Dengue fever is a common mosquito borne viral fever in South Asia, which causes significant morbidity and mortality. Dengue fever is well known to involve the liver, especially in dengue hemorrhagic fever. The hepatic involvement is usually that of a mild hepatitis with transaminase derangement without jaundice. In cases of dengue hemorrhagic fever where shock has ensued, a severe hepatitis with gross derangements of transaminases and bilirubin may occur. These are two rare cases of adult patients with dengue hemorrhagic fever presenting with a cholestatic type of jaundice.This case report describes two female patients aged 30 and 46 years who presented with fever, icterus and biochemical analysis revealed cholestatic jaundice. Evolution of the clinical picture and dropping platelets prompted serological investigations in the form of dengue non-structural protein 1 antigen and dengue immunoglobulin M which confirmed acute dengue infection.These cases highlight the importance of considering dengue fever as a differential diagnosis even in the presence of a cholestatic jaundice, especially in countries where dengue fever is endemic, and in travelers returning from dengue endemic countries. The early diagnosis of dengue fever and timely institution of supportive fluid management is essential to prevent morbidity and mortality. Dengue fever is the commonest mosquito born viral illness known to man, with cases exceeding 2.2 million in 2010 . An incrA 30 year old South-Asian female presented with 5 day history of high fever associated with severe myalgia, headache and yellowish discoloration of the skin. There was no history of contact with muddy water, alcohol consumption, or use of any drugs including antibiotics, anti-pyretics (acetaminophen) or antiemetics. Furthermore history of blood transfusion, sexual promiscuity or any other high risk behavior could not be elicited. She denied any pale stools or pruritus. At the time of admission, general examination revealed she was febrile and icteric but had no conjunctival hemorrhage, pallor, lymphadenopathy, rashes or scratch marks. The respiratory system was unremarkable and the abdominal examination did not elicit right hypochondrial tenderness, palpable gall bladder or abdominal guarding. The cardiovascular examination was normal and she had an initial blood pressure of 90 mmHg systole and 60 mmHg diastole, without a postural drop. She was conscious, alert, well oriented and nervous system examination was otherwise unremarkable.9/L (150–450), a normal white blood cell count (WBC) of 10.4 × 109/L (4–11) with a predominance of lymphocytes (55%) and a hemoglobin of 11.0 g/dL (11–17) . The blood picture mirrored the whole blood analysis and showed only thrombocytopenia, there was no neutrophil left shift or toxic granules and the red cell indices were normal. Liver functions showed a mildly elevated aspartate transaminase(AST) and alanine transaminase(ALT) which were 80 U/L(0–40) and 38 U/L (0–35 ) respectively with an elevated alkaline phosphatase(ALP) value of 450 U/L (100–360). Serum direct and total bilirubin levels were elevated at 6 mg/dL (0–0.3) and 9.1 mg/dL (0.3-1.9) respectively. Serum albumin was found to be reduced with a value of 2.3 g/dL (3.5 -5.5), with a normal international normalized ratio (INR). The renal functions were within normal reference range. The C- reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were also within normal parameters. The following day the platelet count continued to drop and her jaundice deepened. A positive dengue non- structural protein1antigen test (NS1) and a positive dengue IgM antibody ELISA test on day 6 confirmed the clinical suspicion and diagnosis of dengue fever. Hepatitis B surface antigen, hepatitis C antibody, hepatitis A and E antibodies, Ebstein Barr Virus antibody and a retroviral screening were all negative. Leptospira microscopic agglutination test (Lepto MAT) titres were insignificant at 1:200 on day 6 and the titre value remained unchanged when the repeat test was performed on day 12 of the illness (a four -fold rise in titre or a high titre >1:800 is the gold standard for diagnosis). Blood cultures remained repeatedly negative.The initial complete blood count revealed thrombocytopenia with a platelet count of 69 × 109/L (150–450) with rising alkaline phosphatase and bilirubin levels  and a lymphocytic predominant (51%) white cell count of 7.8 × 109/L (4–11) with a haemoglobin value of 11.6 g/dl with a packed cell volume of 34%. The liver enzymes were elevated with the AST more than the ALT which were 242 U/L (0–40) and 120 U/L(0–35) respectively. The alkaline phosphatase was mildly elevated with a value of 400 U/L (100 – 360), with a high direct bilirubin fraction of 12 μmol/L (0–3.4) and total bilirubin of 23 μmol/L (5–21). Albumin was found mildly reduced at 31 g/L (36–48). The INR was normal. The renal functions were normal. The ultrasound scan of the abdomen and chest confirmed an enlarged liver, with a cranio-caudal length of 16 cm. The gallbladder wall was thickened and measured 6 mm, a negative sonographic Murphy’s sign was noted and no gall stones were visualized. Leakage was confirmed as there was mild ascites and a pleural effusion in the right hemithorax. The ESR was 19 mm for the first hour and the CRP was mildly elevated with a value of 7 mg/L (0.0 – 5.0). The dengue IgM antibody was positive. A Lepto MAT was performed twice on day 7 and day 11 of the illness, and showed a titre of 1:400 (a four -fold rise in titre or a high titre >1:800 is the gold standard for diagnosis) without further rise. A hepatitis screen was performed and was found negative. EBV antibody was also negative. This patient was similarly managed with a fluid regimen tailored to her body weight as per guidance, with strict monitoring of hemodynamic parameters and urine output.Whole blood analysis on presentation demonstrated thrombocytopenia with a platelet count of 10 × 109/L (150–450) on day 8 of the illness, and subsequently began to rise. The alkaline phosphatase and bilirubin continued to rise till day 10 of the illness, after which the liver functions began to normalize (Table 9/L (150–450) and a near normal bilirubin level.The platelet count continued to drop reaching a nadir of 5 × 10Dengue infection is usually associated with liver involvement, and this is more frequently seen among women, patients with a primary infection and in those with DHF . This clThe exact pathogenesis of liver injury in dengue is yet to be determined. The liver cells may be damaged through one or more of the following mechanisms: direct cytopathic effect of the virus, killing of virus-infected cells by the host immune response- the activated lymphocytes, antibodies against dengue proteins cross-reacting with host cell proteins and as a nonspecific effect of shock , 14. AnoAlternate causes of cholestatic jaundice should be considered. Ultra-sonographic imaging in our patients failed to demonstrate gall stones, and furthermore the normalizing bilirubin with the recession of jaundice made a sinister cause like carcinoma of the head of the pancreas unlikely. Intestinal flukes such as Fasciola Hepatica may cause an obstructive pattern of jaundice, but the clinical picture seen in acute/liver phase of the infection is different from the characteristic symptoms and biochemical derangement to that of dengue, additionally it is the chronic/biliary phase that classically produces jaundice in Fasciola Hepatica infection . Though Cholestatic jaundice in otherwise uncomplicated DF/DHF has been reported only scarcely in medical literature, once in a neonate with dengue fever  and two These cases highlight that in addition to deranged liver enzymes, cholestatic pattern of jaundice may be an under recognized clinical manifestation of dengue fever. A high suspicion of dengue should be present when other supportive clinical and laboratory criteria are present in addition to that of jaundice. Countries that are endemic to dengue and regions that have epidemic outbreaks should be vary of the different presentations that may mask dengue fever.Written informed consent was obtained from both patients for publication of this Case Report. Copies of the written consent are available for review by the Editor-in-Chief of this journal.


We find that crystallization in individual shear bands preferentially occurs in the regions neighboring the amorphous matrix, where the materials are subjected to compressive stresses demonstrated by our finite element simulations. Our results provide direct evidence that the mechanically induced crystallization kinetics is closely related with the stress state. The crystallization kinetics under compressive and tensile stresses are interpreted within the frameworks of potential energy landscape and classical nucleation theory, which reduces the role of stress state in mechanically induced crystallization in a metallic glass.Stress-induced viscous flow is the characteristic of atomic movements during plastic deformation of metallic glasses in the absence of substantial temperature increase, which suggests that stress state plays an important role in