We report theoretical study of the effects of energy relaxation on the tunneling current through the oxide layer of a two-dimensional graphene field-effect transistor. In the channel, when three-dimensional electron thermal motion is considered in the Schrödinger equation, the gate leakage current at a given oxide field largely increases with the channel electric field, electron mobility, and energy relaxation time of electrons. Such an increase can be especially significant when the channel electric field is larger than 1 kV/cm. Numerical calculations show that the relative increment of the tunneling current through the gate oxide will decrease with increasing the thickness of oxide layer when the oxide is a few nanometers thick. This highlights that energy relaxation effect needs to be considered in modeling graphene transistors. Two-dimensional graphene has a zero band gap and linear energy-momentum dispersion. Graphene’s linear energy − momentum dispersion causes its charge carriers to behave as massless Dirac fermions that can travel at a speed of 10observed . The val devices .m* = 0.012 m0 (m0 is the free electron mass) that is magnetic field independent in monolayer graphene has been obtained from the temperature dependent amplitude of SdH oscillation. It has reported that in a graphene device, the energy relaxation time is about 1 ps for carrier-dense samples [Hot electron phenomena have become important for understanding all modern semiconductor devices. In hot electron transport, elastic collisions dominant its momentum relaxation, whereas inelastic interaction with phonons determines its energy relaxation. Electro-thermal analysis is used for predicting the heat generation of semiconductor devices. The governing equations of electro-thermal analysis consist of the continuity equation, momentum conservation equation, and energy conservation equation . The non samples .2 and SiO2 stacks also needs quantum coupling effects correction [2/SiO2 gate dielectric stacks in experiments is still higher than predictions from motion-coupled quantum model which neglects the energy relaxation of hot electrons [The parabolic band effective mass theory is used to investigate the quantum feature of electrons in the inversion layer of metal-oxide-semiconductor field-effect transistors (MOSFETs), quantum well, and superlattices. And one can notice that most studies assume that electron motions in three spatial directions are described by decoupled Schrödinger equations. It has been pointed out that this decoupled motion approximation results in a large error in evaluating the tunneling current and quantization of the inversion layer when the channel electron velocity is higher than the thermal velocity , 11. It rrection . Howeverlectrons . This im2 layer at 3.5 V, which is the maximal gate-source voltage, is found to be 3 μA/cm2 [2 at the same gate bias of 3.5 V [For graphene FETs, the tunneling current through a 50-nm SiO3 μA/cm2 . This gaof 3.5 V ). Proceeof 3.5 V , 17, whiThe three-dimensional full time independent Schrödinger equation can be written as 1\documenϕ represents the potential energy, m⊥ and mz denote the mass in and perpendicular to the plane of the graphene/gate oxide interface plane at the bottom of the conduction band of graphene, respectively, z is the tunneling direction which is perpendicular to the graphene/gate oxide interface, p⊥ and pz represent the electron momentum operators perpendicular to and parallel with the graphene/gate oxide interface, respectively, and E is the total energy of an electron. To calculate the tunneling current in a graphene FET, the Schrödinger equation for the electron motion in the xy plane keeps unchanged; thus, we need to solve the Schrödinger equation along the z direction. At the same time, one can note that m⊥ and mz (in and perpendicular the plane) change along the z direction because a graphene FET is a multi-layer structure, which has layer dependent effective electron mass along z direction. Therefore, Eq. ϕ(z) represents the potential energy along z axis. Note that x-y plane). According to the conservation of the total energy and transverse momentum of a tunneling electron, the longitudinal energy of a tunneling electron in the graphene is E is the total energy of a tunneling electron). Thus, in the active material (graphene) and the gate oxide  applied to the tunneling electron in the gate oxide mainly depends on the temperature of channel electrons, energy relaxation rate, electron mobility, and effective electron mass.where perature . Usuallyperature . Under tre TL is 5\documenCox and tox are the capacitance and the thickness of the gate oxide, respectively, V is the voltage across the gate oxide layer, ε0 is vacuum permittivity, and ε the dielectric constant of the gate oxide. With setting the bottom of the conduction band of the graphene as zero energy point, the electrons density in the graphene layer of a graphene FET can be obtained asFor graphene FETs, electron or hole density in the channel induced by the voltage across the gate oxide can be written as 7\documenf is the Fermi-Dirac distribution function, N(E) the density of states, T the transmission probability, and v(E) the electron velocity along the tunneling direction.At last, an electron current will form because of the imbalance between the Fermi level of the gate and that of substrate induced by the voltage across the gate oxide, and it can be written as 9\documenN partial sub-barriers. When a voltage across the gate oxide is applied, the potential barrier shape of the gate oxide will change from square to triangular. Thus, the shape of all sub-barriers is trapezoid as this triangular potential barrier is divided into N partial sub-barriers. For trapezoid potential barrier, Airy functions secure an analytical and exact expression of the transmission probability. This is why we use Airy function approach to solve one-dimensional Schrödinger equation to calculate the transmission coefficient of tunneling electrons. Considering the boundary conditions that the wave function and quantum current density are continuous at graphene/gate oxide interface, the transmission probability can be calculated according to the following equation [S11 represents the row 1 and column 1 element of the transfer matrix S which isAfter obtaining the effective potential barrier of the graphene transistors, we can divide the potential barrier into equation , 2410\dowhereMj is a (2 × 2) product matrix, mj* the effective electron mass for jth trapezoid barrier, Fj the electric field across the jth trapezoid barrier, V(xj) the voltage at the position of xj, and Ai (Ai’) and Bi (Bi’) are Airy functions and their corresponding derivatives, respectively.where The abovementioned method has been applied to study high-energy (hot) electron distribution in the graphene layer induced by its linear energy-momentum dispersion and the gate leakage current in graphene FETs . Firstly2 has an electron affinity of 0.9 eV and relative dielectric constant of 3.9. The barrier height used in this study is 3.5 eV. Electron mass of 0.012 m0 in 2D graphene according to [m0 in the gate oxide, was used in all calculations. Because graphene is a 2D crystal, there is no band-structure in the tunneling direction which is perpendicular to the graphene plane. This is the reason why we use free electron mass along such a direction in all calculations. The energy relaxation time of 1 ps [2 V−1 s−1 [In this work, dielectric constant of 2.4 has been used in graphene field-effect transistor . The thirding to , and 0.5How the tunneling current through the gate oxide with and without considering the energy relaxation of channel electrons changes with the gate voltage at different electron mobility is illustrated in Fig. Figure The effects of the electron mobility on the gate leakage current of a graphene field-effect transistor are given in Fig. The reduction in the barrier height caused by the channel electric field and the electron mobility was detailed shown in Fig. The electron energy relaxation time impacting on the tunneling current through the gate oxide at different channel electric fields is illustrated in Fig. The reduction in the barrier height as a function of the electron energy relaxation time at different channel electric fields is depicted in Fig. Figure Figures Figure It is well known that metal contacts  are crucial in the creation of high-performance graphene FETs . The impIn conclusion, energy relaxation effect on the tunneling current through the gate oxide of a graphene field-effect transistor has been theoretically investigated. The quantum coupling among the three electron spatial motion directions can result in a larger reduction in the barrier height. Note that the energy relaxation of electrons may largely increase electron temperature. The energy relaxation due to electron thermal motion along the graphene channel will lower the effective barrier height which enhances the gate leakage current. This study points out that the increase in the electron mobility, channel electric field, and the energy relaxation time always leads to higher leakage current in a graphene field-effect transistor. Based on quantified results, this paper also details how to secure high-performance graphene device and ICs. Devices with energy relaxation of time shorter than 0.1 ps or a working channel field of less than 1 kV/cm must be secured. It is also worth studying how electro-thermal effects affect the performance of multi-stack gated 2X nano-meter or 1X nano-meter MOSFETs and relative study will be reported later.


