A sheet resistance value as low as 100 Ω/□ is reported for a 3-layer stacked graphene:N film.Nitrogen substitutional doping in the π-basal plane of graphene has been used to modulate the material properties and in particular the transition from hole to electron conduction, thus enlarging the field of potential applications. Depending on the doping procedure, nitrogen moieties mainly include graphitic-N, combined with pyrrolic-N and pyridinic-N. However, pyridine and pyrrole configurations of nitrogen are predominantly introduced in monolayer graphene:N lattice as prepared by CVD. In this study, we investigate the possibility of employing pyridinic-nitrogen as a reactive site as well as activate a reactive center at the adjacent carbon atoms in the functionalized C–N bonds, for additional post reaction like oxidation. Furthermore, the photocatalytic activity of the graphene:N surface in the production of singlet oxygen ( Nowadays, however, the good reputation of graphene as a promising material for various applications11, is also due to the possibility to modulate its chemical and physical properties through chemical functionalization and, in particular, doping16.Since the publication on graphene by Geim and Novoselov in 2004, and after more than one hundred thousand publications, there is no doubt that the success of graphene lies in its fascinating properties such as one-atom thickness, transparency, thermally and electrically conductivity, high mobility of electrons and flexibility14. Specifically, when nitrogen atoms are substitutionally incorporated into the π-lattice of the carbon ring, they add resonant electrons, which provide n-doped graphene sheets. Nevertheless, the landscape of the nitrogen functional forms possibly present in nitrogen-doped graphenic materials is very rich19. In CVD nitrogen-doped graphene, depending on the doping procedure, the nitrogen moieties include graphitic-N, combined with pyrrolic and pyridinic nitrogen18. Among these bonding configurations, graphitic-N induces an n-type doping effect, whereas pyrrolic-N and pyridinic-N may result in either weak n-type or p-type doping20. To date, there are quite a number of studies focused on the growth of N-doped graphene by direct or post treatment processes, which show the capability of tuning graphene chemical and physical properties25. However, none of them has reported that the nitrogen functionalities inserted in the basal plane of the carbon lattice may be further modified to further improve the conductivity of the layer. Thus, from a chemical standpoint, N-doped graphene is an appealing graphene type, because nitrogenous functionalities and, in particular, pyridine nitrogen can be a reactive center as well as activate a reactive center at the adjacent carbon atoms in the functionalized C-N bonds for additional post reaction like oxidation27. In addition, the presence of these N-functionalities in the graphene structure, besides changing the carrier density , gives an interesting catalytic activity of the graphene surface, such as the well investigated activated generation of reactive oxygen species (ROS)31. Among these new “catalytic” capabilities, the production of singlet oxygen (1O2) by both (i) activating the dissociation of peroxydisulfate28 and (ii) the photosensitize excitation of molecular oxygen30 is of particular interest. In fact, singlet oxygen ), being a non-radical reactive oxidizing specie, does not intervene on the conjugated double bond as it happens for radical species  and shows higher reactive selectivity also because of its electrophilic nature.Graphene doping can be performed through two methodologies: (i) surface chemical functionalization, both covalent  and non-covalent  and (ii) substitutional doping in the hexagonal lattice of the graphene with heteroatoms, mainly nitrogen and boron, capable to control the n-type and p-type character of the graphene, respectively2 graphene network. As for the CVD growth of N-doped graphene on copper foil, the major challenges are how to control the insertion of the different N-functionalities. Specifically, in order to realize an increase in p-type doping, we need to minimize the graphitic nitrogen in favour of pyrrolic and, even better, pyridinic nitrogen; this is because each nitrogen substituting a graphitic carbon increases the number of conjugated π-electrons, thus improving the n-type conductivity. It is important to underline that N-graphitic atom, like carbon atom in pure graphene, has a low reactivity. Among the different methodologies proposed in literature, we opted for the CVD growth of N-functionalized graphene (graphene:N) via N atoms embedded into Cu substrate and used as a nitrogen solid source24. We show that this approach allows obtaining N-graphene layers with small grain size and therefore a high density of structural defects, such as grain boundaries and carbon vacancies, which are the preferred sites for the inclusion of pyridinic- and pyrrolic-nitrogen. From this point of view, in other words, graphene grain-boundary defects do not damage the material properties but rather improve them32. The post-growth treatment of N-doped graphene to realize chemical modifications at nitrogen reactive sites is performed by photoirradiation with Xe lamp of N-graphene sample in air. The chemistry, involving the interaction of singlet oxygen photo-catalytically activated on N-graphene, is highly selective towards pyridine active sites leaving the basal plane components intact. We show that the pyridine ring almost disappear in favour of pyridine N-oxide and pyridone structures, both having a strong p-doping effect.In this paper, we report on N-doped graphene, grown by CVD, and its subsequent chemical modification to improve the p-type conductivity, taking advantage of the presence of nitrogen functionalities as new active sites for selective reactions, without affecting the spare there possibilities to functionalize the aryl carbon in such a way as to generate new electron-withdrawing groups, thus improving p-doping? The outcomes arising from the present study confirm positively the hypothesis we set out. Figure +-doped graphene with some pyridinic groups converted to pyridine N-oxy and pyridone groups.To enhance the performances of graphene employed as a transparent conductor, a uniquely accessible route is to reduce the sheet resistance by operating a chemical doping. Therefore, it is essential to fingerprint the structure of the graphene layer and to understand the possible chemical modification for achieving high efficiency and stability in the charge transport process. For the nitrogen doped graphene (G:N), since the presence of C-N hetero-bonds into the aromatic platform, the main question is: 3 defects in the C-sp2 basal-plane. Here, it is important to underline that Pyridinic-N and Pyrrolic-N occur mainly at the boundary of C-vacancy sites or, even more, at the edge of the graphene grains19. Therefore, since the Pyridinic-N is responsible for the activation of reactive sites leading to p+-doping, a higher defects density  in G:N introduced during the growth allows for higher p-doping33.Looking ahead, from the side of the growth and the doping chemistries, both Raman and XPS data confirm (provided evidence) that graphene (G:N) incorporates N atoms during the growth and contains the “new” N-functionalities without introducing significantly C-sp35.Figure 13. The size of graphene grains is around 10 µm. Two typical spectra with G (∼1582 cm-1) and 2D (∼2718 cm-1) peaks are observed without apparent D (∼1350 cm-1) peaks indicating the high quality of the graphene layer. The ratio I(2D)/I(G) = 1.8 and the G and 2D peak bandwidths of, respectively, 14 cm-1 and 27 cm-1 are fingerprint of a single layer graphene (blue line). The Raman spectrum with I(2D)/I(G) ∼1 (red line), recorded at the grain centre, is representative of bilayer. Most of the area is covered with single layer graphene, whereas, the presence of the small double layer island in centre of each grain confirms the under-layer nucleation mechanism described by Nie et al.36 The distribution density of double layer islands on graphene can be evidenced by enhanced optical contrast of the microscope image of graphene transferred on 300 nm SiO2/Si reported in Figure S1 together with the relative Raman analysis.Optical microscopy and Scanning Raman spectroscopy were used for quality mapping the graphene layer Fig. c. The mi13. Besides the small contribute of carbon solubilized in copper foil (peak 1 at 283.6 eV)37, there are no significant changes in the deconvolution going through the three samples. The assignment of the peak deconvolution indicates that the largest proportion of C-functionalities are always associated with presence of sp2 carbon with C = C bonds (284.4 eV) and carbon–oxygen as follows: C–OH (285.2 eV), C–O–C (286.9 eV), C = O (287.9), O = C–O (288.8 eV). The presence of carbon-to-nitrogen bonds adds a new peak at 286.0 eV ascribed to N = C bonds that are located in the π-conjugated graphene structure39. On the other side, significant changes are observed on N1s peak deconvolution.Figure 41, it is recognized the presence of the three typical components of pyridinic , pyrrolic  and graphitic/quaternary  nitrogen bonds40. The peak of graphitic nitrogen (Ngr) is much lower than pyridinic (Np ) and pyrrolic (Npr) nitrogen, thus the n-type doping with two p-electrons at the graphene π-cloud is low. Almost equivalent peak intensities are observed for Np and Npr signals for as grown graphene on copper foil. Following the graphene:N single layer transfer on Si/SiO2 substrate and the subsequent Xe-lamp irradiation, the N1s peak deconvolution needs two additional contributions at 400.7 eV and 402.3 eV assigned to Pyridone (Npd) and Pyridine N-oxide (Nox), respectively38. The appearance of these new N-functionalities takes place at the expense of pyridine nitrogen as evidenced by the strong reduction of the Np energy peak, while Npr peak, and, thus, the amount of pyrrolic nitrogen structures remains unchanged.Specifically, in the pristine graphene:N (G:N) grown on Cu substrate, besides the contribute at high binding energy (404.6 eV) due to adsorbed nitrogen42. The proposed chemistry is outlined in the following overall reactions (Eqs. (1O2). Here, graphene:N surface is the catalyst that activate the hydrolysis of ammonium persulfate for the formation of superoxide radicals. Specifically, mainly pyrrole nitrogen acts on the neighbouring carbon atoms by increasing the charge density, thus generating an adsorption and activation center to form singlet oxygen42.The observed evolution of the N1s peaks can be read in the chemical processes involving the interaction of G:N with singlet oxygen. In fact, the formation of singlet oxygen occurs on pristine G:N during both (a) the solubilisation of cupper foil by ammonium peroxydisulfate and (b) the light irradiation by Xe-lamp. As for the generation of single oxygen by interaction of G:N layer (still on TRT) with ammonium peroxydisulfate during the phase of copper solubilisation, the chemistry has been described in detail in previous studies29 