mechanically induced crystallization in a metallic glass. However, it is poorly understood. Here, we report on the stress-induced localized crystallization in individual shear bands of Zr The advantages of cold rolling are that the uncertainties in strain rate and temperature increase can be safely excluded. Furthermore, the influence of contamination on the phase transformation during rolling is minimized with respect to other severe plastic deformation methods . However, it is nearly impossible to directly investigate the stress distributions in individual shear bands (with a width of up to a few micrometers56781560Al15Ni25 bulk specimens. ε of 40% (see its definition in Method section). A bright contrast region, being thinner than the surrounding undeformed amorphous matrix due to its lower resistance to chemical attack during electrolytic thinning, is seen in 59.03Al16.41Ni24.33, being consistent with the nominal composition of Zr60Al15Ni25. The EDS patterns (see The X-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) image see  verify terns see  suggest erns see  indicateerns see  is a difε of 30%. The shear yield stress of Zr60Al15Ni25 bulk metallic glass is approximately 1.0 × 109 Pa  are triggered. The configuration B becomes mechanically unstable and undergoes structural rearrangement to an alternative PEM. It’s reasonably considered that nano-sized STZs are under hydrostatic stress states. Under a hydrostatic compressive effect, the cluster tends to be configured in a PEM  without release of elastic energy. Under the compressive state, the atoms in the STZ become jammed, which enhances the atomic redistribution to form an orderly configuration, as substantiated by the molecular dynamics simulationsC2) by releasing the elastic energy under a hydrostatic tensile effectC2). From C1is considerably lower than that in the PEM C2.Within the framework of potential energy landscape (PEL) theory∆G*) for homogeneous nucleation of a nucleus with a critical radius of r under a hydrostatic pressure can be expressed as17For an amorphous-to-crystalline phase transformation, the thermodynamic energy barrier , 60Al15Ni25 alloy is approximately 14.3 kJ/molγ) between the crystalline Zr2Ni phase and the amorphous Zr60Al15Ni25 phase is calculated to be approximately 0.1 J/m2. The value of ∆Gm is needed to calculate ∆G*. By setting P = 0, Eq. Ec) in a metallic glass can be expressed asGd is the activation free energy for the transfer of flow units (kinetic barrier) that is approximately 120 kJ/mol for Zr-based metallic glassesm and n are 3 and 4, respectively, for the exothermic nucleation of spherical nuclei during heatingEc of Zr60Al15Ni25 metallic glass is determined to be approximately 345 kJ/mol by Kissinger analysis∆Gm is calculated according to expressions of ∆G* and Ec and shown in Based on the fringe spacing  and the G*and P for the amorphous-to- crystalline phase transformation in Zr60Al15Ni25 metallic glass due to rolling can be expressed asBased on the values of parameters shown in ∆G* and P predicted by Eq. 8 Pa and −1.41 × 109 Pa R is the ideal gas constant. Since the viscosity in individual shear bands is similar to that in the supercooled liquid stateT for calculation of the kinetic contribution to the nucleation rate is selected to be 700 K, which is slightly higher than the glass transition temperature of 696 K∆G*  were prepared by suction casting in a copper mold under an argon atmosphere. The amorphous nature of the as-cast specimens is verified by X-ray diffraction (XRD) using monochromatic Co-Kα radiation and transmission electron microscopy (TEM). The as-cast bulk specimens were cut into bars with a cross-section of 1 mm × 2 mm and a length of 10 mm for cold rolling. The strain rates were controlled to be 10−3 ~ 10−4 s−1. The deformation degree was evaluated by the reduction in thickness, i.e., ε = (h0 − h)/h0, where h0 and h were the sample thickness before and after rolling, respectively. The microstructures of shear bands were observed by high-resolution transmission electron microscopy (HRTEM) under an accelerating voltage of 200 kV, equipped with a Gatan Image Filter System (GIF 2000) and an energy dispersive X-ray spectrometer . The TEM foils with a diameter of 3 mm were thinned using a twin-jet thinning electropolisher in a solution of 5% (volume percent) perchloric acid and 95% ethanol at 243 K. The chemical compositions of amorphous matrix and shear bands were checked by EDS and electron energy loss spectroscopy (EELS).Specimens with a nominal composition of Zr60Al15Ni25 bulk metallic glass .