Long interspersed element type 1  is a mobile genetic element comprising about 17% of the human genome, encoding a newly identified ORF0 with unknown function, ORF1p with RNA-binding activity and ORF2p with endonuclease and reverse transcriptase activities required for L1 retrotransposition. L1 utilizes an endonuclease (EN) to insert L1 cDNA into target DNA, which induces DNA double-strand breaks (DSBs). The ataxia-telangiectasia mutated (ATM) is activated by DSBs and subsequently the ATM-signaling pathway plays a role in regulating L1 retrotransposition. In addition, the host DNA repair machinery such as non-homologous end-joining (NHEJ) repair pathway is also involved in L1 retrotransposition. On the other hand, L1 is an insertional mutagenic agent, which contributes to genetic change, genomic instability, and tumorigenesis. Indeed, high-throughput sequencing-based approaches identified numerous tumor-specific somatic L1 insertions in variety of cancers, such as colon cancer, breast cancer, and hepatocellular carcinoma (HCC). In fact, L1 retrotransposition seems to be a potential factor to reduce the tumor suppressive property in HCC. Furthermore, recent study demonstrated that a specific viral-human chimeric transcript, HBx-L1, contributes to hepatitis B virus (HBV)-associated HCC. In contrast, host cells have evolved several defense mechanisms protecting cells against retrotransposition including epigenetic regulation through DNA methylation and host defense factors, such as APOBEC3, MOV10, and SAMHD1, which restrict L1 mobility as a guardian of the human genome. In this review, I focus on somatic L1 insertions into the human genome in cancers and host defense mechanisms against deleterious L1 insertions. HBV integration into the human genome was found in most HBV-related HCC and it has been implicated in the development of HCC. An initial study proposed that HBV integration occurs randomly without preferred integration site   including IFNα and IFNβ have been involved in innate immunity against viruses. In this regard, a recent study reported that L1 induces IFN1 and IFN1, in turn, inhibits L1 retrotransposition, suggesting that IFN1 controls propagation of L1 as well as maintenance of genomic integrity  silence L1 during genome reprogramming in the embryonic male germ line , by the Research Program on Hepatitis from Japan Agency for Medical Research and Development, AMED, and by Takeda Science Foundation.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we demonstrate that OH groups can efficiently induce robust magnetic moments on the basal plane of the graphene sheet. We show that the inducing efficiency can reach as high as 217 μB per 1000 OH groups. More interestingly, the magnetic moments are robust and can survive even at 900°C. Our findings highlight the importance of OH group as an effective sp3-type candidate for inducing robust magnetic moments on the basal plane of the graphene sheet.Inducing robust magnetic moments on the basal plane of the graphene sheet is very difficult, and is one of the greatest challenges in the study of physical chemistry of graphene materials. Theoretical studies predicted that introduction of a kind of  For the edge-type approach, it mainly includes ion irradiation to create point vacancies47891011121415etc. The biggest weakness of this approach is that it can only induce limited edge magnetism at the vacancy or edge sites of the graphene sheet. For example, although a single isolated vacancy can theoretically induce magnetic moment μ ~ μBMagnetic properties of graphene are gaining increasing interest recently for its potential application in spintronics, since its weak spin-orbit interaction and long spin-relaxation time provide ideal conditions to manipulate the spins1234sp3-type defects on the basal plane of the graphene sheet and, thus more defects can be introduced. Therefore, higher spin density is expected by this approach, and which is considered as hitherto the most promising approach to induce robust magnetic moments on the graphene sheet81819202122568181920242525sp3-type alternative that can effectively induce robust magnetic moments on the basal plane of the graphene sheet.Quite differently from the edge-type approach to create defects only at vacancy or edge sites, the basal-plane approach is to introduce μB6276862930632343536Recently, theoretical studies confirmed that the chemisorption of a single OH group on the basal plane of graphene can introduce magnetic moment ~1 μB per 1000 OH groups) and high stability (surviving even at 900°C). Our results demonstrate that OH group is an effective candidate for inducing robust magnetic moments on the basal plane of the graphene sheet. These results have important implications for the synthesis of high-magnetization graphene for future applications of spintronics. In particular, the robust magnetic moments we observe in OHG provide the prerequisite to induce long-range magnetic ordering. Conversely, the ferromagnetic coupling between these basal-plane magnetic moments still needs to be addressed for the realization of ferromagnetic graphene with high magnetization.In this study, we prepared hydroxylated graphene (OHG) with a very high magnetization of 2.41 emu/g by annealing of graphene oxide (GO) to remove the unstable oxygen groups and leave the stable OH groups. We demonstrate that OH groups can effectively induce robust magnetic moments on the basal plane of the graphene sheet. Our results highlight the two great superiorities of OH groups: high magnetic inducing efficiency  oxygen groups present on GO mainly in forms of epoxy, OH, carbonyl, and carboxyl groups; and (ii) most epoxy and OH groups lie on the basal plane of the graphene sheet, and carbonyl and carboxyl groups can only sit at the edge or vacancy sites3815404042μB on the basal plane of the graphene sheet627343536It is known that (i) epoxy is nonmagnetic for it creates equal defects in A and B sublatticeThe atomic force microscope (AFM) image of aGO shows that the heights of most of them are ~0.7 nm see , indicatsp3-type defects  measurements were carried out. As shown in 3434ca. 34%. Therefore, one can conclude that many new OH groups were created with the decomposition of other unstable oxygen groups during annealingsp3-type defect, is predominant among all the oxygen groups in all the OHG samples, and (ii) the dominance increases with the increase of the annealing temperature. Group A samples have a high OH coverage ~8 at.%, while in Group B, the coverage decreases to a steady value ~3 at.%, implying that a number of OH groups decomposed at high temperatures. Even so, the OH coverage of OHG-900 still maintains 2.96 at.%, confirming the high thermal stability of OH group343536ca. 3 to 10 at.% by changing the annealing temperature.According to the O contents and C–OH ratios in oxygen bondings, we calculated the OH coverage (OH/C) of aGO and the OHG samples . The OH M of OHG-500 on temperature T and it fits well with Curie law χ = C/T. χ is the susceptibility and χ = M/H. H is the applied magnetic field. Inset is the corresponding 1/χ – T curve, which demonstrates a linearly, purely Curie-like paramagnetic behavior. The M – T dependences of aGO and other OHG samples were also measured, and they are all Curie-like paramagnetic just like OHG-500. The M – H dependences were also measured. At 300 K, all the samples are linearly diamagnetic , Bk is the Boltzmann constant, N is the number of present magnetic moments, S is the spin angular momentum number, and g is the Landau factor assumed to be 2. The fitted values for all the samples are summarized in sM of 1.27 emu/g, much higher than the value observed in GO with long oxidation duration  located at the vacancies and/or edges. Generally speaking, some factors, such as isolated vacancies814etc, may only contribute to the edge magnetism. However, the vacancies and edges generated by annealing GO are disordered662728We now consider the magnetic source for the robust magnetic moments observed in aGO and the OHG samples. As mentioned above, our OHG is a kind of defective graphene with sM. The C contents are calculated based on the XPS results of C and O atomic ratio. In short, if the O/C atomic ratio of the sample is x and the OH coverage is y, its chemical formula can be represented as COxHy. It is found that the spin density is ~4.42 μB per 1000 C for aGO, which increases up to ~6 μB per 1000 C for the OHG samples of Group A. In view of the fact that the weight loss of aGO by annealing aGO at 200°C is ca. 34%, one can make a reasonable assumption that magnetic contribution of the newly annealing-created OH groups is low. The reason may be that the newly created OH groups prefer to lie at edges or vacancies6μB per 1000 C, this may result from both the decrease of the OH coverage  and inducing efficiency (71.5 μB per 1000 OH groups). It indicates that the magnetic moments are robust despite that some of them vanished with the unavoidable decomposition of original ‘magnetic’ OH groups on the basal plane of the OHG sheet.To take a deeper insight into the magnetic properties of aGO and the OHG samples, we calculated their spin densities  accordincoverage  and the coverage . Surpris7 clusters on the basal plane of the graphene sheet477 cluster can induce a large magnetic moment of 5 μB7 cluster configuration inducing 5 μB was sketched in Bμ ed in μB , close tS = 2 are generated by 1000 C atoms, so on average, a 25 × 25 or 26 × 26 graphene supercell (~666 C atoms) contributes a large magnetic moment. Assuming that the graphene sheet is flat and the magnetic moments distribute uniformly, we obtain that the average distance is at least 6 nm. Since the OH group interaction is negligible when the space is larger than 3 nmInducing high-density magnetic moments on graphene is usually the prerequisite to induce long-range magnetic orderingμB per 1000 OH groups, it is still lower than the theoretical value of 1 μB per single OH group. Namely, most of the OH groups on the aGO and OHG sheets do not contribute magnetic moments. It may attribute to the analogous clustering of OH groups, similar to the case of fluorine on graphene sheet8In addition, we should note that even the highest efficiency observed in aGO is as high as 217 μB per 1000 C by reducing the oxidation duration, and have observed a very high magnetic inducing efficiency of 217 μB per 1000 OH groups. By annealing of aGO to remove the ‘non-magnetic’ unstable oxygen groups and leave the ‘magnetic’ stable OH groups, we have obtained OHG with very high magnetization of 2.41 emu/g. Our results provide the solid experimental evidence that OH groups can induce robust magnetic moments on the basal plane of the graphene sheet. More importantly, we demonstrate that the magnetic moments are highly stable and can survive even at 900°C. Our results provide an effective sp3-type functional group for generating robust magnetic moments in graphene and, thus represent an important step for the potential applications of spintronics.In summary, we have obtained aGO with high spin density of 4.42 3, 48 g KMnO4, and 384 ml condensed H2SO4 was stirred for 1.5 h at 0°C, and then followed by another 2 h stirring at 35°C. Thereafter, 320 ml H2O was added into the mixture within 15 min at a steady flow. Without any delay, the premix of 800 ml H2O and 40 ml H2O2 was added within 6 min at a steady flow. During the process, the temperature