have evaluated the role of graphene functionalization in determining the ROS generation. In particular, they underline that the generation of 1O2 is driven by graphene photoexcitation with energy larger than the excitation energy of O2-ground state (3Σg-) to 1O2 (1Δg), which is of approximately 0.97 eV. This photosensitization mechanism has been reported as the most common means of singlet oxygen generation43. It is detailed that the energy transfer to O2 (3Σg-) from an excited state of a sensitizer, which is formed by the light absorption of light in a specific wavelength region, results in the formation of both excited states O2(1Σg-) and 1O2 (1Δg). And, followed by the very fast spin-allowed 1Σg- → 1Δg deactivation that results in the complete formation of 1O2 (1Δg). Moreover, it has been demonstrated that the graphene doping by heteroatoms, e.g., nitrogen, increase the photocatalytic efficiency44. The photochemical production mechanism of singlet oxygen on graphene:N can be depicted as follows:The graphene:N shows its catalytic action also in the photogeneration of reactive oxygen species (ROS) and, in particular, of singlet oxygen by molecular oxygen. In a recent paper, Yao and coworkers1O2-treatment process is carried out also on substrate-transferred graphene:N (glass or SiO2/Si substrates) by irradiation with Xe lamp. The effectiveness of the photocatalytic aerobic oxidation via singlet oxygen has been exploited by many for the realization of selective oxidative processes towards organic and biological molecules30.Thus, the completion of the 46. In particular, since the high lifetime of 1O2 in the gas phase, reducing singlet oxygen is expected to facilitate the first electron transfer in ORR. The general scheme for the complete reduction of O2/1O2 molecules to OH- is as follows:Furthermore, nitrogen-doped graphene is an active catalyst also in the oxygen reduction reaction (ORR), in which again the singlet oxygen can play an important role27, as schematized in Fig. Nevertheless, the same oxidative processes can occur on the reactive sites present on the graphene surface; among these, the pyridine site is important as it can be oxidized simultaneously by aerobic photoirradiation to N-oxy pyridine and pyridineNp at 398.8 eV) is in favour of N-oxy pyridine (Nox at 402.3 eV) and hydroxyl-pyridone (Npd at 400.7 eV). While the other two pyridone-tautomers (carbonyl and zwitterion) with a protonated nitrogen, which resemble a quaternary nitrogen with a very similar XPS peak position to the graphitic nitrogen, contribute to the observed increased of the peak Ngr at 401.5 eV. The scheme emphasizes the role of the delocalization of the positive charge on the aromatic ring in both the oxidation to N-oxide pyridine and the formation of pyridone by the attachment of the OH group to the carbon atom in α-position to the pyridine nitrogen. The C1s XPS spectra shows that the C–OH, peak 3 at 285.5 eV, slightly increases after transferring on substrates. This confirms that carbon atoms close to pyridine nitrogen are the main active sites, among the different doping configurations of nitrogen, capable of further functionalizing graphene in the direction of p-doping27. Thus, the possibility of maximizing the introduction of N heteroatoms with pyridinic structure in the sp2 carbon lattice would maximize the p-doping effect.The observed changes in nitrogen configurations, as assigned in Fig. 2. The observed slow kinetics is consistent with complex formation pathway of singlet oxygen, whose concentration is determined not only from light irradiation  measurement, i.e. the shift of the Dirac point at high positive voltage (VD = 39 Volts) for the light irradiated graphene:N when compared with pristine graphene (VD = 20 Volts). The evaluation of hole carrier mobility has been done using the formula µh = (L/W)·C-1·VD-1·(dID/dVG), where C = 120 µF·m-2 is the gate capacitance of the silicon dioxide dielectric layer (300 nm thick)49. The measured hole mobility of annealed pristine-G and light irradiated graphene:N are nearly equal and reach an average values of about 1350 cm2/(V·sec). Thus, the reduction of sheet resistance in irradiated graphene:N is mainly ascribed to the increase of hole carrier density.The changes in G:N surface structure in the direction of a p-doping have been monitored by Raman spectroscopy and electrical characterization, both sheet resistance (Van der Pauw) and FET measurements. Figure  see Eq.  in the p-1, from 1580 cm-1 to 1604 cm-1; (iii) the G peak asymmetry and Lorentzian-deconvolution in two narrow peaks (G1 at 1596 cm-1 and G2 at 1604 cm-1) after Xe-lamp irradiation; and (iv) the high-energy displacement of the 2D peak that moves of 9 cm-1, from 2675 cm-1 to 2684 cm-1. Similar results are observed from the Raman analysis of graphene:N transferred on glass before and after Xe lamp irradiation  is confirmed by the Raman analysis of single layer graphene on FET device. Figure n Figure .Figure 648. The observed asymmetry of the G-peak indicates the existence of charge inhomogeneity within the laser probe, i.e., on a scale of 1 µm50.The simultaneous occurrence of these four events can be read in the more general picture of a strong increase in p-type doping (increase in the carrier density of holes) as described in the Raman "master paper" on the doping effect by Das et al.49, from these important Raman parameters we can estimate an hole doping density of about 1.3 × 1013 cm-2 for the irradiated graphene:N sample. This value is consistent with the average value of sheet resistance measured for a single layer of G:N after irradiation. In fact, by applying the simple Drude model of electrical conductivity, Rs (Ω/□) = 1/(n·µ·e), where n = 1.3 × 1013 (cm-2), µ = 1350 (cm2V-1 s-1) and e = 1.6 × 10–19 (A·sec), we extract the sheet resistance of 350 (Ω/□) that matches the measured values shown in Fig. According to ref.13. Figure 13.Multilayer graphene samples on Corning glass substrates have been prepared by using the layer-by-layer procedure described in ref.In summary, using a Nitrogen-doped graphene deposited from a direct CVD method, an efficient p-doping effect has been demonstrated through the selective photochemical activation of pyridinic nitrogen to N-oxy pyridine and pyridone. Notably, the sheet resistance of light-irradiated Nitrogen-doped graphene significantly reduces and can even reach a value as low as 100 Ω/□ for a three-layer graphene.3, SOCl2,…) for the covalent doping or it is required to host molecules or particles for the non-covalent doping, the photochemical doping of graphene:N has the advantage to be easily applicable to any combined graphene-semiconductor device architecture. In addition to the capability of doping graphene on any surface device, this light-driven doping strategy has great potential to realize selective-area doping by photolithography mask or laser patterning.Unlike conventional chemical doping, where the graphene layer is submitted to chemical reaction with strong oxidants (HNO2 size) was inserted in a quartz tube of the thermal-furnace CVD reactor. The quartz-tube was evacuated to a vacuum better than 10-3 torr and heated to 100 °C under an N2 gas flow of 200 sccm (0.2 torr) that was maintained for 20 min after the temperature was stabilized. The copper nitridation process was carried out in the nitrogen plasma downstream under the following conditions: N2 flow rate 200 sccm, pressure 0.2 torr, rf (13.56 MHz) power 250 watts, exposure time 30 min. Subsequently, the temperature was raised to 990 °C under an H2 gas flow of 10 sccm (0.05 torr) that was maintained for 20 min after the temperature was stabilized. The annealing process was followed by the graphene growth: CH4 (5 sccm) was added to the gas feed for a growth time of 20 min. After the growth phase, the furnace was moved from the growth-zone to realize the rapid cooling of the graphene/copper foil.The growth of nitrogen-doped graphene occurs in two phases: the plasma nitridation of copper foil and the catalytic CVD-growth. Both processes were carried out in the same quartz tube (i.d.10 cm) equipped with external capacitive-coupled electrodes for the plasma nitridation process and furnace for the CVD growth. The copper foil  using the thermal release tape (TR-tape) procedure. The tape was placed on top of the graphene/copper foil and pressed by the laminator. The copper was etched away in an ammonium peroxydisulfate, ((NH4)2S2O8) solution (20 gr/liter) and the floating sheet of graphene/TR-tape was rinsed in DI water and air-dried. Hot-pressing (at ≈100°C) performed the graphene transfer onto substrates, thus the tape was then peeled off. Multilayer graphene samples on Corning glass for sheet resistance measurements were fabricated by the layer-by-layer transfer procedure described in ref.13.The graphene layer was then transferred to the substrate  with maximum wavelength emission at 470 nm. During light irradiation, the sheet resistance was monitored in real time to ensure the completion of the variation process.. The laser power was kept at 1.0 mW to avoid laser-induced heating effect.Raman spectra of the pristine graphene:N layers and of the photochemical modified layers were recorded with a LabRam HR (Horiba JY) system. Spectra on glass and SiO2/Si were measured at room temperature with 532 nm laser light and captured using 100x objective lens magnification with a focusing laser spot less than 1µm in diameter. Spectra of graphene on copper foil were collected using 473 nm laser2 graphitic component of the C1s spectrum (284.4 eV) was used as internal standard for charging correction. The high-resolution spectra were fitted with mixed Gaussian-Lorentzian peaks after a Shirley background subtraction. The standard deviation in the peak position was ± 0.1 eV.The surface chemical composition of doped samples was investigated by X-ray Photoelectron Spectroscopy (XPS), using a Theta Probe spectrometer (Thermo VG Scientific) equipped with a monochromatic Al Kα X-ray source (1486.6 eV) operated at 15 kV and a spot size of 400 µm, corresponding to a power of 70 W. Survey (0–1200 eV) and high-resolution  spectra were recorded in FAT  mode at pass energy of 150 and 50 eV, respectively. All spectra were acquired at a take-off angle of 37° with respect to the sample surface. A flood gun was used to balance the surface charging. The C1s signal for the spA Keithley 2400 Source Meter was used to monitor the electrical properties of graphene layers. Sheet resistance was measured in the 4-probe Van der Pauw configuration.2 / Si substrates  were used for the field effect measurements. The FET device was fabricated by gold evaporation of the source and drain contacts . Measurements were performed in air and in vacuum (better than 10-3 torr) with a Keithley 2400 analyzer at room temperature. A back-gate bias (Vg) ranging from −80 to +110 Volts was applied on the Si side of the SiO2/Si substrate. Before measurement, the FET device was kept under vacuum overnight and treated by thermal annealing @ 200 °C. This allows to desorb surface species on graphene.Single layer graphene on 300 nm SiOSupplementary Information.