Enhancing the critical heat flux (CHF) of industrial boilers by surface texturing can lead to substantial energy savings and global reduction in greenhouse gas emissions, but fundamentally this phenomenon is not well understood. Prior studies on boiling crisis indicate that CHF monotonically increases with increasing texture density. Here we report on the existence of maxima in CHF enhancement at intermediate texture density using measurements on parametrically designed plain and nano-textured micropillar surfaces. Using high-speed optical and infrared imaging, we study the dynamics of dry spot heating and rewetting phenomena and reveal that the dry spot heating timescale is of the same order as that of the gravity and liquid imbibition-induced dry spot rewetting timescale. Based on these insights, we develop a coupled thermal-hydraulic model that relates CHF enhancement to rewetting of a hot dry spot on the boiling surface, thereby revealing the mechanism governing the hitherto unknown CHF enhancement maxima. Cooling hot surfaces by boiling water is widely practiced, but the amount of heat transfer is normally constrained by vapour layer formation at sufficiently high temperatures. Here, the authors report the maximum in the critical heat flux on textured hydrophilic surfaces at an intermediate texture density. Boiling is a ubiquitous phenomenon of vital importance in a broad range of industries such as power generation, desalination, chemical processing, refrigeration and electronics thermal management12341per se that enhances CHF, more recent studies29et al.23Despite being an active area of research for over half a century, the boiling crisis is one of the least understood among known thermal and hydraulic instabilities1510121461618191218161819In this paper, we present data showing that the CHF of a textured hydrophilic surface neither increases monotonically with texture density162916a∼10 μm wide and h∼12.75 μm high square micropillars on a 650-μm-thick silicon substrate. Silicon was chosen as the test surface due to its compatibility with standard cleanroom microfabrication processes such as deep reactive ion etching and piranha cleaning. The micro-textured surface is composed of plain micropillars, whereas the nano-textured surface is composed of micropillars covered with an ∼100 nm length scale nano-texture called nanograss , the CHF of both the micro- and nano-textured surfaces is almost equal to the corresponding surface without the micropillars. A further reduction in micropillar spacing leads to a gradual increase in CHF, starting somewhere between b=25 μm and b=50 μm for the micro-textured surface and between b=50 μm and b=200 μm for the nano-textured surface. This initial result agrees qualitatively with the static force balance model16r, defined as the ratio of the actual surface area to the projected surface area. The logic behind their argument is that surface roughness increases the effective length of the three-phase contact line, thereby increasing the capillary forces that help to prevent the boiling crisis by keeping the surface wetted with liquid. For a given measured nanograss roughness of rng=3.43 (rm=1+4ah/(a+b)2) and nano-textured (rn=rng[1+4ah/(a+b)2]) surfaces increases monotonically with decreasing micropillar spacing b, and therefore one might expect CHF to exhibit a similar monotonic trend.To characterize the effect of surface texture on the boiling crisis, the heat flux ater see . It was ater see , which e  and nano-textured (199.5±11.5 W cm−2) surfaces  of the infrared-opaque Ti heater on the back of the substrate, we calculate the boiling surface temperature Tb using Fourier's law: Tb=Th—q″·ts/ks. Here q″ is the applied heat flux, ts∼0.6 mm the substrate thickness and ks∼105 W m−1 K−1 the thermal conductivity of the substrate. There are two approximations associated with this approach, which have a minimal affect on our analysis: (i) Steep spatial temperature variations on the boiling surface  can get smoothed out over a distance on the order of substrate thickness ts (∼0.6 mm), which fortunately is much smaller than the bubble/dry spot diameter we observe (∼5 mm). (ii) Sudden temperature fluctuations on the boiling surface require a thermal diffusion time Tb, which again is much smaller than the typical lifetime of a bubble/dry spot (∼50 ms).To justify our hypothesis for the boiling crisis, we first analysed the thermal behaviour of the boiling substrate at different heat fluxes to identify and characterize a hot dry spot. A real-time temperature map of the boiling substrate and boiling images was obtained using high-speed infrared and optical cameras, respectively, for boiling on the micro-textured surface with 10 μm-spaced micropillars . It shou−2, we obtain a single periodic bubble that grows and departs from the surface (39Tc because of evaporation of the liquid microlayer37At a low heat flux of 20 W cm surface . The bubTc increases to ∼137 °C as the dry spot grows to its maximum size (points 2→3). This behaviour is quite different from that of the single bubble at low heat fluxes, where there was a substantial drop but almost no rise in Tc  that develops across the advancing liquid–vapour front