committee is composed of two principal investigators (TM and LB) plus seven international experts on ventilation who contributed to the design and revision of the study protocol. Each participating center will indicate a local investigator in charge of the study. The principal investigators are responsible for administrative management and communication with the local investigators and for helping the participating clinical sites in trial management, record keeping and data management. The local investigators provide structural and scientific leadership. They guarantee the integrity of data collection and ensure timely completion of the case report forms.https://sighprotection.digitalcrf.eu), with protected individual access for each participating center. Patient data will be anonymous and coded according to a number. The eCRF includes tools to promote data quality, such as range checks for data values. Data monitoring will be performed by means of queries on the database done by statisticians and analyzed to identify abnormalities and inconsistencies.Collected data will be entered in an electronic case report form (eCRF) available online at a dedicated website  to standard PSV for lung protection able to favor assisted ventilation.Mortality remains considerably high (around 40%) in intubated patients with AHRF . The cli2 (FiO2) [2; decreases the tidal volumes by decreasing the patient’s inspiratory drive; increases the EELV by regional alveolar recruitment; decreases regional heterogeneity of lung parenchyma; decreases patients’ inspiratory efforts limiting transpulmonary pressure and improves regional compliance [Key determinants of VILI in patients with AHRF undergoing assisted ventilation include elevated ventilation pressures and inspired fraction of O2 (FiO2) ; heterog2 (FiO2)  and stre2 (FiO2) . To thismpliance –16, 26. mpliance . In concWe anticipate the PROTECTION trial to be highly feasible, as study procedures are mainly limited to ventilation settings and do not include complex interventions, with limited burden on daily activities. PSV + sigh is an easy-to-implement ventilation mode and, for the present study, we will use high-performance ICU ventilators already available in each clinical unit, dramatically decreasing the study costs.Even though PROTECTION might be a largely pragmatic study, the protocol specifically suggests adjustments of ventilation settings and weaning procedures. Moreover, criteria for switch to controlled ventilation, use of rescue therapies and reintubation are pre-defined by the study protocol. Since return to controlled mechanical ventilation can occur due to reasons not related to failure of assisted ventilation , the protocol stresses the importance of periodic reassessment (at least every 8 h) to restore assisted ventilation as soon as clinical conditions permit. To this end, failure of assisted ventilation is defined when controlled ventilation is maintained for at least 24 h. In summary, the PROTECTION study protocol limits discretional settings and clinical decisions that might interfere with the primary outcome.One important limitation of the PROTECTION trial is that blinding is not possible due to the nature of the intervention, and this could induce bias. However, all the processes that can influence the primary outcome are regulated by the protocol and all the analyses will be performed in a blinded fashion. Second, even though a lower sigh rate might be regarded as more physiological , in the In conclusion, PROTECTION is a pilot randomized controlled trial to test the feasibility of long-term addition of sigh to PSV. Its results could provide a ready-to-use treatment enhancing successful application of assisted ventilation. Moreover, PROTECTION will be the basis for planning a future larger trial investigating the use of sigh as a strategy to improve hard clinical outcomes in patients with AHRF undergoing assisted ventilation through enhanced lung protection.The PROTECTION trial is currently recruiting patients. The protocol was approved by the coordinating center Institutional Review Board on 12 June 2017. The first patient was enrolled on 20 December 2017. Sixteen centers already received approval from the local ethical committees and are actively recruiting patients. Recruitment is expected to be completed in November 2019.Additional file 1:SPIRIT 2013 checklist: Recommended items to address in a clinical trial protocol and related documents. (DOC 121 kb)