Meanwhile, no evidence of malignancy was found, and pathological manifestations led to the subsequent six months of anti-tuberculosis treatment. Combined with the fact that standard anti-tuberculosis showed no improvement, and the patient’s condition was stabilized by corticosteroid treatment alone, a final diagnosis of sarcoidosis was made by an MDT (multidisciplinary consultation). Reported herein is the first case of a hyper vascularization condition within the non-typical asymmetrical sarcoidosis lesions, which should help to establish that the uptake of 3PRDG2 in sarcoidosis can avoid imaging pitfalls.Sarcoidosis is a multi-system disease of unknown etiology that typically occurs in middle-aged adults, often presenting as the formation of granulomas in various organs, including the lungs. Non-typical pulmonary sarcoidosis is rare, and it isnecessary to distinguish its imaging features from lung cancer and tuberculosis. They may appear as an irregular mass with multiple nodules on thoracic computed tomography (CT). In this case, primary lung cancer was suspected in a 57-year-old papillary thyroid carcinoma patient, as the pulmonary lesions were non-radioiodine avid and progressed shortly afterward. The asymmetrical focal uptake that was demonstrated in integrin receptor imaging (


The present study investigated the intimate partner femicide (IPF) and intimate partner femicide–suicide (IPFS) perpetrators’ individual, relational, and contextual characteristics by analyzing, within the ecological approach, femicide cases that occurred in Italy from 2010 to 2019. On the topic, to date, scant studies examined possible differences between IPF and IPFS risk factors, and no studies have analyzed these factors by adopting an ecological systems model perspective. To this aim, archival research was carried out. Of a total of 1.207 femicides, 409 were IPF, and 227 were IPFS. Perpetrators’ age, level of employment, law enforcement membership, mental and/or physical illnesses, use of psychoactive substances, previous crimes, previous violent relationships, presence of children, previous violence in the couple, inability to accept the end of the relationship, quarrels and conflict, jealousy and the psychophysical illnesses of both authors and victims, as well as the use of firearms and victim’s request for help were analyzed. The results underlined the existence of different risk factors contributing to the IPF perpetrators’ decision to commit suicide such as perpetrators’ age, law enforcement membership, and firearm availability. These findings stress the need for specific risk assessment and management strategies for IPFS perpetrators. Considered the misogynous killing of women by men, motivated by hatred, contempt, pleasure, or a sense of ownership of women, femicide is the most extreme form of gender-based violence leading premature death of women  and is iIntimate partner femicide (IPF) is a pervasive form of violence against women and represents a serious social, criminal justice, and public health issue. It could be defined as “the killings of a woman by her intimate male partner, including current or former legal spouses, common-law partners, or boyfriends”  (p. 78).Previous empirical results highlighted that femicide committed by an intimate current or ex-partner in some cases, is followed by the perpetrator’s suicide ,7,8,9,10Unfortunately, while in recent years data on IPF have increased and have been widely acknowledged ,16,17, dPrevious studies on IPFS perpetrators’ risk factors showed that it is almost always characterized by an intimate male perpetrator and a female victim ,35,36,37At the individual level, existing studies focused on gender, age, race, alcohol/drug use, mental illness, and previous crimes committed by the author ,36,37,41Regarding IPFS perpetrators’ alcohol and drug use, instead, there are contrasting results ,30,36. AContrasting results also emerged regarding the presence or absence of the perpetrators’ illness, only sometimes reported in research ,12,21,41At the relational level, one of the most important predictors of IPFS was prior episodes of IPV: 72.0% were preceded by previous forced sexual intercourse, stalking, strangulation, and abuse during pregnancy . DespiteRegarding age, a study carried out in Ghana on 35 cases of IPFS , found tConcerning the presence of children, having one or more children in the household, who were not fathered by the perpetrator, was more common in IPFS cases , while aAt the contextual level, the most considered factor in IPFS was firearm availability from the perpetrator, ranging across different studies from 47.0% to 92.0% ,40,44,55Considering the devastating effects of these phenomena , to bettThe ecological approach is the most widely used model for understanding IPV and the factors associated with variations in its prevalence, e.g., ,62,63, a(1)Do male IPFS perpetrators, compared with only IPF perpetrators, differ from each other in terms of individual, relational, and contextual risk factors?(2)Are some individual, relational, and contextual risk factors predictive of the perpetrators’ suicide after IPF?In particular, we try to answer the following research questions:The current study was part of broader research on femicides called FATHERS , approved by the Departmental Ethics Committee (protocol nr. 18/2016).For the study, we conducted archival research by consulting online newspapers and collecting all femicide cases in Italy, from January 2010 to December 2019 following the Vienna Declaration definition . The anaWe considered IPFS in each case of IPF followed by the perpetrators’ suicide regardless of the amount of time elapsed between IPF and the suicide. In line with other studies and methodologies, information about IPF and IPFS cases and their characteristics were investigated by consulting databases of local, regional, and national online media and newspapers; those were the most suitable sources for collecting our data of interest and were able to provide us with as much information as we needed on these phenomena ,6,65, es. A total of three thousand ninety-two online articles were conferred using keywords . Furthermore, the authors monitored each IPF/IPFS case over time to detect any possible missing and additional information. From 1.207 femicides in Italy in the last decade, 636 were included and considered for further analyses as identified as IPF or IPFS cases.During data collection, every effort was made to record additional information about victims and perpetrators’ individual, relational, and contextual risk factors, the homicide and/or the subsequent suicide, and consulting NGO reports and the 27ora website Geographical data .Characteristics of the victims .Characteristics of the author .Characteristics related to the author/victim relationship .Criminological characteristics .For the present study, the following set of variables was analyzed:The perpetrators’ individual-level risk factors included socio-demographic information such as gender, age, level of employment , law enforcement membership, mental and physical illness, alcohol/drug use, previous crimes, and violent relationships.Among the relational risk factors that concern both the perpetrators and the victims were included: perpetrator-victim age gap, presence/absence of sons and/or daughters both in common and not in common, previous violence in the couple, inability to accept the end of the relationship, quarrels and conflict, jealousy and mental and/or physical illness both of perpetrators and victims.Finally, the contextual-level risk factors included the previous request for help from the victims and the use of a firearm as a weapon to commit IPF/IPFS.Data have been collected and codified according to a Fatality Review Protocol , developp < 0.05) was corrected according to the Bonferroni procedure in Step 1, Step 2, and Step 3.The data collected within the database were analyzed using the SPSS statistical package . Descriptive statistics were carried out to assess IPF/IPFS prevalence and characteristics. Then, we compared individual, relational, and contextual risk factors to investigate their possible role in some perpetrators’ decision to commit suicide after IPF by using odds ratios (ORs) because they are not influenced by sample size  ,68. FurtOf a total of 1.207 femicides that occurred in Italy in the last decade, 707 were IPF. A total of 636 cases were included in our sample, as no information about the perpetrators’ suicide intentions was found for the remaining 71 cases see .The perpetrator’s suicide followed 35.7% of IPF cases. In 88.5% of cases, perpetrators committed suicide within 24 h after the IPF. In contrast, in the remaining part of IPFS, perpetrators committed suicide in a time-lapse ranging from one day to seven months after IPF.Perpetrators’ age ranged from 18 to 97 years , while victims were aged between 16 and 93 years , 75.8% were older than their victims. On average male offenders were 7.04 (SD = 5.49) years older than their victims.Concerning IPF victims’ nationality, 77.4% were Italian. Regarding their occupational level, 27.2% resulted unemployed or retired, 27.0% had a low specialization level , and 4.2% had a high specialization level. A total of 9.3% of victims of IPF were affected by mental and/or physical illness.A total of 35.2% of perpetrators were unemployed or retired, 50.0% had low specialization occupations , and 9.7% were law enforcement officers. A total of 81.4% of perpetrators were Italian, 17.6% committed previous crimes, 12.6% used psychotropic substances such as alcohol, cocaine, or marijuana, and 8.8% of perpetrators were affected by a mental and/or physical illness. In 1.6% of IPF/IPFS cases, both the perpetrator and the victim suffered from a mental and/or physical illness.Regarding the characteristics of the relationship, in 84.3% of IPF cases, the perpetrator was the victim’s husband or ex-husband; in 57.7% of IPF cases, the perpetrator had a child in common with the victim, while in 21.5% of the cases, the children were only the victims. The most common motives for IPF were the perpetrator’s inability to accept the end of the relationship (35.7%) and jealousy (19.5%).A total of 38.5% of perpetrators had already been violent against the victim, and 29.6% used a firearm to commit IPF.We examined the possible existence of significant differences in terms of individual, relational, and contextual risk factors across IPF and IPFS perpetrators see .p < 0.001]. Respectively, 9.7% and 6.2% of suicide perpetrators used substances and committed previous crimes. However, perpetrators’ prior use of substances and/or involvement in criminal behaviors seem to be protective factors for suicide. Additionally, the history of violent relationships was a predictive risk factor only for IPF.At the individual level, the results showed that, respectively, 43.3% of low-specialized workers, 39.0% of highly specialized professionals, and 16.7% of law enforcement committed suicide after IPF. Only being part of law enforcement was a significant predictive factor for suicide after IPF . Among motives for IPF, the perpetrator’s and/or the victim’s mental and/or physical illness significantly increased the perpetrators’ likelihood of suicide .At the relational level, 64.8% of perpetrators with sons/daughters in common with the victim committed suicide after killing the women. To have sons/daughters in common was found to be a significant risk factor for perpetrators’ suicide after homicide .Concerning the contextual level, 55.5% of perpetrators used a weapon as a firearm for IPFS, compared to 15.2% for IPF. The use of a gun emerged as a significant predictive risk factor IPFS [OR = 6.98, 95% CI (4.79–10.17), A hierarchical logistic regression analysis was conducted to establish the predictive role of some individual, relational, and contextual risk factors in the IPF perpetrators’ decision to commit suicide.p < 0.01), being a member of law enforcement , and with suffering mental and/or physical illness . Interestingly, perpetrators suicide was negatively associated to substances use , to previous involvement in criminal behaviors  and to previous violent intimate relationships . At the relational level, perpetrators’ suicide emerged to be significantly associated to being older than the victims , having children in common , having children not in common  and with the presence of perpetrator and/or victim mental and/or physical illness (r = 0.20. p < 0.01). On the contrary, perpetrators’ jealousy  and quarrels and conflicts  were negatively associated to suicide after IPF. Regarding the contextual level risk factors, only using a firearm as a weapon to commit IPF was significantly associated with suicide .First, simple correlations were calculated between different risk factors and perpetrators’ suicide after IPF to analyze the relationship between variables. Regarding individual-level risk factors, the