of the micro-imbibition layer and the viscous pressure drop (Pv) associated with the flow of liquid between the micropillars  and/or by imbibing it onto the surface of the dry spot. Here, we focus on the process of liquid imbibition into surface textures, which is dictated by a balance between capillary and viscous forces414243444546opillars . TheoretPc, we note that the liquid–solid contact angle at different levels of the surface texture hierarchy will be different (θ2=θ. At the micro-texture level (level 1) the apparent liquid–solid contact angle is θ1=cos−1[min], whereas at the topmost surface level it is given by θ0=cos−1[min], where rng and r=rng[1+4ah/(a+b)2] are the nanograss and overall-texture areal surface roughness, respectively. Considering the work done by the capillary forces on the advancing liquid front, an effective capillary pressure can be calculated using44Pc=−ΔE/ΔV, where ΔE is the net change in surface energies associated with the imbibition of liquid into a unit cell (V=h[(a+b)2–a2] +4ah]rng(σsl–σsv)+b(2a+b)σlv, where σsl is the solid–liquid, σsv the solid–vapour and σlv the liquid–vapour interfacial surface tension. Using the Laplace equationσsl–σsv=−σlv cosθ, and noting that the capillary force responsible for imbibing the liquid between the micropillars maximizes for θ1=0, we getTo calculate the capillary pressure ifferent . The nannit cell  of the mPc>0.Note that the liquid will imbibe into the micro-textures as long as The viscous pressure drop associated with the flow of liquid between the micropillars can be written asμ is the liquid dynamic viscosity, vm is the mean imbibition velocity, L is the length of the imbibed layer and Kv is the in-plane permeability of the surface texture. Using a simple scaling analysis (Kv=[3/h2+24a/b2(a+b)]−1. Equating the capillary pressure and the viscous pressure drop (and ignoring the hydrostatic pressure because the Bond number is low), we can solve for the imbibition time τi required for the liquid to imbibe to a length L:where analysis , we can (θ∼0) and the imbibing liquid front was tracked using a high-speed camera (σlv), liquid viscosity (μ) and the liquid–solid contact angle (θ), we can easily extrapolate the silicone oil imbibition results to other liquids including water . For larger micropillar spacings, the thickness of the imbibed liquid microlayer ti is expected to be less than the height of the micropillars. Indeed, the model agrees with the data for b>10 μm assuming ti∼4 μm. Second, the occurrence of the minimum in imbibition time τi at the same micropillar spacing (b∼10 μm) as the observed CHF maxima indicates that a higher CHF for textured surfaces is in-part due to enhanced rewetting of a hot dry spot by the micro-imbibition layer, which further strengthens our fundamental hypothesis for the boiling crisis. Representing the nanograss using square nanopillars where a=b=h∼100 nm, we can also verify that a liquid will imbibe into the nanograss much slower  than into the micropillars . The inset in This imbibition model was verified using imbibition experiments, where silicon micro-textured samples were dipped in 10 cSt silicone oil d camera . For expng water . Figure D∼Dmax/2 and average height H∼D, which results in the formation of a dry spot of maximum radius λ∼D on the boiling surface  is equal to To. As our hypothesis relates the boiling crisis to rewetting of a hot dry spot on the boiling surface, we are interested in the following two timescales: (i) a heating timescale τh, in which the temperature at the centre of a fully grown dry spot increases from To to the critical dry spot temperature Tcrit, and (ii) a rewetting timescale τw, in which the surrounding liquid is able to completely rewet the dry spot. Below, we will postulate that the boiling crisis occurs when τh<τw.Having developed an understanding of the surface heating characteristics and the imbibition process in the previous two sections, we now present the dynamic model that predicts the boiling crisis. We focus on an idealized spherical vapour mass with an average diameter  surface . At heaton study , where aτh can be estimated using a one-dimensional thermal lumped capacitance model. Ignoring any lateral conduction of heat in the thin solid substrate For a thin boiling substrate, the heating timescale ρs is the density, Cs the specific heat and ts the thickness of the substrate. To determine τh, we need a value for Tcrit–To, the permissible rise in substrate temperature at the centre of the dry spot during its rewetting phase, which we call the critical dry spot superheat. Previously, we saw that at a heat flux close to CHF, the dry spot centre temperature of the micro-textured surface with 10-μm-spaced micropillars, rose from ∼137 °C to a maximum value of ∼149 °C  and uniform vapour pressure inside it, the pressure at the base of the sloshing liquid front should be equal to the pressure at the top of the bubble minus a surface wetting-related pressure reduction term ΔPr for partially wetting surfaces (θ0>0). Starting from the comparatively stationary liquid near the top of the bubble, the average velocity of the sloshing liquid front vg can be obtained using the Bernoulli's