of the solution fixed at 35°C. To avoid deeper oxidation, the mixture was immediately transferred to other container and diluted with 2000 ml H2O. The obtained solution was washed for 13 times by repeated 30 min centrifugation at 13000 rpm. The sediment was redispersed into water and centrifugated at 6000 rpm for 20 min, and only the supernatant was left to get GO sheets with high few-layer ratio. Finally, the lightly oxidized aGO was obtained by drying the GO solution in vacuum freeze drier at 50°C. To avoid other extra source which may induce magnetic moments, no ultrasonication was adopted. The OHG samples were synthesized by annealing aGO in Ar atmosphere at different temperatures for 1 h.The as-prepared GO was synthesized by chemical exfoliation of 8 g natural flake graphite powder (500 mesh)−8 emu , and all data have been corrected for the diamagnetic contribution by subtracting the corresponding linear diamagnetic background at room temperature. The 3 d impurity elements of all the samples are measured by inductively coupled plasma (ICP) spectrometry .The morphologies of the samples were investigated by TEM  and AFM . XPS measurements were performed on PHI5000 VersaProbe  using Al Ka radiation. Raman images were performed on inVia confocal Raman microscope  using a laser excitation of 532 nm. The magnetic properties of the samples were measured using SQUID magnetometer with a sensitivity less than 10Our calculation was performed by density function theory as implemented in the SIESTA codeN.J.T. proposed and supervised the project, T.T. and N.J.T. designed the experiments, T.T. and Y.L. carried out the experiments, Y.P.Z. performed the DFT calculations, T.T., X.G.W. and N.J.T. analyzed the data and wrote the manuscript. Y.P.Z., X.G.W., Y.L., F.C.L., Q.H.X. and Y.W.D. had valuable discussions and edited the manuscript. All the authors participated in discussion of research.Supplementary Information


Onchocerca volvulus transmission by Simulium guianense s.l. in 16 previously hyperendemic Yanomami communities in southern Venezuela after 15 years of 6-monthly and 5 years of 3-monthly mass ivermectin treatment.The World Health Organization (WHO) has set goals for onchocerciasis elimination in Latin America by 2015. Most of the six previously endemic countries are attaining this goal by implementing twice a year  mass ivermectin (Mectizan®) distribution. Elimination of transmission has been verified in Colombia, Ecuador and Mexico. Challenges remain in the Amazonian focus straddling Venezuela and Brazil, where the disease affects the hard-to-reach Yanomami indigenous population. We provide evidence of suppression of Baseline and monitoring and evaluation parasitological, ophthalmological, entomological and serological surveys were conducted in selected sentinel and extra-sentinel communities of the focus throughout the implementation of the programme.S. guianense flies tested by PCR none had L3 infection (heads only). Prevalence of infective flies and seasonal transmission potentials in 2012–2013 were, respectively, under 1 % and 20 L3/person/transmission season. Serology in children aged 1–10 years demonstrated that although 26 out of 396 (7 %) individuals still had Ov-16 antibodies, only 4/218 (2 %) seropositives were aged 1–5 years.From 2010 to 2012–2015, clinico-parasitological surveys indicate a substantial decrease in skin microfilarial prevalence and intensity of infection; accompanied by no evidence  of ocular microfilariae in the examined population. Of a total of 51,341 We report evidence of recent transmission and morbidity suppression in some communities of the focus representing 75 % of the Yanomami population and 70 % of all known communities. We conclude that onchocerciasis transmission could be feasibly interrupted in the Venezuelan Amazonian focus.The online version of this article (doi:10.1186/s13071-016-1313-z) contains supplementary material, which is available to authorized users. Onchocerca volvulus Leuckart and transmitted among humans through the bites of blackfly species of the genus Simulium Latreille. The embryonic stages of the parasite  migrate through the skin and cause severe itching, skin disease, and ocular lesions, with the severity of the clinical manifestations depending on the length of exposure to blackfly bites and the density of mf in the skin [Onchocerciasis is a chronic and cumulative skin and ocular disease caused by infection with the filarial nematode the skin , 2. Visuthe skin .In the Americas, the infection was formerly prevalent in 13 endemic foci distributed in 6 countries , where 565,232 persons were considered at risk of infection . In VeneSimulium guianense sensu lato (s.l.) Wise, S. incrustatum Lutz, and S. oyapockense s.l. Floch and Abonnenc are the main vectors in the Southern Focus of Venezuela, with the former species being the most competent for O. volvulus and the predominant human-biting blackfly in most of the hyperendemic areas of the focus [Simulium incrustatum, with a lower vector competence than S. guianense s.l., contributes to the transmission of onchocerciasis in mesoendemic and some hyperendemic areas, whereas S. oyapockense is the main vector in hypoendemic communities with low intensity of transmission [he focus , 10. Simsmission .O. volvulus, and interruption of transmission by 6-monthly mass administration of ivermectin , delivered by mobile teams with a therapeutic coverage ≥85 % of eligible population in all the endemic communities of the region, including hypoendemic areas [The strategy adopted by the Onchocerciasis Elimination Program for the Americas (OEPA) since its commencement in 1993 has included elimination of new (ocular) morbidity caused by ic areas , 14.  communities localized in different geographical areas of the southern Venezuelan focus after 15 years of 6-monthly and 5 years of 3-monthly mass drug administration (MDA) of ivermectin.The present work reports on the progress towards onchocerciasis elimination in southern Venezuela, according to the protocols proposed by the World Health Organization (WHO), which include in-depth parasitological, entomological and serological surveys, as well as guidance on operational thresholds . SpecifiThe parasitological, clinical, entomological and serological studies received ethical clearance from the Ethics Review Committee of CAICET . All the participants signed an informed consent form before undergoing any examination, testing, or agreeing to help as human attractants for entomological collections. Additionally, there was active participation of Yanomami volunteers (and their organization “Horonami”) in the identification of new communities and distribution of ivermectin across the focus.Text S1. Geographical and environmental characteristics of the Venezuelan part of the Amazonian onchocerciasis focus). The main onchocerciasis transmission seasons (regardless of the Simulium vector species present), occur during the dry to rainy (February–April) and rainy to dry (September–November) transitions [Onchocerciasis transmission in southern Venezuela occurs in the lowlands  and uplands 500–1200 m asl) of the Upper Orinoco, Upper Siapa and Upper Caura River basins (in the Amazonas and Bolivar States), which are part of the ancient Guayana Shield of northern South America, the oldest (3600 million years) region of the world .The Venezuelan part of the focus encompasses 12 geographical areas: Padamo; Ocamo; Mavaca; Platanal; Guaharibos; Orinoquito; Parima; Chalbaud; Ventuari; Uasadi; Caura, and Siapa. Within these 12 main areas, 31 geographical sub-areas have been described  Yanomami at-risk population has been estimated as 13,231 people, living in 241 ‘shaponos’ scattered deep in the forest, and practicing shifting cultivation, hunting, fishing and gathering of forest products . Their sAccording to OEPA guidelines , those cS. guianense s.l. in most of the localities [The pre-treatment levels of endemicity across all geographical areas were very heterogeneous Table , with socalities , 22.The criteria for selection of sentinel and extra-sentinel communities for regular monitoring and evaluation activities, consisting of in-depth epidemiological evaluations included: a) hyperendemic status, b) relative ease of accessibility by the mobile teams that conduct such evaluations, c) existence of historical, baseline epidemiological data prior to wide-spread ivermectin distribution; d) illustrative of the simuliid species composition of the focus. Accordingly, eight communities were selected as sentinel communities, namely : (1) Hasupiwei  in Guaharibos–Unturán; (2) Awei  and (3) Pashopëka  in Upper Ocamo–Shitari; (4) Koyowë (= Coyowë) , (5) Waharafitha—previously Fubalema— and (6) Matoa  in Orinoquito; and (7) Kanoshewë  and (8) Niayopë—previously Niyayowë— in Parima–Shamatari. Since the size of Yanomami communities is generally small, to increase sample size an additional 8 villages were selected as extra-sentinel communities. These were: (9) Yaurawë  in Guaharibos–Unturán; (10) Masiriki  and (11) Toumawei  in Parima (A); (12) Arokofita  and (13) Okiamo  in Parima (B); (14) Warapawë  in Parima (C); and (15) Kakarama  and (16) Pokoshiprare  in the Parima–Shamatari sub-area within the Parima area, the latter two communities derived from Yoreshiana A and Yoreshiana B  iliac crests were taken from each examined individual with a 2-mm Holth corneoscleral punch, and incubated for 24 h in buffered saline solution; the emerging mf were counted under an inverted microscope and snips were weighed to express results as mf/mg [Parasitological surveys were carried out in the sentinel and extra-sentinel communities of the focus at baseline 1981), pre-ivermectin MDA , and during ivermectin (twice a year and subsequently quarterly) MDA . The skin snip method was used to determine the prevalence and intensity of 1, pre-ivas mf/mg . When itas mf/mg , 26.Text S4. Calculation of parasitological indices).Ophthalmological evaluations were carried out during similar periods . The prevalence of microfilariae in cornea (MFC) and/or in the anterior chamber (MFAC) was determined by an ophthalmologist experienced in conducting onchocerciasis ocular evaluations for OEPA (HM). Ocular examinations were conducted with a slit-lamp in a darkened area after patients were asked to sit with their head between their legs for 5 min to allow any mf present in the anterior chamber to settle in a visible position in order to determine MFAC , 27. TheO. volvulus L3 larvae and the mean number of L3 larvae per fly were evaluated using classical dissection methods, examining the abdomens, thoraces, and heads of flies [S. guianense s.l. were calculated according to [Detailed entomological evaluations were carried out between 1982 and 2000 in two sentinel communities (Koyowë (=Coyowë) and Niayopë (=Niyayowë) from the hyperendemic geographical areas of Orinoquito and Parima, respectively. In these communities, the proportion of flies with of flies . The basrding to .S. guianense s.l. females were collected throughout the high O. volvulus transmission seasons (January–March/February–April or September–November) by using collecting protocols adjusted to the local transmission conditions previously established in the area and known periods of highest biting activity by parous flies [In a selection of sentinel  and extra-sentinel (Arokofita) communities, and during several consecutive collection