perpetrators’ suicide after IPF was found to be significantly associated with perpetrators’ age  and law enforcement membership were statistically significant individual risk factors for perpetrators’ suicide after IPF. In the second step, relational level risk factors were entered, significantly increasing the variance explained, through only quarrels and conflicts and jealousy were negatively associated with the IPF perpetrators’ suicide. In the third step, using a firearm to commit IPF was entered, indicating that holding and using a gun to kill the victim, being older, and being a law enforcement member were significant risk factors that predict IPFS. Additionally, quarrels/conflicts and jealousy were significantly and negatively associated with the IPF perpetrators’ suicide. The full model explained 26.0% of the total variance of the IPF perpetrators’ suicide.Intimate partner femicide is a widespread phenomenon worldwide , represeIn some cases, it could be followed by the perpetrator’s suicide ,32. HoweTo our knowledge, no studies analyzed and compared the IPF and IPFS perpetrators’ risk factors according to the ecological system theory ,60,61. STo this aim, we carried out an archival study on IPF in Italy in the last decade, of 636 cases of IPF, 227 were IPFS (35.7%).Following our first research question, the comparison between IPF and IPFS perpetrators’ risk factors highlighted, in line with prior studies, that using illicit drugs ,44,48,49Consistent with other studies on IPFS, the existence of a significant age gap between the perpetrator and the victim (7.04 years more for the perpetrator on average) ,12,37 anLikewise, as underlined by a previous study , no prioAmong motives for both IPF and IPFS, jealousy, quarrels and conflicts, and incapability to accept the end of the relationship were quite spread (even if they have not been found as risk factors for themselves) ,35,37,52Furthermore, it would seem that only the presence of the perpetrator’ and/or the victim’s psychophysical illnesses ,37,41,42About the second research question, the hierarchical logistic regression model results confirmed the perpetrators’ age role ,6,12 andThe present study has certain limitations. As in most archival research, information about IPF and IPFS was collected by searching and reading online newspaper articles, causing a high rate of missing cases as in the sample considered in the current study. Despite this, archival research using online newspaper articles is one of the methods adopted in this field ,6,65, esCausing the death of approximately two individuals, IPF and IPFS represent the most lethal forms of intimate partner abuse, leading to far-reaching effects on public health, families, friends, neighborhoods, and entire communities. This study contributes to the scant literature on the differences between IPF and IPFS fatal events and the need for prevention and intervention efforts. Consistent with other studies ,12, our Our results summarize the suicidal killer profile as an ordinary man without a history of criminal records or violent relationships, nor a previous history of drug or alcohol use . HoweverIf on one side, our results evidenced a suicidal killer as an ordinary man, and on the other side, they seem to suggest the need to better consider and investigate, consistently with Dawson , the posOur findings underline that law enforcement members and those who possess a firearm are more likely to commit suicide after the IPF.Regarding IPFS prevention, it could be crucial to focus on the employees’ psychological health, especially those equipped with a firearm. In fact, despite few studies, law enforcement members showed the presence of some job-related risk factors  that neeConsidering this, it could be helpful to develop, implement and evaluate the effectiveness of Behavioral Health Training functional to intercept signs, symptoms, and risk factors of mental health issues . BecauseFurthermore, further studies are needed considering the scant literature on the topic and the few variables considered to explain the phenomenon. Future studies should identify the critical risk factors related to the IPF perpetrator’s suicide and investigate which individual, relational and contextual dimensions operate as protective factors for the IPF perpetrators, by adopting quantitative and qualitative methods.For example, future research could focus on the contributing role of some cognitive and emotional dimensions associated with IPF perpetrators’ choice to commit suicide. IPFS perpetrators should have more to lose and be characterized by high levels of apprehension, fear of social disapproval, social margination, stigmatization of the consequences, and remorse related to killing the victim ,9,72,73.Finally, future research should be designed to collect multiple data sources , for exa


Graphene has unique properties that can be exploited for radiofrequency applications. Its characterization is key for the development of new graphene devices, circuits, and systems. Due to the two-dimensional nature of graphene, there are challenges in the methodology to extract relevant characteristics that are necessary for device design. In this work, the Thru-Reflect-Line (TRL) calibration was evaluated as a solution to extract graphene’s electrical characteristics from 1 GHz to 65 GHz, where the calibration structures’ requirements were analyzed. It was demonstrated that thick metallic contacts, a low-loss substrate, and a short and thin contact are necessary to characterize graphene. Furthermore, since graphene’s properties are dependent on the polarization voltage applied, a backgate has to be included so that graphene can be characterized for different chemical potentials. Such characterization is mandatory for the design of graphene RF electronics and can be used to extract characteristics such as graphene’s resistance, quantum capacitance, and kinetic inductance. Finally, the proposed structure was characterized, and graphene’s resistance and quantum capacitance were extracted. Two-dimensional 2D) materials have attracted researchers due to their unique properties. Their optical, electronic, and mechanical properties indicate that these materials have great potential for novel applications in electronics and optoelectronics ,2,3,4. AD materiaTo perform RF characterization, the first mandatory step consists of the vector network analyzer (VNA) calibration to shift the measurement plane to the device under test (DUT). That can be achieved through several available alternatives such as short-open-load-through (SOLT), line-reflect-match (LRM), and TRL . The TRLIn this work, the issues of implementing the TRL calibration applied to the characterization of 2D materials, such as graphene, is presented. The impact on the RF parameter extraction due to the design of the calibration structures, such as contact thickness, substrate loss, contacting graphene, and backgate design, are analyzed. Finally, resistance and quantum capacitance are extracted for graphene and processed using standard commercial 8-inch wafers processing equipment.This section discusses the implementation of the TRL calibration. It focuses on the design of the calibration structures, their frequency span, and the characteristics of the substrate. In this case, coplanar waveguides were used so that it is possible to contact them with ground signal ground RF probes and fabricate the device in small dimensions so that it can be used with graphene. Then, the determination of the characteristic impedance is discussed since the calibration is related to the impedance of the transmission lines. Finally, the extraction of the parameters used in this work is presented.To explore this calibration, it was necessary to define the calibration structures as well as the device under test. The number of lines used depends on the frequency span for which the calibration is used. The optimal line length is ¼ wavelength, and each thru/line pair has a usable bandwidth of 8:1 (frequency span/start frequency) ,19. Sinc3 Ω.cm, and the silicon oxide had a dielectric constant of 3.9 and loss tangent of 0.01. The metal used was copper, with a conductivity of 5.8 × 107 S/m. The dimensions , forward directivity (FD), and forward source match (FSM). It is now possible to obtain the S-parameter matrix [Three values obtained from the scikit-RF package  are usedr matrix :(4)SCPW=It is now possible to extract the characteristic impedance of the lines by transforming the S-parameters to T-parameters and through Equations (5) and (6) :(5)G=T12This method, however, is only accurate at lower frequencies. To obtain a more accurate approximation, the propagation constant that is given by the TRL calibration can be used by solving the capacitance and substrate conductance per unit length:The capacitance and conductance values are obtained from the lower frequency results of Equations (7) and (8), respectively, given that the method is more accurate in this range . Since tThe goal is to extract the series resistance, quantum capacitance, and kinetic inductance of graphene, since that information is necessary to develop interconnects, components, devices, and systems. Depending on the device geometry and number of graphene layers used, it is expected that the graphene device can have a capacitive or inductive behavior. To perform the TRL calibration, the NISTMultilineTRL procedure from the scikit-RF package  was usedThe proposed calibration method is carried out and tested in this section. Then, it is used to demonstrate different factors that can negatively impact or even prevent accurate measurements from being obtained. These are the metal thickness of the calibration structures and the substrate loss. Then, a calibration with thick copper contacts and high resistivity substrate is demonstrated, as well as including thin gold contacts and a buried gate for the characterization of graphene. Since the inclusion of the gold contacts and the buried gate are a modification of the original TRL calibration, their influence on the characteristic extraction is reported.The procedure starts with a typical calibration (such as SOLT) of the measurement system to shift the measurement planes to the transmission lines of the TRL calibration. Then, the TRL calibration structures are measured, and their characteristic impedance is extracted through the steps described in the previous section. In this case, the simulation software is used to simulate only the calibration structures, mimicking the initial step (SOLT and structure measurement for characteristic impedance extraction). After obtaining the characteristic impedance of the transmission line, the calibration can be carried out without any pre-calibration of the VNA, and the desired RF characteristics can be extracted. This corresponds to including two 2 m long, 53 Ω coaxial cables in the simulation, one for each measurement port. This demonstrates that the calibration can be performed even with slightly mismatched coaxial cables.Implementing this calibration with 2D materials can be a challenging task due to their physical dimensions and characteristics. Another constraint related to characterizing two-dimensional materials is their inherent high series resistance. Graphene has a sheet resistance of 0.5 kΩ to 1.5 kΩ . Since tAs described in , when grAn example of a graphene contact would be using a 20 nm thick Au contact, given that it allows for a small thickness to prevent graphene from rupturing and allows for a good contact resistance. In this case, to extract the RF characteristics, the TRL calibration method is evaluated. To test the effect of such small thickness on the calibration accuracy, the calibration was performed with 20 nm of Au. An accurate calibration would yield results close to 1 NH for the imaginary part and 1 kΩ for the real part. However, it is possible to observe that the inductance changes from approximately 0.2 nH to 0.7 nH, and the resistance is around 600 Ω. Therefore, it is possible to conclude that very thin contacts compromise the implementation of the TRL calibration and extraction of the device’s properties.Since the characteristics of the transmission line can influence the accuracy of the calibration procedure, the substrate conductivity can be another inaccuracy source. To demonstrate the impact of the substrate conductivity, a standard clean room doped silicon wafer with 100 Ω.cm is used. The measurement structures are as previously described, and the contacts are made of 5 µm thick copper to remove the previously described effect of the metallic resistance of the calibration accuracy. It is possible to observe that the substrate loss impacts the extraction of the RF characteristics of the device. The series resistance is in the 800 Ω to 900 Ω range when it should be around 1 kΩ, and the inductance is in the −165 nH to 0.8 nH range when 1 nH is expected. This is due to the increased transmission line losses that are induced by the substrate conductivity.As demonstrated, using thin metallic contacts and lossy substrate cause the calibration to provide very unreliable results. This is due to the extremely high losses that are introduced in the transmission line. To overcome these issues, two approaches must be taken: reduce the contact resistance and reduce the substrate conductivity. To reduce the contact resistance, copper was used, which has one of the highest conductivities, and its thickness was increased to 5 µm to further improve the contact. For the case of substrate conductivity, a commonly available 10 kΩ.cm silicon substrate was used.The problem of using 5 µm thick contacts is that these will break graphene, and contact will not be made. Therefore, a 20 nm gold contact with a length of 35 µm is included to bridge the connection from the 2D material to the copper contact. 