days , host-seeking us flies , 30. AllO. volvulus-specific DNA probes has been generally applied to examine pools of flies in the OEPA region [O. volvulus using a species-specific PCR assay [HBR), seasonal biting rate (SBR), seasonal transmission potential (STP) and annual transmission potential (ATP) .Polymerase chain reaction (PCR) using A region . TherefoCR assay  . According to the results of the most recent epidemiological evaluation (2008–2009 for Awei and 2013–2015 for the remainder), 8 out of 16  had 0 mf in skin (and eyes), and 7 out of the 8 remaining communities had CMFL <1 mf/ss. A striking decline was also observed in the prevalence of MFC , with prevalence of MFC as high as 12 % Fig. . Of thesS. guianense s.l. in two sentinel communities prior to ivermectin MDA. Biting rates were high, particularly in Koyowë, where the monthly biting rate (MBR) reached values up to 23,997 bites person−1 month−1 and the monthly transmission potential (MTP) up to 576 L3 person−1 month−1. Seasonal biting rates (during the higher transmission months of January through March/April plus October through November) were in excess of 100,000 bites per person per transmission season. As these flies were processed by manual dissection, it was possible to determine both the proportion of infective flies and the number of L3 in the flies, with the latter ranging from 0.001 to 0.036 L3/fly  by dissection and 0.38 % (95 % CI 0.19–0.69 %) by PCR are in agreement with the value of 0.37 % for the entire baseline study period , in contrast with the 7.4 (0.37 %) infective flies per 2000 dissected flies recorded at baseline . In Hasupiwei, Pashopëka and Arokofita, after 2–3 years of quarterly ivermectin treatment, all 8085, 6464 and 12,793 flies respectively tested in 2012–2013 were PCR negative (with the upper 95 % CI for STP of 3–4 L3/person/transmission season).Results of the entomological evaluations carried out from 2006 to 2013 in Hasupiwei, Pashopëka, Koyowë and Arokofita are presented in Table O. volvulus Ov-16 for this age group  in 2009 to 100 %  in 2015. By 2015, CMFL and MFAC have become, respectively, negative in 7/16 44 %) and 5/7 (71 %) of the communities examined for these indicators. Communities of the Orinoquito sub-area  are still positive for 4 % and 5O. volvulus infection  in children have been the WHO criteria followed to certify focal interruption of parasite transmission [O. volvulus transmission by the most competent vector of the focus, S. guianense s.l., in areas formerly hyperendemic to holoendemic. The dramatic decline in the seroprevalence among children and the lack of evidence of parasite-vector contact suggest that four times per year treatment has successfully suppressed transmission in many of the endemic communities. It will now be necessary to maintain pressure on the parasite population, continuing to suppress transmission, until the existing adult female parasites are either killed or rendered sterile by repeated ivermectin treatments [In the OEPA region, absence or near absence of L3 larvae in the head of blackfly vectors , a 99 % reduction in the intensity of transmission , and the absence of detectable smission . Prior tsmission , 34. Hereatments .S. oyapockense s.l. and/or S. incrustatum [R0 ≥ 1 [O. volvulus, but also on the human blood index  of the various blackfly species and populations therein. At present, this parameter is unknown for the simuliid species prevailing in the Amazonian focus, but field and theoretical studies on S. damnosum s.l. in West Africa indicate that the HBI can be highly variable [SBRs) of S. guianense s.l. lower than 50,000 bites/person/transmission season after at least 4 rounds of treatment with coverage ≥85 % of eligible population. In these communities 133 children under 5 years of age from Pashopëka (Jénita–Putaco); Hasupiwei (Guaharibos); Arokofita, Kanoshewë, Niayopë and Okiamo (Parima B); Warapawë (Parima C); Masiriki and Toumawei (Parima A), and Kakarama (Shamatari) were negative for Ov-16 , where 4/85 (5 %) children aged 1–5 years were seropositive in 2013.Ivermectin is expected to have a faster impact in those areas of the Amazonian focus with lower vector competence blackfly species . These variable  and possvariable . Given tModelling studies conducted with the EPIONCHO transmission model in African savannah settings  have sugOnchocerca parasites along the reticular nature of the Yanomami use of space may indeed be very diffuse, necessitating intensive treatment in all sub-areas; however, if particular networks could be identified as being responsible for most transmission, a more targeted approach could be beneficial towards the goal of achieving elimination.More likely, in the Amazonian focus, and due to the Yanomami network of kinship ties, alliances and hostilities , 22, oncThe semi-nomadic characteristics of the human population, the remoteness of the Yanomami territory, the holoendemic status of some areas, and the ongoing identification of new endemic communities in the Venezuelan part of the Amazonian focus constitute the main challenges for the elimination of onchocerciasis in the Amazonian focus. From 2009, ivermectin treatment frequency has been increased to four times per year in 80 % of the hyperendemic communities in an attempt to hasten interruption of transmission in areas showing slow progress or in communities recently identified and incorporated into the programme at later stages. Sustaining a high geographical and therapeutic coverage for each treatment round is essential. Only the first round of the 2012–2014 quarterly distributions reached the ≥85 % goal. Besides, since the sentinel and extra-sentinel communities included in this study were partly selected because of relative facility of access, there is the possibility that more remote communities received a lower treatment coverage and/or frequency. Currently, there is a total of 72 communities identified as being remote , with a population of 3359 individuals . This represents approximately 25 % of the total population. Most of these communities are receiving regular treatment, and many of them have received more than 8–12 treatment rounds . However, there are communities located in the Upper Siapa and Upper Ocamo-Parima sub-areas that are only accessible by helicopter . This represents only 11 % of the total population and 12 % of all the communities under treatment. Treatment is delivered to these communities when helicopter support is available. Depending on their endemicity status and their connectedness with well-controlled areas, these less accessible communities could pose a risk of infection re-introduction. The use of high spatial-resolution satellite data to identify remote communities in the rainforest is a strategy currently used in an attempt to delineate the extent of the focus, the distribution of transmission zones , and theOur results contribute to the ongoing success of the OEPA strategy , 37, 38.


Two-dimensional (2D) atomic crystals and their heterostructures are an intense area of study owing to their unique properties that result from structural planar confinement. Intrinsically, the performance of a planar vertical device is linked to the quality of its 2D components and their interfaces, therefore requiring characterization tools that can reveal both its planar chemistry and morphology. Here, we propose a characterization methodology combining (micro-) Raman spectroscopy, atomic force microscopy and time-of-flight secondary ion mass spectrometry to provide structural information, morphology and planar chemical composition at virtually the atomic level, aimed specifically at studying 2D vertical heterostructures. As an example system, a graphene-on-h-BN heterostructure is analysed to reveal, with an unprecedented level of detail, the subtle chemistry and interactions within its layer structure that can be assigned to specific fabrication steps. Such detailed chemical information is of crucial importance for the complete integration of 2D heterostructures into functional devices. The properties of 2D materials such as graphene can vary according to the quality and, for vertical devices, the interfaces between materials. Here, the authors report a method using TOF-SIMS, micro-Raman spectroscopy and atomic force microscopy to give high levels of detail of vertical 2D heterostructures. Our characterization approach is able to detect, identify, localize and quantify the various contaminants introduced during fabrication, thus leading to improved manufacturing processes and, therefore, better-performing devices.Here, TOF-SIMS is applied, for the first time to our knowledge, to a 2D atomic crystal heterostructure fabricated by common sequential layer transfer, to study its planar chemical composition at the atomic level, with significant implications for understanding the influence of manufacturing and transfer processes. As a system of choice, the conductor-on-insulator stacking of graphene on h-BN shows promise as a base for high-quality nanoelectronics11132 substrate that was partially covered by mechanically exfoliated h-BN flakes, was first characterized by (micro-) Raman spectroscopy (−1 and AFM height topography confirmed large (tens of microns), continuous graphene patches extending over the h-BN flakes and SiO2 substrate . However, atop SiO2 the graphene D peak is disentangled from any substrate-related signal and presents a relatively low intensity compared with the G peak , thus confirming a relatively small defect density of the graphene domains .To evaluate its final configuration, the 2D system, composed of a CVD-grown, wet-transferred graphene onto a SiOtroscopy  and AFM troscopy . Initialrate  sputtering. As deposited  that is better or comparable to micro-Raman (−1), we can progressively study the outer adsorbed species, the top graphene layer, the interfacial contaminants and the underlying h-BN and SiO2., before  and afte, before  ∼60 s ofeposited , the traro-Raman , TOF-SIM2 interfaces and (ii) estimate sputtering rates for the species of interest, the surface topography of the 2D heterostructure was investigated by AFM before (+ at 500 eV energy. Both height distributions (histograms) shown in 2 substrate (Z≈−65 nm) and h-BN flake surface (Z≈150 nm). An obvious change in shape from Gaussian to Lorentzian and a significant reduction of the width (FWHM) for the two main histogram peaks suggest that Cs+ sputtering substantially decreases the surface corrugation of both the SiO2 substrate and h-BN flake surface , both the acquired data (discrete markers) and their 1-point spline interpolations (continuous curves), representing single-layer graphene, adventitious organic material chemisorbed at the h-BN surface and the h-BN flake, respectively. Owing to the discontinuous nature of the transferred graphene (tens-of-microns-sized patches), the depth profiles on both the graphene/h-BN and graphene/SiO2 systems were reconstructed from the initial data using regions of interest that ensured graphene coverage . For depth profiling, polyatomic species such as C3− and 10BB− were chosen to avoid the intrinsic artefacts that monoatomic species such as C− and B− have due to residuals from oxides or other chemisorbed adventitious species. An estimation of the atomic mixing length between graphene and h-BN or SiO2 can be obtained by applying the so-called mixing–roughness–information (MRI) model3− profile , we