7 S/m was included. These structures were simulated, and the characteristics of the lumped model were extracted. It is possible to observe that the accuracy of the measurements has increased when compared with the scenario where the structures have thin contacts, and the scenario where the substrate has a high conductivity. The extracted inductance is now around 0.9 nH to 1.1 nH, and the resistance is between 900 Ω to 1 kΩ, when 1 nH and 1 kΩ were expected, respectively. For example, when comparing with the high conductivity substrate scenario, where the inductance was much lower than 0.6 nH for frequencies lower than 25 GHz and the resistance was between 800 Ω to 900 Ω, this demonstrates that using 5 µm thick copper contacts and 10 kΩ·cm silicon substrate provides a reliable calibration for measuring 2D materials. Furthermore, 20 nm gold contact with the length of 35 µm and with a conductivity of 4.1 × 10Most applications of graphene use its conductivity that depends on its chemical potential. Such property can be modified through a voltage gating and has been explored for tunable applications . Therefo5 S/m and a thickness of 20 nm, resulting in 500 Ω/square. The DUT area corresponds to two squares in series, which totals 1 kΩ series resistance. To determine the characteristics of the mimic device, the TRL calibration without the backgate was performed as well as with a backgate made of gold. The previous simulations used a lumped element to mimic the extraction of the properties of graphene. That approach, however, does not account for the signal propagation in the device as well as its interaction with the backgate contact. Therefore, to mimic graphene, the lumped element was replaced by the best possible approach allowed by the simulator—a material with 1 × 104 S/m. An alternative could be implementing a backgate design that has a high impedance, which would prevent the RF signal propagation from being influenced by the backgate. However, this design would need to be fit for a frequency range from 1 GHz to 65 GHz. Since the purpose of the backgate is transmitting a DC voltage to gate the 2D material, a simpler solution would be to increase its resistance. The only constraint is that the gate oxide must be much more resistive than the series resistance of the backgate itself, so that the applied voltage at the gate is close to the applied voltage at the gate contact. Therefore, the same backgate design can be used if the material’s conductivity is modified. Since the highly conductive backgate contact impacts the parameter extraction, by reducing the backgate conductivity, it is possible to circumvent this problem. The extraction structures, with the same backgate design were simulated with a material with a conductivity of 2 × 10The proposed measurement and calibration structures were fabricated. In this case, the 2D material was graphene, and the device fabricated is shown in The fabricated structures were measured with the Keysight E5071C VNA , the GSG |Z|-Probes , and the CS-5 calibration substrate . Then, the previously proposed calibration procedure was undertaken, allowing us to extract graphene’s characteristics. These measurements were performed for 0 V, 5 V, 10 V, and 15 V of backgate bias. The extracted resistance and quantum capacitance of graphene are shown in 2V−1s−1, and the Dirac point is in the range of 6 to 8 V when it is gated with 15 nm of Al2O3, meaning that in this case, it should be from 50 to 67 V.The extracted resistance changes from around 450 Ω at 1 GHz down to around 250 Ω at 8.5 GHz for the bias voltage of 0 V. It then increases with the increase of the backgate voltage up to a maximum resistance of around 750 Ω at 1 GHz to around 400 Ω at 8.5 GHz, obtained at 15 V. The quantum capacitance changes from close to 0.55 pF at 1 GHz down to around 0.2 pF at 8.5 GHz for the bias voltage of 0 V. This capacitance reduces with the increase in voltage, achieving a capacitance of around 0.23 pF at 1 GHz down to around 0.1 pF at 8.5 GHz for the bias voltage of 0 V. Both resistive and capacitive behavior are as reported in the literature ,27. The Graphene is a promising material for several fields such as electronic and RF applications. The TRL calibration provides one of the highest calibration accuracies. In this work, the application of the TRL calibration for the extraction of the RF characteristics of graphene was explored. The implementation of the calibration was described, as well as the parameters that negatively affect its accuracy such as thin contacts and lossy substrate. To obtain an accurate calibration, thick contacts and low loss substrate should be used to reduce the losses in the transmission line. In this case, considering the characteristics of the transmission line, the series resistance is around 0.8 Ω, the substrate conductivity 10 kΩ·cm, and the backgate contact around 730 kΩ, which can be used as a reference point for the calibration. Since thick contacts will damage graphene, a short length thin contact was added, and its impact on the extraction of the RF characteristics of graphene was demonstrated to be negligible. Furthermore, since graphene’s conductivity changes according to its chemical potential, a backgate was added to characterize it for different potentials. Finally, the proposed structures were fabricated and measured, and the properties of graphene were extracted, from 1 GHz to 8.5 GHz. Its resistance at 1 GHz changed from around 450 Ω to 750 Ω at 0 V and 15 V, respectively; at 8.5 GHz, it changed from around 250 Ω to 400 Ω at 0 V and 15 V, respectively. Its quantum capacitance at 1 GHz changed from around 0.55 pF to 0.23 pF at 0 V and 15 V, respectively; at 8.5 GHz, it changed from around 0.2 pF to 0.1 pF at 0 V and 15 V, respectively. The development of such calibration procedure to characterize graphene will allow us to further improve the knowledge of the graphene’s RF characteristics, paving the way to obtain tunable electronics either through biasing or biological functionalization. Finally, the proposed calibration and extraction method are not limited to graphene and can be used with other 2D materials.


The persistence of inflammatory mediators in tissue niches significantly impacts regenerative outcomes and contributes to chronic diseases. Interleukin-4 (IL4) boosts pro-healing phenotypes in macrophages (Mφ) and triggers the activation of signal transducer and activator of transcription 6 (STAT6). Since the IL4/STAT6 pathway reduces Mφ responsiveness to inflammation in a targeted and precise manner, IL4 delivery offers personalized possibilities to overcome inflammatory events. Despite its therapeutic potential, the limited success of IL4-targeted delivery is hampered by inefficient vehicles. Magnetically assisted technologies offer precise and tunable nanodevices for the delivery of cytokines by combining contactless modulation, high tissue penetration, imaging features, and low interference with the biological environment. Although superparamagnetic iron oxide nanoparticles (SPION) have shown clinical applicability in imaging, SPION-based approaches have rarely been explored for targeted delivery and cell programming. Herein, we hypothesized that SPION-based carriers assist in efficient IL4 delivery to Mφ, favoring a pro-regenerative phenotype (M2φ). Our results confirmed the efficiency of SPION-IL4 and Mφ responsiveness to SPION-IL4 with evidence of STAT6-mediated polarization. SPION-IL4-treated Mφ showed increased expression of M2φ associated-mediators  when compared to the well-established soluble IL4. The ability of SPION-IL4 to direct Mφ polarization using sophisticated magnetic nanotools is valuable for resolving inflammation and assisting innovative strategies for chronic inflammatory conditions. Inflammation is a dynamic process triggered by the immune system in response to injury or infection. However, the uncontrolled activation of immune cells results in persistent inflammatory signals, with deleterious consequences for tissue regeneration. Resolving inflammation is thus a challenge to be overcome with impact in the management of chronic inflammatory diseases , autoimmInterleukin-4 (IL4) is a key regulator in humoral and adaptive immunity and a well-known promoter of alternatively activated M2 macrophages (M2φ) . The preMagnetically assisted technologies ,14,15 ofAlthough IL4 is a well-established M2φ switch, there is a lack of research on IL4 delivery using particulate carriers. Such systems would enable cell and receptor magnetic targeting strategies for the in situ modulation of the macrophage phenotype to locally discourage abnormal inflammatory signals and reestablish pro-healing environments. Therefore, we hypothesized that superparamagnetic iron oxide nanoparticles (SPION) decorated with IL4 (SPION-IL4) favor M2φ via magnetically guided IL4 presentation. By targeting Mφ, which has a coordinated action over inflammatory cascades, SPION-IL4 could assist in the contactless control of inflammatory cues in injured or pathological environments. Here, SPION-IL4 were fabricated and investigated downstream of the IL4 receptor in Mφ. The activation of STAT6 and the gene and protein inflammatory mediators were assessed with different concentrations of SPION-IL4 and compared with carrier-free IL4 (Exo IL4) exogenously supplemented to the culture medium. We foresee improved and more effective SPION-mediated therapeutics in precision Mφ targeting for the immunomodulation of persistent inflammatory environments.w/w and that of the organic component was 22 ± 9% w/w, confirming the presence of dextran-COOH in the shell of the SPION  carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry. The magnetic response of SPION-IL4 to EMF was evaluated, and SPION-IL4 exhibited magnetic responsiveness A. The inhe SPION .A stable amide (-CONH-) covalent linkage was selected for the magnetic presentation of IL4 to macrophages. This conjugation method has been used for the functionalization of nanomaterials with proteins, peptides, antibodies, and nucleic acids ,17,18,19−1, which is due to the stretching vibrations of the carbonyl bond of the carboxylic acid group (COOH) in dextran. When comparing the SPION to the SPION-IL4 spectrum, this band was replaced by two bands at ~1650 cm−1 and ~1550 cm−1. The band at ~1650 cm−1 is due to carbonyl bond stretching vibrations, whereas the band at 1550 cm−1 represents N–H bond bending vibrations. Thus, the substitution of a COOH representative band in SPION by two amide bond representative bands indicates the successful functionalization of SPION with IL4. The absence of a 15 kDa band associated with IL4 molecular weight in the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)  spectroscopy D. The FTDS-PAGE)  in the SDS-PAGE) E. DespitDS-PAGE) .2 [p > 0.05) were observed in cellular viability  administration to Mφ. Thus, we estimated a 646 fg IL4/cell in SPION-IL4/30, and a 1940 fg IL4/cell in both SPION-IL4/100 and Exo IL4 conditions. Hence, SPION-IL4/100 corresponded to the highest SPION concentration to be administered to cells in the present study, with an equivalent number of IL4 molecules to Exo IL4, namely 1940 fg IL4/cell.Unidirectional formation of highly oriented rods of SPION-IL4 was observed in the SEM images A, suggesp < 0.0001). When SPION-IL4 or soluble IL4 binds to the IL4 receptor, the receptor is no longer available for immunostaining. Thus, the signal detected was related to IL4 receptors that were not previously occupied by IL4. Since SPION-IL4/30 is related to a lower concentration of both SPION and IL4 in comparison to SPION-IL4/100 and Exo IL4, more free receptors are available, and thus the signal intensity is higher. Despite the successful delivery of IL4 via SPION-IL4 to the IL4 cognate receptor, the concentrations of SPION-IL4 used did not saturate all the IL4-receptors available in the cells.To investigate the SPION-IL4 formulation, interactions with IL4 receptor cells were immunostained with an antibody against IL4 receptor alpha (IL4Rα) B. SPION-p < 0.05 and p < 0.01) (p > 0.05) independently of the iron concentration used  , and ind < 0.01) D-II. Theion used D-II. Conion used E.Overall, SPION-IL4 was more effective in the transport and delivery of IL4 participating in the activation of the IL4/STAT6 pathway. The production of IL4 at the gene and post-transcript levels was more efficient when magnetic nano-vehicles were applied, especially with a lower concentration of SPION-IL4 (30 µg/mL), suggesting that higher concentrations of IL4, either provided by soluble IL4 (Exo IL4) or in SPION-IL4 nanocarriers, may lead to suboptimal levels of IL4 mediated signals in THP1-derived macrophages.