use the forward calculation procedure3− data points. The fitting procedure searches for the minimum deviation between the simulated profile and the C3− profile points . This is a result of the chemisorbed species (identified below) accumulated in graphene during the transfer process and which are chemically interacting with the SiO2 substrate 2S2O8  equates to about 0.4% of the total mass (<0.08 atomic %). However, given its depth localization, the copper residue appears to be spatially decoupled from the graphene overlayer and thus may not contribute to its defect density. Moreover, it is virtually undetectable by other spectroscopic techniques, for example, XPS, accounting for <0.03% of the mass (<0.006 atomic %) in the first 10 nm of the graphene/h-BN surface 2S2O8 . An oxide system . The cor− and CB−) are a result of chemisorbed adventitious sulfur or carbon, respectively, with h-BN and are formed before the addition of graphene. Most of the physisorbed adventitious species  at the graphene, h-BN and SiO2 surfaces are likely diffused to the sides of the graphene domains by the capillary forces exerted at the graphene/h-BN and graphene/SiO2 interfaces or uniformly spread at the graphene surface− species that appears to be located in two layers, one at the very surface corresponding to the physisorbed water (due to air exposure) and another seemingly chemisorbed in the graphene overlayer  recorded in high lateral resolution (∼200 nm) mode with a Bi3+ (30 keV) analysis ion beam. These maps are recorded after ∼0.3 nm of the surface has been removed by Cs+ sputtering (500 eV ion energy) and represent the main species of interest related to the graphene overlayer (C3−), chemisorbed copper solvent residues (C2N−), chemisorbed water (OH−), PMMA/acetone residues partial underlayer (C2O−), chemisorbed adventitious organic material at the h-BN flake surface (CB−) and the SiO2 substrate (SiO2−). The dark areas visible in the C3− maps correspond to the space between the graphene islands that are inherent to the transfer process. Having the same in-plane localization, the C3−, C2N−, S− , within the transferred graphene. In addition, as the depth profiles of C2N− and S− extend over the depth profiles of C3− and C2O− , respectively. As a clear indication of being a PMMA/acetone marker, the C2O−map follows both the C3− and SiO2− maps, as expected for a solvent that was used after the deposition of the PMMA/graphene system onto the h-BN/SiO2 substrate. Given that the physisorbed species (∼0.3 nm) were previously removed by sputtering, the secondary ion map of the OH− species represents the chemisorbed water that appears uniformly distributed within the plane of the graphene overlayer and the exposed SiO2 substrate. Additional contaminants, if present, were below the detection limit or their signal was too low to produce a reasonable image, as in the case of Cu−. Albeit impossible to spatially map in plane, the copper residue is most probably isotropically chemisorbed at the bottom of the graphene overlayer in the lower ammonia persulfate residual layer, as inferred in C2N−, S−  and C2O−and C2O− , we conc2 substrate, and (ii) consists of three atomic-like layers: a single layer graphene and two partial atomic underlayers that combine residues from the solvents used in the transfer process and the initial copper foil support for the CVD graphene. All residues detected as a part of the graphene system are chemisorbed in or at the bottom of the graphene overlayer, thereby having a passivation effect and possibly acting as an electronic decoupling layer from the substrate. Located in the lower organic residual (passivation) layer, the copper residue (< 0.08 atomic %) appears spatially decoupled from the graphene overlayer thus not contributing to the actual graphene defect density. Finally, traces of water are identified both physisorbed at the very surface of the 2D system and chemisorbed in the graphene overlayer, confirming previous SPM observationsTo summarize, in depth AFM and TOF-SIMS analysis show that the transferred graphene system (i) is chemically inert with respect to h-BN but interacts with SiOAs shown above, all detected surface and interface species can be traced back to the transfer or CVD growth processes, thus TOF-SIMS profiling can directly measure the effects of synthesis, handling, and fabrication processes on the final 2D materials and heterostructures. Such detailed information could be critical towards understanding their device function. TOF-SIMS can also be applied to other ultra-thin layered structures, providing detailed, vertical chemical analysis at the atomic level . We thus2 (285 nm thick) on a Si wafer4)2S2O8); Sigma-Aldrich) solution2/Si substrate and the top PMMA was removed using acetone, giving the final graphene/h-BN/SiO2 configuration. We deliberately used common growth, processing and transfer techniques to produce 2D heterostructures that are typical of other published results.The substrate was prepared by depositing mechanically exfoliated h-BN flakes from h-BN powder  atop SiO2 system. The instrumentation consisted of a Raman spectrometer WITec alpha300 using a laser wavelength of 488 nm, an AFM Asylum Research MFP3D and a TOF-SIMS ION-TOF GmbH TOF.SIMS 5 configured with 500 eV Cs+ sputtering and 30-keV Bi1+/Bi3+ analysis ion beams. Depending on the acquisition mode, the TOF-SIMS analysis ion gun was set in either high-current bunched mode (Bi1+ at 30 keV ion energy and 18 ns pulse duration) for depth profiling or burst alignment mode (Bi1+/Bi3+ at 30 keV ion energy and pulse duration of 100 ns without or 18 ns with bursts) for high lateral resolution. The raster area, beam sample currents and specific settings for both the analysis and sputtering beams are indicated in the main text and supporting information. The high-current and burst alignment (with bursts) modes allowed a mass resolution better than 5,000 (m/δm) for all analysed masses. All detected secondary ions had negative polarity. The TOF-SIMS data were acquired at a base pressure of ∼7.5 × 10−10 Torr.Raman spectroscopy and mapping, AFM and TOF-SIMS were used in conjunction to characterize the structure, morphology and layered chemical composition, respectively, of the graphene/h-BN/SiOHow to cite this article: Chou, H. et al. Revealing the planar chemistry of two-dimensional heterostructures at the atomic level. Nat. Commun. 6:7482 doi: 10.1038/ncomms8482 (2015).Supplementary Figures 1-10, Supplementary Notes 1-7 and Supplementary References


The aim of this manuscript is to describe radiological findings of extra-pulmonary sarcoidosis. Sarcoidosis is an immune-mediated systemic disease of unknown origin, characterized by non-caseating epitheliod granulomas. Ninety percent of patients show granulomas located in the lungs or in the related lymph nodes. However, lesions can affect any organ. Typical imaging features of liver and spleen sarcoidosis include visceromegaly, with multiple nodules hypodense on CT images and hypointense on T2-weighted MRI acquisitions. Main clinical and radiological manifestations of renal sarcoidosis are nephrolithiasis, nephrocalcinosis, and acute interstitial nephritis. Brain sarcoidosis shows multiple or solitary parenchymal nodules on MRI that enhance with a ring-like appearance after gadolinium. In spinal cord localization, MRI demonstrates enlargement and hyperintensity of spinal cord, with hypointense lesions on T2-weighted images. Skeletal involvement is mostly located in small bone, showing many lytic lesions; less frequently, bone lesions have a sclerotic appearance. Ocular involvement includes uveitis, conjunctivitis, optical nerve disease, chorioretinis. Erythema nodosum and lupus pernio represent the most common cutaneous manifestations encountered. Sarcoidosis in various organs can be very insidious for radiologists, showing different imaging features, often non-specific. Awareness of these imaging features helps radiologists to obtain the correct diagnosis.Systemic sarcoidosis can exhibit abdominal, neural, skeletal, ocular, and cutaneous manifestations.• T2 signal intensity of hepatosplenic nodules may reflect the disease activity.• Heerfordt’s syndrome includes facial nerve palsy, fever, parotid swelling, and uveitis.• • In the vertebrae, osteolytic and/or diffuse sclerotic lesions can be found.Erythema nodosum and lupus pernio represent the most common cutaneous manifestations.•  Sarcoidosis is a multi-systemic disorder of unknown cause, pathologically characterized by the accumulation of inflammatory cells forming non-caseating granulomas. Lesions can be located in any organ but in about 90 % of patients, granulomas affect the lungs or the related lymph nodes .According to Geize et al., sarcoidosis can be encountered in extra-pulmonary locations, in approximately 30 % of cases . MoreoveThe systemic location of the disease can be very insidious for radiologists: a wide variety of imaging features are found, often non-specific, which can mimic other pathological conditions , 7. TherThe disease affects young men and women aged between 25 and 40 years, without major differences in ethnicity. Some studies do show a greater incidence in females . In abouNormally, sarcoidosis shows a variable clinical course; it may also exhibit different extra-thoracic manifestations. These vary widely according to ethnicity: for example, involvement of the eye, liver, bone marrow, extra-thoracic lymph nodes, and skin has been more statistically associated with African Americans than with Caucasians , 5.Cardiac and ocular diseases are frequently encountered in Japanese patients, erythema nodosum in northern Europeans, and ocular and granulomatous skin involvement in black patients –13. In aExtra-thoracic sarcoidosis involves females more frequently than males .Extra-pulmonary sarcoidosis has been found in 30 % of patients with the disease . The mosThe cutaneous system and the eyes are involved with a frequency of 25 % , 15. FolThe following sections below describe more in detail clinical and radiological features of systemic sarcoidosis Table , focusinHepatic sarcoidosis occurs with a prevalence of 1-40/100,000; it generally involves young people . The disCommonly, imaging demonstrates hepatomegaly, with homogeneous appearance of parenchyma ; very ofGranulomatous lesions are located in the portal and periportal spaces of hepatic sinuses and generally exhibit “an identical stage of maturation” , 23. TheSplenic sarcoidosis is generally associated with lung disease; however, normal chest radiography is observed in one quarter to one third of patients with splenic sarcoidosis . SplenicPatients can be asymptomatic, or complain of fever, weight loss, and malaise , 32. In Splenic sarcoidosis can be present in a homogeneous fashion or in a nodular pattern , 27, 33.On MRI acquisitions, nodular lesions show low signal in all sequences ; the visParenchymal lesions show increased FDG uptake on PET-CT : low-denAmong abdominal sarcoidosis, autopsy revealed only a small percentage of cases (5 %) located in pancreatic tissue, intestinal tract, and testes . The gasThe biliary tree may be involved in extra-hepatic or intra-hepatic pattern. Granulomatous lesions develop in the portal triad, causing a cholestatic pattern.Extra-hepatic involvement of the biliary tree can be caused by multiple small granuloma of the wall; the radiological appearance in these cases may simulate a cholangiocarcinoma; in many cases, the biliary tree may be compressed by enlarged abdominal nodes.According to Koyama et al., in 6 % of cases parotid glands may have sarcoidosis involvement; generally, the disease is found bilaterally, with enlargement of glands . An inhoLymphatic abnormalities have been already described as abdominal manifestations of sarcoidosis, consisting of “lymphadenopathy or increased number of normal sized nodes” ; lymph nEnlarged nodes can be observed in different locations: in the periportal region, close to the liver hilus Fig. , or betwLymphatic involvement is frequently associated with pulmonary disease, and this can be very helpful for radiologists in order to achieve a correct diagnosis. When the disease is limited to the lymphatic system in mediastinum and in the abdomen, a differential diagnosis from other disorders, e.g. lymphoma, may be very difficult.Peritoneal sarcoidosis is extremely rare , 38–41. In sarcoidosis, the real incidence and prevalence of renal involvement is difficult to establish, because the disease can occur in a variable manner; in most cases, it has been found in 35 to 50 % of patients . Main clThe increased levels of 1,25-dihydroxyvitamin, produced by mononuclear cells trapped in pulmonary alveoli, determines a greater absorption of calcium: this leads to hypercalcemia and consequential nephrolithiasis and nephrocalcinosis .The prevalence of interstitial nephritis in sarcoidosis oscillates from 7 to 27 % , 45. TheEnhanced CT images may show signs of interstitial nephritis , 47, 48 Rarely, radiological features of renal sarcoidosis can be represented by granulomatous pseudotumour; these lesions are generally incidentally discovered during CT examination. They can appear as hypo-, iso-, or hypeattanuating areas on unenhanced CT scans; after contrast administration, they are hypodense to the surrounding renal parenchyma Fig.  48]. Di. Di48]. The urinary manifestations of sarcoid nephritis are nonspecific: the alterations of the urine analysis are similar to those of chronic tubular pathologies. Biopsy is the only way to make a diagnosis, showing normal glomeruli with interstitial infiltration of mononuclear cells and non-caseating granulomas; occasionally, it may be also depicted with glomerular disease , 50.Central nervous system (CNS) involvement is reported in 25 % of cases at autopsy; in a post-mortem series, neural sarcoidosis was detected with a frequency of 14–27 % –54. SympThe disease shows a special predilection for the basal cisterna of the brain; cranial nerve involvement is frequent, influencing the clinical manifestation of disease. Facial nerve palsy, for example, is a typical manifestation of brain sarcoidosis in young adults . It may Other symptoms associated with neural sarcoidosis are headache, seizure, and signs of meningeal irritation , 56. InvDiagnosis of sarcoidosis can be performed following the criteria reported in the study by Pawate et al., in which the presence of neurosarcoidosis is evaluated as definite, probable, and possible. According to their criteria , the disLesions appear hyperintense on T2-weighted MRI images, located in the superficial and deep white matter; sometimes, these imaging features may resemble lesions of multiple sclerosis . Also grDural thickening is generally isointense to the grey matter on T1-weighted acquisitions, hypointense on T2-weighted sequences, with contrast enhancement after gadolinium injection .Leptomeningeal localizations are more visible after contrast injection, showing increased enhancement Fig.  58]. As. As58]. Nodules can be solitary or multiple, showing enhancement after contrast injection with a ring-like appearance in the activity phase: this appearance can simulate glioblastoma or metastases. An intracranial mass presentation has been also reported in the literature, simulating a brain neoplasm .Spinal sarcoidosis is reported in about 4–28 % of cases; it occurs predominantly in old people and has a poor prognosis . The disMRI features of stage 1 and 2 disease include leptomeningeal thickening, with hyperintense signal on enhanced T1-weigheted images . In cervThe skeletal system represents a rare localization of sarcoidosis . RadioloThe disease generally involves hands and feet, which have been reported in several reviews as the most affected locations , 64. On Large bones and axial skeleton involvement is uncommon; however, the disease may be encountered in ribs, long bones, skull and vertebrae. Axial skeleton disease, without pulmonary lesions, is rarely reported .Lesions of large bone and axial skeleton can be detected as radiolucent Fig.  or sclerIn patients with sarcoidosis, skeletal FDG uptake can be observed Fig. ; these iOcular involvement occurs in approximately 25–60 % of patients with systematic sarcoidosis, more frequently in the third decade (first peak of incidence) and between the sixth and the seventh decade (second peak of incidence) . This maIn most cases (about 20 %), uveitis is the first common manifestation: symptoms that patients complain of include tearing, photophobia, pain, infection, lacrimation, redness. However, these symptoms can be absent: “silent uveitis” is very insidious because it may produce permanent ocular damage before treatment. The common type of uveitis is often anterior in black patients, posterior in white patients, specifically in elderly female patients –74. BlinIn the anterior segment, conjunctivitis occurs in 7–70 % of the patients with ocular sarcoidosis and, together with lacrimal gland involvement, is usually asymptomatic. The sarcoidosis granulomas in the eyes have the appearance of yellow “millet-seed” nodules. When the disease involves the posterior part of the eyes, it is frequently associated with neurological manifestations: optic nerve disease, cranial nerve palsies, encephalopathy, and disorders of the hypothalamus and pituitary gland , 78.Other manifestations of ocular sarcoidosis include granulomatous iridocyclitis, retinal periphlebitis and chorioretinis; foci of retinal involvement are generally revealed by fluorangiography as “punched-out” lesions Fig. . In 7 toIn sarcoidosis, cutaneous involvement occurs in 20–35 % of patients . The cutErythema nodosum is the most common nonspecific cutaneous lesion of sarcoidosis. In most cases it occurs with subcutaneous erythematous nodules Fig. , often oThe term “specific” is misleading, because clinically lesions are not specific to sarcoidosis, and the only way to make a diagnosis is biopsy. Papule Fig.  is the mA characteristic clinical manifestation of cutaneous sarcoidosis is lupus pernio Fig. : it appeThe diagnosis of cutaneous sarcoidosis is performed with histological examination of a cutaneous biopsy, which shows the presence of sarcoidal granuloma without any cornoid lamella.Cardiac involvement with clinical manifestations occurs in 2–7 % of patients, but the occult form is much more present –86.Sarcoidosis characteristic granulomatous lesion manifests in any part of the heart, but the myocardium is most frequently involved . In ordeGadolinium-enhanced cardiac MRI allows us to detect differences between normal tissue and altered tissue, the latter displaying areas of focal enhancement, particularly in the myocardial wall or subepicardial region Fig.  92, 93], 9392, 9Radionuclide scans with gallium, citrate, and thallium are used for diagnosis and follow-up in patients with cardiac sarcoidosis , 95. PETAbdominal, neural, musculo-skeletal, cutaneous, ocular and cardiac sarcoidosis has extremely variable clinical features; their imaging findings can often simulate other diseases, namely malignancies. Sarcoidosis should be considered in the differential diagnosis of multiple lesions of brain, abdomen and bones in patients with suspected or proved disease.In patients with pulmonary sarcoidosis, a careful evaluation of patients’ symptoms is mandatory in order to identify other locations of the disease. Radiologists, correlating imaging and clinical-pathologic findings, play an important role in diagnosis and follow-up of the disease, in order to reduce its morbidity and mortality.


Pan troglodytes) use tools to probe for out-of-reach food, both in the wild and in captivity. Beyond gathering appropriately-sized materials to create tools, chimpanzees also perform secondary modifications in order to create an optimized tool. In this study, we recorded the behavior of a group of zoo-housed chimpanzees when presented with opportunities to use tools to probe for liquid foods in an artificial termite mound within their enclosure. Previous research with this group of chimpanzees has shown that they are proficient at gathering materials from within their environment in order to create tools to probe for the liquid food within the artificial mound. Extending beyond this basic question, we first asked whether they only made and modified probe tools when it was appropriate to do so (i.e. when the mound was baited with food). Second, by collecting continuous data on their behavior, we also asked whether the chimpanzees first  modified their tools prior to probing for food or whether such modifications occurred after tool use, possibly as a by-product of chewing and eating the food from the tools. Following our predictions, we found that tool modification predicted tool use; the chimpanzees began using their tools within a short delay of creating and modifying them, and the chimpanzees performed more tool modifying behaviors when food was available than when they could not gain food through the use of probe tools. We also discuss our results in terms of the chimpanzees’ acquisition of the skills, and their flexibility of tool use and learning. Am. J. Primatol. 77:162–170, 2015. © 2014 The Authors. American Journal of Primatology Published by Wiley Periodicals Inc.Chimpanzees ( Pan troglodytes) using tools to probe for out-of-reach food, both in the wild and in captivity [e.g., Boesch & Boesch, There are numerous accounts of chimpanzees . The group was comprised of three males and four females, with an average age of 10 years (range: 4–19 years). The chimpanzees inhabited an expansive indoor/outdoor exhibit that included climbing structures and deep-mulch bedding and an off-exhibit holding area. The indoor exhibit space also featured an artificial termite mound [Bonnie et al., This study was approved by the Lincoln Park Zoo Research Committee, which is the governing body for all animal research at the institution. No social group manipulations occurred as the result of this project. Food substances, amount, and frequency were reviewed and approved by veterinary and nutrition staff prior to the start of the project. No modifications were made to standard animal care routines. This research adhered to legal requirements in the United States of America and to the American Society of Primatologists’ Principles for the Ethical Treatment of Nonhuman Primates.For this study, liquid foods were presented to the chimpanzees via an artificial termite mound, mimicking the look of a natural termite mound found in wild ape habitats . The mouCrataegus spp.), serviceberry trees , red oak trees (Quercus rubra), and a bur oak tree (Q. macrocapra). Unfortunately, due to the degradation of the video footage collected in 2004 when these data were collected, for these current analyses, it was not possible to identify which species of plants were used by the chimpanzees to