+ cells was observed after 24 h in SPION-IL4 conditions. The decreased availability of the CD11b receptor on cells treated with SPION-IL4 was accompanied by an elongated morphology (white arrow), which has been associated with a M2φ phenotype [According to the literature, integrin αM (CD11b) has been implicated in the activation of pro-inflammatory transcription factors, such as nuclear factor kappa B (NFKB) , as wellhenotype .The cocktail of cytokines/chemokines released by macrophages is indispensable for characterizing their polarized states. The cytokine/chemokine profile of SPION-IL4 treated cells was reported for pro- and anti-inflammatory mediators B. IL8 isThe concentration of SPION-IL4 seemed to have a particular effect on the secreted chemokines CCL5 (C-C Motif Chemokine Ligand-5) and CCL2 (C-C Motif Chemokine Ligand-2). The production of CCL5, whose signaling contributes to M1φ polarization and can be inhibited by IL4 , was detp < 0.05) also supports an M2φ shift.Since M2φ polarization is associated with decreased inducible nitric oxide synthase (iNOS) expression and increased ARG1 levels , the ratIn this study, we investigated the promising role of magnetic nanoparticle-mediated delivery of IL4 aiming at M2φ polarization. SPION-IL4 contributed to IL4-mediated actions and to the expression of STAT6 responsive molecules such as CCL2 and ARG1. In particular, ARG1 together with STAT6 has been described to modulate Mφ phenotypes and to accelerate inflammation resolution . Studies3O4) and a dextran -COOH shell were purchased from micromod GmbH . SPION were functionalized with IL4 (SPION-IL4) by carbodiimide coupling chemistry using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)  and N-hydroxysuccinimide (NHS) . All production steps were performed at room temperature (RT) unless stated otherwise. SPION were mixed with EDC and NHS in 0.5 M MES buffer  (pH 6.3) for 45 min. Then, activated SPION were washed by magnetic separation with 0.01 M phosphate buffered saline (PBS)  (pH = 7.4), and mixed with 10 µg of Recombinant Human IL4  for 3 h. Following a washing step, 25 mM glycine  in 0.01 M PBS was added, and the mixture was continuously stirred for 30 min. SPION-IL4 was washed three times and resuspended in 1 mL of ultra-pure water. SPION-IL4 was further purified by dialysis  against water for 24 h and stored at 4 °C.SPION composed of a magnetite core  and after functionalization (SPION-IL4). An ultrapure water background was used before the experiments. SPION-IL4 morphology were analyzed using scanning transmission electron microscopy (STEM) . The elemental composition of SPION-IL4 was determined using Energy Dispersive X-ray spectroscopy (EDS) . The hydrodynamic diameter and zeta potential of SPION-IL4 were determined using a Malvern NanoZS .Fourier transform infrared (FTIR) spectra were recorded with an IRPrestige 21 spectrometer (Shimadzu) using the attenuated total reflectance (ATR) mode over the range of 4000–600 cm®20 detergent  and incubated with Anti-Rabbit IgG secondary antibody  at RT for 1 h. After washing in TBST, the signal was developed using an AP Conjugate Substrate Kit  according to the manufacturer’s instructions. For the western blotting assay, the supernatant of the SPION-IL4 solution (S) after brief centrifugation was also included as a control for IL4 binding to SPION. Membrane images were acquired using a digital scanner  in conjunction with Epson Scan software ver. 3.9.2.1US. IL4 bands were further quantified using ImageJ 1.50i  software.The functionalization efficiency of IL4 for SPION was evaluated using western blotting. Samples: SPION, SPION-IL4, supernatant from the IL4 incubation solution with SPION (S1), and supernatant from the first washing step with PBS (S2) were analyzed, as well as standard IL4 solutions (0 to 25 µg/mL). A mixture of Laemmli buffer  and the samples (1:1) was incubated at 95 °C for 5 min before running on a 12.5% separation gel and 4% stacking gel . The PageRuler Plus Prestained Protein Ladder  was used as a molecular weight marker. The gel was stained with Coomassie Brilliant Blue  for protein visualization. The gel was transferred to a nitrocellulose membrane  using a Pierce Power System . The membranes were incubated for 1 h with a blocking solution, followed by overnight incubation with recombinant anti-IL4  at 4 °C. Membranes were washed in Tris-Buffered Saline containing 0.1% Tween2 atmosphere. THP1-derived macrophages were differentiated with 100 nM phorbol 12-myristate 13-acetate  for 24 h, followed by 24 h incubation with a PMA-free medium. Adherent THP1-derived macrophages were cultured and expanded in RPMI medium. Two time-points (1 h or 24 h) and up to four conditions were investigated: THP1 cells cultured with (i) SPION-IL4 at 30 µg/mL or (ii) SPION-IL4 at 100 µg/mL of iron  and non-functionalized SPION at (iii) 30 µg/mL or (iv) 100 µg/mL of iron . Conditions (iii) and (iv) act as experimental controls for IL4 functionalization. Magnetically stimulated THP1 cells exogenously supplemented with soluble IL4 were used as a positive control (hereafter referred to as Exo IL4) whereas cells cultured on tissue culture polystyrene (TCPs) were used as a negative control. The concentration of IL4 per cell used in the SPION-IL4/100 and Exo IL4 conditions was the same. EMF was provided by a magnefect nano device  (350 mT/well) under all conditions.Human monocytes ) were cultured in Roswell Park Memorial Institute medium (RPMI)  supplemented with 1% L-Glutamine  in a humidified 5% CO× 105 cells per well , followed by incubation with SPION-IL4 at 37 °C under magnetic stimulation. For LDH experiments, cells were seeded at 1 × 104 cells per well . Live/dead cell double staining: Cells were washed with PBS and incubated with 2 µM calcein AM in PBS  and 4 µM propidium iodide (PI)  in the dark for 30 min at 37 °C. Cells were rinsed in PBS, and the fluorescence signal was acquired using a transmitted and reflected light microscope with Apotome 2 . Cell viability was assessed using both 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and LDH assays. MTS: THP1 cells were incubated in MTS solution  for 3 h at 37 °C in a 5% CO2 atmosphere and protected from light. The supernatant was transferred to a new 96-well plate and the optical absorbance was measured at 490 nm . LDH: At the respective time-points, cytotoxicity was measured in each sample medium using a CyQuant LDH Cytotoxicity Assay kit , following the manufacturer’s instructions. LDH activity was measured at 490 nm/680 nm excitation/emission wavelengths using a microplate reader .To determine the effect of SPION-IL4 on cells, THP1-derived macrophages were seeded at 1 ® RNA Isolation Reagent  following the manufacturer’s instructions. RNA was quantified using a Nanodrop® ND-1000 spectrophotometer  at 260/280 nm. First-strand complementary DNA was synthesized from 1 µg of RNA from each sample  in a 20 µL reaction using a Mastercycler® ep realplex gradient S machine (Eppendorf). Transcripts were quantified by quantitative polymerase chain reaction (qPCR) using the PerfeCTA SYBR Green FastMix kit  following the manufacturer’s protocol in a Real-Time Mastercycler ep realplex thermocycler (Eppendorf). Primer sequences  phosphorylation (pSTAT6) used to screen the effects of SPION-IL4 in THP1-derived macrophages was assessed using a cell-based phospho-STAT6 (pTyr641) ELISA kit  following the manufacturer’s instructions.IL4 Receptor Alpha (IL4Rα) is the alpha chain of the IL4 receptor and is involved in the regulation of alternative activation of macrophages and, consequently, IL4 secretion levels, whereas CD11b is an integrin family member involved in the regulation of leukocyte adhesion and migration to mediate the inflammatory response. To detect these molecules, THP1-derived macrophages were fixed in 4% neutral buffered formalin  overnight and stored in PBS at 4 °C until use. Subsequently, the cells were incubated with 0.025% Triton-X100  in PBS, and blocking was performed using 2.5% normal horse serum . Cells were incubated overnight at 4 °C with anti-IL4Rα , anti-IL4 , or anti-CD11b  diluted in 0.1% bovine serum albumin diluted , followed by incubation for 1 h at RT with the secondary antibody . The samples were washed three times with PBS before staining with DAPI  for 10 min. Immune detection was then assessed by confocal laser scanning microscopy, and 3D reconstruction images obtained from the z-stacks were obtained using TCS SP8 software . IL4Rα and IL4 fluorescence intensities were quantified using ImageJ 1.50i software .In the case of CD11b, the number of cells positive for CD11b was determined by flow cytometry. The cells were permeabilized and incubated with anti-CD11b  for 20 min, followed by incubation with the secondary antibody (Anti-Rabbit IgG Alexa Fluor 488) for 30 min in the dark. Cells were then washed with PBS and resuspended in acquisition buffer  before acquisition on a FACSAria III sorter equipped with blue and red lasers . The cells were identified using forward and side scattering. A minimum of 5000 cells were acquired and analyzed using the FACS Diva software version 7. Unstained cells were used to establish cell autofluorescence, and cells incubated only with secondary antibodies were used as controls. The population of cells positive for CD11b was expressed as a percentage.Scanning electron microscopy (SEM) was used to visualize the physical distribution of SPION-IL4 in the cells. THP1-derived macrophages were fixed in 4% neutral buffered formalin  overnight and dehydrated using ascending concentrations of ethanol (from 30 to 100%), followed by a 5 min immersion in hexamethyldisiloxane . The samples were then air-dried overnight and sputter-coated  with gold. Images were acquired using scanning electron microscopy .× 105 THP1-derived macrophages per well were cultured in 24 wells plates  before supplementing with SPION-IL4 and IL4. After 24 h, the supernatants were collected and analyzed using a Proteome Profiler™ Human Cytokine Array Kit  following the manufacturer’s instructions. The spot intensity was detected using the Odyssey Fc Imaging System 2800 (LI-COR®) and quantified using ImageJ 1.52o  software. Supernatants were also analyzed for the quantification of secreted cytokines using commercially available enzyme immunoassay kits for ARG1  and IL4  following the manufacturer’s instructions.For the cytokine arrays, 3 (n = 3), except for flow cytometry analysis of CD11b, which is representative of two independent experiments (n = 2), with a minimum of 5000 cells analyzed per experiment. Statistical analysis was performed using SPSS statistical software (version 27.0.1.0). First, a Shapiro–Wilk test was used to ascertain data normality and Levene’s test for homogeneity of variances. Normality and variance homogeneity were rejected, and non-parametric tests were used . Different degrees of confidence were considered, p < 0.05, p < 0.01, p < 0.0001, and are represented by symbols * and # for p < 0.05, **, ## for p < 0.01, and **** for p < 0.0001.Results are expressed as the mean ± standard error of the mean (SE), representative of three independent experiments This study showed that SPION-IL4 is a suitable IL4 presenter, anticipating the modulation of cell behavior with insights into macrophage-priming strategies mediated by EMF-actuated SPION. SPION-IL4 treated Mφ showed an increased expression of genes such as IL10 and ARG1 and proteins (CCL2 and IL1Ra) typically associated with the M2φ phenotype, with unique advantages over carrier-free administration. Together with the therapeutic effect, the magnetic memory of SPION-IL4 comprises multimodal possibilities, including cell tracking and in vivo identification of IL4-targeted populations.In view of the presence of macrophages in almost all tissues and organs, SPION in combination with IL4 and/or other cytokines could hold wide applicability as phenotype modulators and, consequently, as magnetically responsive switches of pro- and anti-inflammatory activities. Either in the form of magnetic delivery systems or magnetically responsive elements incorporated into tridimensional matrices, these systems could render immunomodulatory actions foreseeing improved therapeutic solutions for chronic inflammation conditions and tissue regeneration.


Cardiovascular disease is prevalent in idiopathic pulmonary fibrosis (IPF), yet the extent of left-sided heart failure (HF) burden, whether this has changed with time and whether HF impacts mortality risk in these patients are unknown. The aims of this study were therefore to determine the temporal trends in incidence and prevalence of left-sided HF in patients with IPF in England and compare these to published estimates in the general population and those with comparable chronic respiratory conditions such as chronic obstructive pulmonary disease (COPD), as well as determine the risk of all-cause and cause-specific mortality in patients with comorbid left-sided HF and IPF at population-level using electronic healthcare data.Clinical Practice Research Datalink (CPRD) Aurum primary-care data linked to mortality and secondary-care data was used to identify IPF patients in England. Left-sided HF prevalence and incidence rates were calculated for each calendar year between 2010 and 2019, stratified by age and sex. Risk of all-cause, cardiovascular and IPF-specific mortality was calculated using multivariate Cox regression.From 40,577patients with an IPF code in CPRD Aurum, 25, 341 IPF patients met inclusion criteria. Left-sided HF prevalence decreased from 33.4% (95% CI 32.2–34.6) in 2010 to 20.9% (20.0–21.7) in 2019. Left-sided HF incidence rate per 100 person-years (95% CI) remained stable between 2010 and 2017 but decreased from 4.3 (3.9–4.8) in 2017 to 3.4 (3.0–3.9) in 2019. Throughout follow-up, prevalence and incidence were higher in men and with increasing age. Comorbid HF was associated with poorer survival (adjusted HR (95%CI) 1.08 (1.03–1.14) for all-cause mortality; 1.32 (1.09–1.59) for cardiovascular mortality).Left-sided HF burden in IPF patients in England remains high, with incidence almost 4 times higher than in COPD, a comparable lung disease with similar cardiovascular risk factors. Comorbid left-sided HF is also a poor prognostic marker. More substantial reduction in left-sided HF prevalence than incidence suggests persistently high IPF mortality. Given rising IPF incidence in the UK, this calls for better management of comorbidities such as left-sided HF to help optimise IPF survival.The online version contains supplementary material available at 10.1186/s12890-022-01973-5. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease resulting in irreversible lung scarring. Due to diagnostic challenges, evolving diagnostic criteria as well as differences in study methodologies, estimates of IPF disease burden show substantial heterogeneity, ranging from a prevalence of 0.57–4.51 per 10,000 persons in Asia, 0.33–2.51 in Europe, and 2.40–2.98 in North America . For insHowever, while the high burden of CVDs such as IHD , pulmonaWhile there is evidence that IPF and left-sided HF co-exist, there are few population-level studies examining the epidemiology of left-sided HF in IPF patients, with generalizability of findings from existing studies made challenging by use of small cohorts from registries or single centres, frequent study of HF in only hospitalised patients, and lack of clear HF case definition . FurtherThe aim of this study was therefore to investigate temporal trends in incidence and prevalence of left-sided HF in IPF patients in England, compare this to published estimates in the general population and those with comparable chronic respiratory conditions such as chronic obstructive pulmonary disease (COPD), as well as explore the risk of all-cause, cardiovascular and IPF-specific mortality in England using population-level electronic healthcare data. This is the first European study to explore temporal patterns of the burden of left-sided HF in IPF and one of few studies to date to investigate left-sided HF epidemiology in IPF using nationally representative population-level data.Data were obtained from the Clinical Practice Research Datalink (CPRD) Aurum, a nationally- representative database of de-identified primary care electronic healthcare records (EHR) covering approximately 19% of the UK population . Use of CPRD Aurum data for eligible patients registered in England was linked to secondary care data from Hospital Episode Statistics (HES), mortality data from Office of National Statistics (ONS) and socioeconomic data from the 2015 Index of Multiple Deprivation (IMD). Data linkage between sources was conducted by NHS Digital, a statutory trusted third party.https://github.com/NHLI-Respiratory-Epi/IPF_HF_codelists.Consistent with previous studies exploring IPF epidemiology by means of diagnostic codes in EHR , 36, we st January 2010–31st December 2019). For each patient, start of follow-up was defined as the latest of: 12 months post registration date with a general practice, start of study, patient’s 40th birthday or date of IPF-CS diagnosis. The end of follow-up was defined as the earliest of: transfer out of practice, end of study, last data collection date or patient’s date of death.We used a retrospective cohort study design with rolling entry and a 10-year study period  and based on previously published work were used to identify baseline covariates. Smoking, alcohol status and body mass index (BMI) were determined closest to start of follow-up. BMI data in the 3 years prior to start of follow-up was used, with BMI values calculated from height and weight data closest to start of follow-up, where available, in event of missing BMI data. Presence of baseline cardiovascular comorbidities and left-sided HF risk factors were determined at any point prior to start of follow-up, while cardiovascular medication use was defined as at least one prescription in the 12 months preceding start of follow-up. All codelists used, including those for ascertaining outcomes below, are accessible at: Codelists were also used to identify prevalent and incident left-sided HF. Codes denoting right HF were excluded.Cause of death was ascertained from ICD-10 codes using the derived cause of death field in linked ONS records. All-cause mortality was defined as death from any cause, with cardiovascular mortality defined as death due to myocardial infarction, ischaemic stroke or HF.Baseline characteristics are presented as absolute numbers and frequencies (%) for categorical data and medians with interquartile ranges [IQR] for non-normally distributed continuous variables. Proportions of missing data are described as frequencies (%) for each covariate.Left-sided HF prevalence and crude incidence rates were calculated annually for the 10-year study period, stratified by age  and sex. Incidence rates are reported per 100 person-years at risk, with associated 95% confidence intervals (CI).p < 0.05 level of statistical significance was used in all analyses. All analyses were conducted in Stata v16.0 .Multivariate Cox regression was used for mortality analyses. Covariate inclusion in the multivariate models was based on directed acyclic graphs (DAGs) created using clinical knowledge and existing literature to identify possible confounders  removing patients from the cohort diagnosed with IPF-CS post 1st September 2018 when ATS/ERS/JRS/ALAT IPF diagnostic guidelines changed (2) removing patients with non-specific IPF-CS diagnostic codes such as fibrosis of lung, pulmonary fibrosis, diffuse pulmonary fibrosis, Hamman-Rich syndrome, O/E-fibrosis of lung; (3) removing patients if they had evidence of another cause for pulmonary fibrosis or interstitial lung disease other than IPF, based on ICD-10 codes denoting an autoimmune condition, occupational lung disease, hypersensitivity pneumonitis or sarcoidosis in mortality data; and (4) removing patients diagnosed with IPF-CS when younger than 50 or 60 years old to exclude possible hereditary or familial IPF cases.For mortality analyses, ethnicity and IMD were also added as possible confounders in the multivariate models for all-cause and cardiovascular mortality.25,341 IPF-CS patients from 1379 general practices in England were included in the cohort  had HF diagnosed prior to IPF-CS, 3.7% were diagnosed concurrently with IPF-CS and 38.9% following IPF-CS.Those with comorbid IPF-CS and left-sided HF were more likely to be older, male, more socioeconomically deprived, have a higher BMI, be an ex-smoker, have a history of CVD or HF risk factors and be on cardiovascular medications at start of follow-up Table .Table 1BLeft-sided HF incidence rate per 100 person-years (95% CI) remained relatively stable between 2010–2017 but decreased modestly from 4.3 (3.9–4.8) in 2017 to 3.4 (3.0–3.9) in 2019  in 2010 to 20.9% (20.0–21.7) in 2019  than COPD patients [ref] and having comorbid HF is poor prognostic marker which increases risk of both all-cause and cardiovascular mortality, with a comparatively higher risk of the former than the latter.The modest reduction in HF incidence could reflect improved management of cardiovascular risk factors over time. Indeed, prescriptions for CVDs increased by 78% in England between 1991 and 2014 . By contWe were unable to ascertain from the data whether IPF patients with HF had HFrEF or HFpEF, although this would be useful to characterise. While HF prevalence has been noted to be increasing globally, many studies show a stable or decreasing HF incidence. An increasingly ageing population and improved HF survival due to treatment advances may explain increased prevalence while better treatment of acute coronary syndromes due to the advent of revascularisation therapies may explain reduced incidence of HF . FurtherAs discussed earlier, the treatment landscape of IPF has changed significantly in the last decade and it is unclear how these changing treatment patterns have influenced the burden of comorbidities such as left-sided HF in IPF. As antifibrotics are prescribed in tertiary care, we did not have access to antifibrotic use data in CPRD Aurum and therefore were unable to explore this potential association. Interestingly however, antifibrotics have been shown to have cardioprotective properties within the context of HFpEF, albeit this has not been explored specifically in the context of comorbid IPF and HF , 29. To Our HF incidence estimates are significantly lower than those reported by a US study, the only other population-based study which has explored the burden of left-sided HF in an IPF patient population. Using claims data between 2001 and 2008 for over 9000 patients, this study reported an incidence rate of 67.5 cases per 1000 person-years . Given tBy contrast, our prevalence estimates are higher than those from smaller cohort studies which range from 8 to 23% , 23–26. Consistent with other studies, male gender was found to be a significant risk factor for having a higher HF burden . AlthougBased on our estimates, left-sided HF prevalence is approximately 30-fold higher and incidence is almost 12-fold higher in IPF patients than in the general UK population . AssuminOur all-cause mortality estimates are in-keeping with two studies showing an increased mortality risk with comorbid IPF and HF, although our estimates are 29% lower than those in an Italian study , and thrWhile this study cannot explain why left-sided HF burden is so high in IPF relative to COPD patients, there are several possible theories. Shared risk factors such as smoking and ageing or shared pathological mechanisms such as telomere dysfunction and molecular and cellular fibrosis in both IPF and HF may contribute , 31. AltStrengths of this study include use of nationally representative population-level data linked to national mortality and secondary care data to explore left-sided HF epidemiology in IPF patients. This contrasts previous studies to date which have largely explored cohorts from single centres or registries. Although there is recognised heterogeneity in IPF coding terms in electronic healthcare records, left-sided HF incidence and prevalence estimates remained robust despite several sensitivity analyses optimising IPF case ascertainment. While there is potential risk of case misclassification due to dependency on codes entered in primary care, we draw confidence in the validity of ascertainment of both our exposure and outcome due to high positive predictive values (82–95%) of both HF and IPF diagnoses in primary care records , 34.Nevertheless, this study has several limitations. The cohort was predominantly Caucasian with patients residing in England, limiting generalisability of findings to other populations. Almost 80% of patients in the cohort also had non-specific codes for pulmonary fibrosis and although we used proxy measures in the sensitivity analyses to optimise IPF-CS case ascertainment such as exclusion of those with evidence of a potential non-idiopathic cause for pulmonary fibrosis based on mortality data, there is nonetheless a possibility that some cases were misclassified. A more comprehensive study of the validity of IPF codes in CPRD Aurum is currently in progress and would inform confidence in the results from this study. Furthermore, we did not have access to imaging data to determine IPF disease severity. This is important to ascertain as studies have shown IPF patients with a rapid disease course were more likely to be diagnosed with HF post IPF diagnosis . FurtherLastly, there was a significant proportion of missing data for alcohol intake, ethnicity and BMI. As missing data for these variables has been shown to not be missing at random in CPRD Aurum, we conducted a complete-case mortality analysis, but acknowledge this may have introduced some bias. For instance, patients with missing alcohol or BMI data are more likely to not have engaged with healthcare providers as often and by extension may have been less compliant with their medications or monitoring investigations for their IPF or HF. Therefore, it is possible that the patients excluded from the mortality analyses due to missing data may have generally been less healthy and may have had more severe disease severity of both their IPF and HF. The exclusion of these patients due to missing data may have therefore underestimated the risk of all-cause and cause-specific mortality in those patients with comorbid IPF and HF.This study shows temporal changes in both incidence and prevalence of left-sided HF in IPF patients in England, with modest reduction in incidence possibly reflecting improved screening and management of cardiovascular risk factors. However, despite decreasing incidence and prevalence, left-sided HF burden is considerably higher in IPF patients than in patients with other comparable chronic respiratory diseases and confers a higher mortality risk. Further studies are needed to explore HF phenotypes in IPF patients, whether there is a temporal relationship between the two diseases, what the aetiological mechanisms driving this association are, what drives the noted gender differences and whether HF treatment modifies the mortality risk.Additional file 1.Supplementary material.