create their probe tools. However, contemporary observations of this group of chimpanzees revealed that they create tools from a number of species, including willow and poplar that are now provided to them by keeper staff, and they also use the young shoots of serviceberry trees that grow in their outdoor exhibit, which they modify by fraying the ends of the sticks , for this analysis we only include the chimpanzees’ responses in the first two months of the baited phase to enable a clearer comparison across the two periods. These data represent a subset of data previously collected at Lincoln Park Zoo as part of a wider investigation of ape social learning and tool use. The original findings from this study were published previously by Lonsdorf et al. , but theThe first two months (56 days) of the study period represented the “Baseline” phase. Behavioral data were collected on 35 of these 56 days (i.e. Monday–Friday every week) from June 7th 2004–August 2nd 2004. These Baseline sessions were spread evenly throughout the Baseline period and all Baseline sessions were filmed for later analysis. During this period, the termite mound was never baited with food. The chimpanzees had no prior experience with obtaining food from this artificial mound and so this period represented a true behavioral baseline . Spread evenly throughout the Experimental period were 19 “Bait” sessions, on which the tubes in the termite mound were baited with desirable viscous food (e.g. ketchup), at randomized times between 1100 and 1400 hr (Monday–Friday). No more than one Bait session was conducted on a given day. For Bait sessions, the chimpanzees did not know prior to testing what the termite mound was baited with, although, once baited, the chimpanzees could inspect the holes through smell and could also sample the food stuffs directly with probe tools [c.f. Bonnie et al., Video footage of the chimpanzees’ behavior, on, or within 1 m of, the artificial termite mound, was recorded for 35 Baseline sessions, 19 Bait sessions, and 19 Control sessions. Video footage was recorded using a stationary, ceiling-mounted security camera which captured the entire mound and a 1 m perimeter around the mound. This camera was connected to a time-lapse VCR (Panasonic AG-RT650) that was pre-set to record each session. Data were scored directly from the video footage in 2004–2006, wherein each group member’s behavior was scored continuously, as described by Lonsdorf et al.  (Table1)The recorded data captured each session entirely but the data presented here represent the first 15 min of each session. For these analyses, we focused on the ‘probe’ and ‘tool-modification’ behaviors , all behaviors were converted to average rates/session. Friedman’s tests and Wilcoxon’s signed-ranks tests were used to compare the chimpanzees’ responses across conditions. Firstly, we compared the rate of fishing and tool-modification behaviors in the Baseline phase to (a) the days on which the termite mound was baited (Bait sessions) and (b) days within the experimental phase when the termite mound was not baited (Control sessions). Secondly, once the chimpanzees had experienced food in the artificial mound (Bait sessions), to determine whether tool modifications were functional, we compared the chimpanzees’ rates of tool modification and tool use across the Control and Bait sessions. To further investigate the interplay between tool-modification and probing behaviors, we ran a Pearson’s correlation between rates of tool modification and fishing behaviors. All analyses were conducted in IBM SPSS, Version 20 .Z-test and likelihood ratio tests (LRTs) [Bates et al., We were also interested in determining if tool modification was more likely than other behaviors to be followed by tool usage (i.e. probing). For the purposes of this analysis, we simplified the continuous sampling dataset by noting whether or not, for each pair of behaviors observed, the first behavior was modification and whether or not the second behavior was tool use. All other behaviors described in TableThere were significant differences across all three phases  regarding both the rate of tool-modification behaviors as well as the rate of probing behaviors recorded  and we cX2(2) = 11.273, P = 0.04). The chimpanzees performed more tool modification when the termite mound was baited (Bait) than during the Baseline  or Control  sessions. While none of the chimpanzees ever modified tools in the Baseline sessions, six of the seven chimpanzees performed one or more tool-modification behaviors during the Bait phase  and all did so on the first Bait session . Emphasizing the low rate of tool-modification behaviors in Control sessions, there was no difference in the number of tool-modification behaviors seen during the Baseline and Control sessions, but they showed a trend towards performing more tool modification in the Control sessions  = 14.00, P = 0.001). Comparable with their tool-modification behaviors, the chimpanzees performed more probing behaviors when the termite mound was baited (Bait) than during the Baseline  or Control  sessions. The chimpanzees were observed to probe more during the Control sessions compared to during Baseline sessions , indicating comparably low levels of probing behaviors in both phases.During the Baseline sessions, five of the seven chimpanzees performed one or more probing behaviors and the first chimpanzee to do so was a five-year-old female during the 3rd Baseline session (June 10th 2004). In contrast, during the Bait sessions, all seven chimpanzees probed the artificial mound . There was a significant difference in the rate of probing behaviors observed across the three experimental phases , such that those chimpanzees that probed more, also performed more tool-modification behaviors. There was no significant correlation, however, between rates of probing behavior and tool modification in Control sessions .Chimpanzees were only recorded to modify tools in Bait and Control sessions. During the Bait sessions, there was a significant positive correlation between rates of probing and tool-modification behaviors (Pearson’s X2(1) = 862.32, P < 0.0001) and sessions  = 91.61, P < 0.0001), although among-individual variance was substantially higher than among-session variance . After accounting for this variation, the fitted values revealed that probing behaviors occurred more frequently after modification (47.6%) than after other behaviors (14.9%), and this difference was statistically significant . Indeed, in Bait sessions, the average delay between a chimpanzee modifying a tool and then performing a probing behavior was only 4.5 sec (range = 1–247 sec). The degree to which modification predicted probing did not significantly differ among chimpanzees or sessions .Overall, probing behaviors comprised 24.1% of the total behaviors observed. The rate of probing behaviors varied significantly among both chimpanzees  from a novel ‘artificial termite mound’ in their enclosure and, if so, to identify under what circumstances they modified tools for fluid dipping behaviors. Specifically, we wished to record whether the chimpanzees modified their tools prior to using them, and this is what we found. Expanding upon a previous study, which had investigated the learning of tool-use behavior by chimpanzees [Lonsdorf et al., Although captive chimpanzees have been documented to create and modify functionally-relevant tools, these studies also report individual differences across chimpanzees as a function of age or rearing history [Bania et al., Of the six chimpanzees that modified their tools, four did so in Control sessions when no food was available, perhaps due to “carry over” effects from Bait sessions . Indeed, Price et al.  noted thSix subjects began modifying tools as soon as there was available food (in the first Bait session), and because these chimpanzees made their first tool modification within 60 sec of each other, it is most likely that these behaviors were already known, but only elicited when it was appropriate for them to do so. However, we might consider too that the chimpanzees were able to  learn tool-modification skills rapidly. We evaluate both of these proposals in turn. First, although the task of probing an artificial termite mound was novel to this group of chimpanzees, it is likely that they had prior experience with probing given that this is a near-universal behavior among chimpanzees [Whiten et al., sensu Tennie et al., sensu Hrubesch et al., If probe tool manufacture and modification is within chimpanzees’ ‘Zone of Latent Solutions’ [Pongo pygmaeus abelii, Lehner et al., Corvus moneduloides, von Bayern et al., In our study, tool modification predicted tool use. Regardless of whether the subjects in our study already knew how to create, modify, and use probe tools, or if they had to acquire the skills at the start of the study, they all had to adapt and apply their skills to this novel task. This demonstrates their flexible foraging abilities. This is perhaps not surprising as, when presented with novel problem-solving tasks, chimpanzees [Hopper et al., We defined tool modification broadly, but a specific form of probe tool modification that is commonly reported is tool fraying. While even early studies of chimpanzee probing behavior reported chimpanzees’ proclivity to fray the end of probe tools [e.g., Teleki,


The aggregation of RNA-binding proteins is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBM45 is an RNA-binding protein that forms cytoplasmic inclusions in neurons and glia in ALS and FTLD. To explore the role of RBM45 in ALS and FTLD, we examined the contribution of the protein’s domains to its function, subcellular localization, and interaction with itself and ALS-linked proteins. We find that RBM45 forms homo-oligomers and physically associates with the ALS-linked proteins TDP-43 and FUS in the nucleus. Nuclear localization of RBM45 is mediated by a bipartite nuclear-localization sequence (NLS) located at the C-terminus. RBM45 mutants that lack a functional NLS accumulate in the cytoplasm and form TDP-43 positive stress granules. Moreover, we identify a novel structural element, termed the homo-oligomer assembly (HOA) domain, that is highly conserved across species and promote homo-oligomerization of RBM45. RBM45 mutants that fail to form homo-oligomers exhibit significantly reduced association with ALS-linked proteins and inclusion into stress granules. These results show that RMB45 may function as a homo-oligomer and that its oligomerization contributes to ALS/FTLD RNA-binding protein aggregation. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder resulting from the death of upper and lower motor neurons. Approximately 90% of all ALS cases are considered sporadic and of unknown etiology. Multiple cellular mechanisms have been proposed to contribute to motor neuron degeneration in sporadic ALS, including altered RNA processing, protein aggregation, mitochondrial defects and oxidative stress3456Studies of the normal and pathological functions of RNA-binding proteins such as TDP-43 and FUS have provided insight into the role of these proteins in ALS and FTLD. TDP-43 and FUS are multi-functional RNA-binding proteins that contribute extensively to the regulation of gene expression. Both proteins influence pre-mRNA splicing, participate in RNA transport, and modulate RNA stability910TDP-43 and FUS genes of patients with familial forms of ALS and FTLDStructure-function studies of TDP-43 and FUS have provided considerable insight into the mechanisms by which these and other RNA-binding proteins exert their normal functions, associate with stress granules, and aggregate into inclusion bodies in disorders such as FTLD and ALS. Both TDP-43 and FUS contain RNA-recognition motifs (RRMs) that are necessary for the nucleic acid binding functions of the protein1315One such protein is RBM45, an RNA-binding protein we found in inclusions in the cells of patients with ALS and FTLD. A proteomic analysis of cerebrospinal fluid (CSF) from sporadic ALS and healthy control subjects identified increased levels of RBM45 in the CSF of sporadic ALS patientsIn this study, we identify structural determinants of RBM45 for subcellular distribution and protein-protein interactions. Our results show that RBM45 localizes predominately to the nucleus via a bipartite nuclear-localization sequence located at the C-terminal region. In addition, we have identified a novel and evolutionary conserved structural element located within the linker region between RRM2 and RRM3 that is essential for self-association and oligomerization of RBM45. Importantly, the oligomerization of RBM45 mediates its association with TDP-43 as well as inclusion within stress granules.RBM45 was previously detected in cytoplasmic inclusions of neurons and glia in ALS and FTLD patient tissueTo identify the sequence(s) responsible for nuclear localization, we analyzed the amino acid sequence of RBM45 using the computational program cNLS MapperWithin the second NLS candidate (454–472) are additional basic residues  that may contribute to RBM45’s nuclear localization . To examin vivo2627ALS-linked proteins TDP-43 and FUS contain prion-like domains and are intrinsically prone to aggregationWe next sought to map the domain responsible for RBM45 self-association by screening a series of truncated HA-tagged RBM45 fragments for interaction with the full-length FLAG-RBM45 protein co-expressed. We initially focused on the uncharacterized linker region between RRM2 and RRM3 by expressing four HA-RBM45 truncation constructs, Linker-1, -2, -3 and -4 , in a HEin vivo cross-linking with disuccinimidyl suberate (DSS) to capture native RBM45 assemblies in live cells. DSS is an irreversible, cell membrane permeable cross-linking reagent containing bifunctional amine-reactive NHS-estersTo determine if RBM45 self-association leads to dimerization or oligomerization, we performed To determine the stoichiometry of RBM45 homo-oligomers, we analyzed the size of the cross-linked product of a truncated HA-RBM45 variant (107–474) that lacks RRM1. By SDS-PAGE, the monomer of the full-length and truncated HA-RBM45 migrated at 59 and 46 kD, similar to the calculated 56 and 45 kD, respectively  to visualize the co-localization of RBM45 and its binding proteins. PLA visualizes protein-protein interactions To further determine if RBM45 and TDP-43 associate in the cytoplasm, we co-expressed FLAG-TDP-43 together with the HA-RBM45-NLS mutant that predominately accumulates in the cytoplasm, followed by co-IP to detect physical interactions between RBM45 and TDP-43. We observed significant association between the RBM45-NLS mutant and TDP-43 . SimilarWe next asked whether the oligomerization of RBM45 is also necessary for its association with TDP-43 and FUS. Anti-FLAG co-IP analysis using cells co-expressing FLAG-TDP-43 and HA-RBM45 deletion mutants, D1 and D4, showed that wild-type and the D1 mutant HA-RBM45 co-purified with FLAG-TDP-43 . HoweverSince the RBM45-TDP-43 association is RNA-mediated, we examined the importance of the three RRM domains of RBM45 for TDP-43 association. We performed co-IP assays of FLAG-TDP-43 with different RBM45 constructs that lack individual RRM domains . Interesin vivo findings that RBM45 was found in the TDP-43-containing but not FUS-containing inclusions, in neurons of ALS and FTLD patientsOxidative stress has been implicated in the pathogenesis of ALS and FTLD233910In both ALS and FTLD, aggregation of RNA-binding proteins contributes to disease pathogenesis and generates neuropathologic inclusions within the brain and spinal cord. RBM45 was first described as a neuronal RNA-binding protein expressed under spatiotemporal control in the rat brainWe identified a novel HOA domain (residues 258–318) within RBM45 that is responsible for self-association and oligomerization . The HOAet al. reported that RBM45 binds to overexpressed C-terminal fragment (CTF) of TDP-43 in mammalian cellsMany known RNA chaperones contain RRM domains363739PARK2, SMYD3 and KCNIP4) are important for neuronal function and are down-regulated in sporadic ALS motor neurons harboring TDP-43 pathology, suggesting a shared mechanism of neurodegeneration caused by dysregulation of TDP-43 or FUS. As RBM45 and TDP-43 interactions are RNA-dependent, it suggests that RBM45 and TDP-43 share the regulation of specific RNA targets. Future studies are needed to identify the RNA targets bound by RBM45 to determine RNA targets co-regulated by RBM45 and TDP-43.Both TDP-43 and FUS bind pre-mRNA with long introns424344in vitro studies, future studies will determine the presence or absence of this alternative protein isoform in ALS and other neurologic diseases.Although RBM45 is a nuclear protein, overexpressed wild-type RBM45 can form cytoplasmic granules that resemble the inclusions observed in ALS/FTLD patient tissues. Disruption of the RBM45 NLS and cellular stress further exacerbate cytoplasmic accumulation and lead to increased granule formation that co-localize with TDP-43 and stress granule marker TIAR . In cell713We have identified structural domains in RBM45 that contribute to its function, subcellular localization, homo-oligomerization and its interactions with the ALS-associated proteins TDP-43 and FUS. Homo-oligomerization of RBM45 is mediated by a novel HOA domain that is conserved across species and important for its protein-protein interactions and association with cytoplasmic stress granules. These results provide new insights into the role that protein assemblies play in the normal and pathological functions of RNA-binding proteins and provide further evidence of the association of RBM45 and TDP-43 in both the nucleus and cytoplasm, a phenomenon first observed in cytoplasmic inclusions in ALS/FTLD patients.The plasmids of RBM45, TDP-43 and FUS cDNA clones, cGST-hRBM45 (HsCD00356971), pDONR221-TDP-43 (HsCD00079870), and pDNR-Dual-FUS (HsCD00002882), were obtained from the DNASU Plasmid Repository at Arizona State University, Tempe. The cDNA was amplified by PCR using Phusion High-Fidelity DNA Polymerase (NEB) and sub-cloned into the pcDNA3 vector (Invitrogen). The 3xFLAG tag (DYKDHDGDYKDHDIDYKDDDDK) or 2xHA tag (DYPYDVPDYAGGAAYPYDVPDYA) was appended to the N-terminus of specific proteins to generate the 3xFLAG- or 2xHA-tagged construct. The EGFP-RBM45 construct was generated by inserting the EGFP gene into the N-terminus of RBM45 gene in pcDNA3. The mutations were introduced by site-directed mutagenesis using overlap extension PCR2. SHSY5Y cells were cultured in DMEM/F12 medium with 15% FBS, 1% non-essential amino acids and 1% Pen-Strep at 37 °C with 5% CO2. Transfection was performed using the Lipofectamine 2000 (Life technologies) and transfected cells were harvested 48 or 72 hours post-transfection. Stable cell lines were selected in the presence of 500 μg/ml G418 (Life Technologies).HEK293 , Neuro2A (ATCC) and SK-N-SH (Sigma) cells were cultured in DMEM medium with 10% FBS and 1% Pen-Strep at 37 °C with 5% COCells were grown on 6-well plates till confluent and harvested. The cell pellet was gently resuspended in 150 μl cytoplasmic extraction buffer  and incubated on ice for 5 min. The cells were spun at 600 × g for 4 min at 4 °C and the supernatant was transferred to a new tube as crude cytoplasmic fraction (~150 μl). The pellet was gently washed with 100 μl cytoplasmic extraction buffer without NP40 and spun at 600 × g at 4 °C for 4 min. The supernatant was discarded and the pellet was resuspended with 150 μl nuclear extraction buffer  and incubated at 4 °C with rotation for 15 min. The nuclear extract and the crude cytoplasmic fraction were cleared by centrifugation at 16,000 × g at 4 °C for 10 min. Equal volume of the nuclear and cytoplasmic fractions were analyzed by SDS-PAGE gel and immunoblot. The band intensity was quantified with Image Studio software (LiCOR) and the relative abundance of the nuclear RBM45 in total RBM45 was calculated as [(nuclear fraction)/(nuclear fraction + cytoplasmic fraction)] × 100%. Statistical analysis was performed using two-tailed paired t-test.Cells were grown on Poly-D-Lysine coated coverslips to 60% confluent. Fixation was performed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with 0.2% Triton at room temperature for 10 min. Cells were blocked with Super Block (ScyTek) at room temperature for 1 hour. Primary antibodies see  were dilCells were fixed, permeabilized, and blocked as described above. A pair of primary antibodies see  producedProtein samples were mixed with 4× SDS loading buffer and heated at 90 °C for 7 min, resolved on the Bolt 4–12% Bis-Tris Plus Gel (Life Technologies) or 3–8% Tris-Acetate Gel (Life Technologies), and transferred to Immobilon-FL PVDF membrane (Millipore). The membranes were blocked with Odyssey Blocking Buffer (LiCOR) for 1 hr. The antibodies see  were dilDisuccinimidyl suberate (DSS) in-cell crosslinking was performed as previously described28Formaldehyde in-cell crosslinking was performed prior to immunoprecipitation. Formaldehyde is a mild and reversible crosslinker with very short spacer length (2.3–2.7 Å) and cross-links only closely associated proteinsCells were lysed with NP40 lysis buffer  at 4 °C for 15 min. The lysates were cleared by spinning at maximum speed at 4 °C for 10 min and subsequently used for IP. FLAG-IP was performed using anti-FLAG M2 Affinity Gel (Sigma A2220). IgG-IP control was performed using Mouse IgG-Agarose (Sigma A0919). IP was performed at 4 °C for 2 hr and the beads were washed in NP40 lysis buffer for six times. The proteins were eluted with SDS sample buffer and heated at 95 °C for 20 min to reverse formaldehyde crosslinking. For fractionation-IP, the KCl concentration in the cytoplasmic fraction was adjusted from 60 mM to 430 mM to approximate the salt concentration in the nuclear extract.Freshly harvested cells were permeabilized with 100 μl PBS containing 0.15% Triton, 3 μl RNase A (Sigma R4642) and proteinase inhibitor for 10 min at room temperature. 400 μl 0.25% formaldehyde/PBS was added to the 100 μl permeabilized mixture so that the final formaldehyde concentration is 0.1%. The subsequent crosslinking and immunoprecipitation were performed as described above.The RBM45 sequences from human (NP_694453.2), chimpanzee (XP_515938.2), bovine (AAI05263.2), mouse (AAH57890.1), rat (NP_695218.1), chicken (NP_001026423.1), lizard (XP_008116425.1), African clawed frog (NP_001080090.1), rainbow trout (CDQ81595.1), zebrafish (NP_001120874.1), sea urchin (XP_785181.2), honey bee (XP_006566988.1), fruit fly (AAF47879.2), and mosquito (XP_315504.4) were aligned with ClustalW algorithm using the BioEdit software.How to cite this article: Li, Y. et al. RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules. Sci. Rep.5, 14262; doi: 10.1038/srep14262 (2015).