Graphene-based materials have recently gained attention for regenerating various tissue defects including bone, nerve, cartilage, and muscle. Even though the potential of graphene-based biomaterials has been realized in tissue engineering, there are significantly many more studies reporting in vitro and in vivo data in bone tissue engineering. Graphene constructs have mainly been studied as two-dimensional (2D) substrates when biological organs are within a three-dimensional (3D) environment. Therefore, developing 3D graphene scaffolds is the next clinical standard, yet most have been fabricated as foams which limit control of consistent morphology and porosity. To overcome this issue, 3D-printing technology is revolutionizing tissue engineering, due to its speed, accuracy, reproducibility, and overall ability to personalize treatment whereby scaffolds are printed to the exact dimensions of a tissue defect. Even though various 3D-printing techniques are available, practical applications of 3D-printed graphene scaffolds are still limited. This can be attributed to variations associated with fabrication of graphene derivatives, leading to variations in cell response. This review summarizes selected works describing the different fabrication techniques for 3D scaffolds, the novelty of graphene materials, and the use of 3D-printed scaffolds of graphene-based nanoparticles for bone tissue engineering. There is a growing demand to engineer functional tissue using three-dimensional 3D) biological substitutes. Tissue engineering is a field composed of many scientific disciplines including biomedical engineering, cellular molecular biology, material science, and biochemistry. The concept of tissue engineering evolved in the 1990s whereby stem cells and materials could be implanted in vivo to restore injured tissues ,3,4. HowD biologiAlthough 3D printing is revolutionizing personalized treatment, the material needed to print the scaffold is a long-debated topic that depends on the desired tissue source to be repaired. These materials range anywhere from hydrogels, to nanoparticles, bio-metals, bio-ceramics, and bio-degradable polymers, each of which exhibit specific physicochemical properties. There are many material properties that influence tissue regeneration such as porosity, wettability, stiffness, strength, elasticity, biodegradability, and cytocompatibility. For example, a weight-bearing bone may require a stronger material that mimics the strength of the native tissue as the new bone regenerates, in comparison to a non-weight-bearing bone. Materials must withstand water absorption without rapid deterioration, yet gradually degrade overtime so that (1) new tissue can grow and function independently and (2) does not create a permanent implant. Additionally, many tissues require a 3D porous structure that allows blood vessel infiltration for constant nutrient transport as cells are building new tissue . The optFinally, tissue engineering materials must demonstrate properties of cytocompatibility, including cell adherence, cell viability, and stimulation of cell differentiation. Studies have shown that 3D scaffolds support cytocompatibility better than their 2D control counterpart ,26,27,28Carbon-based nanomaterials have gained attention for treating various tissue defects ,30,31. NCarbon nanomaterials include fullerenes, carbon nanotubes, nanodiamonds, carbon-based quantum dots, and graphene . Of thesPristine graphene, which is graphene in its original form, is hydrophobic (due to hydrocarbon contamination following air exposure) thereby lacks dispersion in water which raises aggregation/toxicity concerns when delivered in vivo . This lireducing the number of oxygen atoms found in GO. Reports estimate that rGO restores 80% sp2 structure with the remaining sp3 bonds derived from residual oxygen (C:O = 13:1) . S. S55]. SInterestingly, Nalesso et al. reported in vivo bone regeneration in critical sized defects using 3D printed scaffold of PCL and graphene nanoplatelets. In fact, the team fabricated electroactive scaffolds which enhanced bone regeneration in a rat calvarial bone defect model. Electroactivity of the scaffolds was modulated by varying the amount of graphene used . Two recAlternatively, other laboratories have coated graphene onto 3D scaffolds to enhance mechanical strength and cytocompatibility ,83. For It has thus, been established without a doubt that 3D construction of graphene scaffolds is the next step for clinical translation in tissue engineering. This research is important as the last decade of traditional 2D cell culture systems have shown that graphene substrates support stem cell differentiation into various lineages. These cell lineages are influenced by the concentration of graphene, its functionalization, shape, and the stem cell source ,86. But Although graphene materials have rapidly emerged as bone substitutes, few studies have examined the mechanisms behind its ability to induce osteogenesis. Some theories suggest the carbon arrangement imitates an organic bone ECM microenvironment, attracting cells to attach, self-renew, and differentiate . An ECM Despite this excitement, a major question is determining the best concentration and form of graphene that specifically sustains bone differentiation without collateral damage. BM-MSCs cultured on GO (0.1 µg/mL) had increased proliferation rates; however, at high GO concentrations (10 µg/mL), the BM-MSCs shrank and subsequently had reduced cell proliferation after just 3 days of culturing . SimilarDespite this ongoing challenge, graphene materials have versatile ways in influencing bone regeneration. For example, graphene can indirectly support bone regeneration as a delivery vehicle that controls the release of potent BMP2 growth factors ,106,107.Even though the potential of graphene and its derivatives in biomedicine has been realized, and, as described in the above section, 3D-printed scaffolds are important for the success of a bone tissue engineering strategy, practical applications of 3D-printed graphene scaffolds are still limited. Despite the availability of various 3D-printing techniques including fused deposition modeling, direct ink writing, selective laser sintering, and stereolithography, the use of 3D-printed scaffolds in human or veterinary medicine is limited. This can be attributed to variations associated with graphene manufacturing processes, leading to variations in cell response. For instance, the flaky texture of pristine graphene resembles sawdust particles, and thus is not a candidate for direct printing. In other words, the flaky texture draws challenges in creating pure graphene scaffolds via 3D-printing. This limitation is addressed by incorporating graphene flakes within a polymer  ink that sustains a 3D shape upon printing ,115,116.Despite the excitement of graphene materials and their use as a 3D scaffold for tissue engineering, their therapeutic use is still a novel idea and has yet to undergo a human clinical trial. As with any new substance, the primary question to address will always be safety. Since graphene nanoparticles exist in a variety of sizes (1 to 100 nm in length and 1–10 nm in thickness), and derivatives (oxidized or reduced with varying oxygen content), each form exhibits unique physicochemical properties, which can pose occupational and environmental risks to humans and animals. Information ,118 regaThere are a growing number of in vivo reports regarding the toxicity of graphene materials ,125,126.Wang et al., 2011, evaluated GO toxicity in mice after 30 days of exposure to one of three concentrations: 0.1 mg, 0.25 mg, or 0.4 mg . ResultsA similar study from a separate laboratory also examined GO toxicity in mice following a low dose (1 mg/kg body weight) and a high dose (10 mg/kg body weight) via IV injection . After 1With growing pulmonary toxicity concerns, Singh et al., 2012 compared the lungs of mice injected with either GO or graphene that was functionalized with amine groups (G-NH2) . After 1Schinwald et al., 2012 evaluated the risk of graphene nanoplatelets (GP) (average thickness of ~10 nm) following either inhalation or intrapleural injection in mice . For inhAmrollahi-Sharifabadi et al., 2018 studied graphene oxide nanoplatelets (GONs) and how various doses measured using hematological and histological parameters after 21 days in vivo. They concluded that four cumulative doses of 50 (200 mg/kg) and 150 (600 mg/kg) did not show any toxicity effects in the serum levels, liver, kidney, spleen, lung, intestine, brain, or heart. However, 500 (2 g/kg) showed a significant change in all levels except the heart .Most recently, Tabish et al., 2018 studied the toxicity of graphene nanopores (GNP) after a single IP injection or multiple injections  over 27 days at doses of 5 mg/kg or 15 mg/kg in a rat model . All dosConventional strategies of repairing tissue defects have relied on exogenous stem cells and 2D substrates. However, stem cell-based therapies have many limitations with future strategies turning to 3D structures that both support and attract cell differentiation within the injury site. Graphene, a carbon-based biomaterial, is under thorough research for repairing various tissues such as bone, cartilage, nerve, and heart. However, in vitro work of graphene has mainly been studied as a 2D monolayer or a 3D foam, whereby scaffold morphology is poorly controlled. With the revolution of 3D-printing technology, questions have asked whether graphene scaffolds can be 3D printed. Currently, there is a paucity of studies that have attempted 3D-printed graphene scaffolds for tissue engineering. These studies have mainly surfaced in the last few years, but it is expected that more developments will evolve in the future. Finally, 2D-graphene substrates have predominantly been studied in supporting new bone differentiation. Therefore, 3D-printed graphene scaffolds are the next step for clinical application in bone tissue engineering. However, understanding the optimal concentration of all graphene derivatives that balances both bone differentiation and minimizes toxicity is necessary prior to transplantation. Overall, there is great excitement over 3D-printed graphene scaffolds, but much work is necessary before standardization within tissue engineering.