equationPr∼2σ(1–cosθ0)/H. Noting that the effect of ΔPr is limited to the scenario where ΔPr<<ΔρgH (for hydrophobic surfaces with large ΔPr the sloshing liquid can just roll onto the surface), we can calculate the dry spot rewetting timescale as follows:At the same time that the substrate temperature is increasing, the surrounding liquid moves in to rewet the dry spot under the influence of gravity and aided by the micro-imbibition layer that develops between the micropillars . The boitance fλ . Assuminτw,g denotes the gravity-induced component of the rewetting timescale and the non-dimensional term τr=ΔPr/2ΔρgD accounts for the resistance of partially wetting surfaces to rewetting by the sloshing liquid front. In the absence of liquid imbibition, the dry spot is wetted purely by gravity, in which case f=1 and τw=τw,g. Substituting water properties at 100 °C, we get a value of the gravity induced rewetting timescale for flat silicon of the same order (∼13 ms) as the corresponding heating timescale  and the average imbibition liquid front velocity vi is larger than vg, then part of the dry spot is rewetted by this imbibed layer (f)λ:If the surface texture is favourable to the formation of a micro-imbibition layer . The rewetting timescale can therefore be written as:where Pc is negative (non-imbibing textures). In b for the micro-textured surfaces and see that it agrees quite well with an experimental timescale obtained by extrapolating the silicone oil imbibition results to water  and heating (τh) timescales at the experimentally observed CHF values for all the micro- and nano-textured boiling samples in τw and τh is plotted against the micropillar spacing b using experimentally measured values of micropillar width (a), height (h) and spacing for each sample , the rewetting of the dry spot is purely gravity-induced because either the liquid does not imbibe into the micropillars (Pc<0 for micro-textured samples) or the imbibition liquid-front velocity vi is smaller than the velocity of the sloshing liquid front vg (nano-textured samples). In this regime, the slightly higher CHF for the perfectly wetting (θ0=0) nano-textured surface is explained by the non-zero sloshing-liquid pressure reduction term (ΔPr) for the partially wetting micro-textured surface (θ0∼30°). From b∼200 μm to b∼50 μm, the CHF increases for both the surfaces, albeit due to different reasons. The smaller increase for the micro-textured surface is due to a reduction in ΔPr (decreasing θ0), whereas the larger increase for the nano-textured surface is because of imbibition-induced rewetting of the dry spot becoming active (vi>vg). For b<50 μm, imbibition-induced rewetting becomes active for both the surfaces with the CHF enhancement larger for the nano-textured surface because of a smaller micro-texture level liquid–solid contact angle θ1. Below a micropillar spacing of ∼10–20 μm, CHF for both surfaces starts to decrease with further reductions in b. This is because reducing b now increases the viscous pressure drop Pv to a larger degree than the imbibition capillary pressure Pc, resulting in a reduced imbibition liquid-front velocity vi. For small micropillar spacings (b<<a and b<<h), the capillary pressure scales as Pc∼1/b, whereas the viscous pressure drop scales as Pv∼1/b2 (or Kv∼b2).θ>0), both the micropillars and nanopillars enhance CHF to a small degree by decreasing the surface-level liquid–solid contact angle θ0, which enhances gravity-induced rewetting of a dry spot. (ii) The micropillars (but not the nanopillars) also lead to an additional larger enhancement in CHF because of the supplementary role played by the micro-imbibition layer in rewetting of the dry spot. A nano-imbibition layer is not feasible because of the comparatively lower rate of imbibition. (iii) For θ>0, the presence of nanopillars on top of the micropillars enhances CHF further by decreasing the micro-texture level liquid–solid contact angle θ1, which enhances the micro-imbibition layer-induced rewetting of the dry spot.We can summarize the role of the micropillars and the nanograss (or nanopillars) in CHF enhancement as follows: (i) For a partially wetting substrate (As there are two distinct mechanisms (gravity and imbibition-induced surface rewetting) by which texture influences CHF, we can identify two non-dimensional surface texture parameters that dictate CHF enhancement. Normalizing the expression of CHF obtained in φg=(1–τr)/ is the gravity-induced and φg normalizes the CHF enhancement effect of nanograss at larger micropillar spacings, φi accounts for the imbibition-induced enhancement because of texture at both length scales. In φi as predicted by τr, Kv and Pc can be calculated or experimentally determined for other surface micro-texture morphologies.where ρsCsts), below a minimum value of the boiling substrate thickness ts. The expression for the heating timescale developed in ts (∼0.6 mm) being less than the characteristic thermal diffusion length ts>LD, the heating of the substrate during the dry spot rewetting phase should be limited to a depth LD, making CHF independent of ts for thicker substratesIn addition to correctly predicting the effect of surface texture on the boiling crisis, the coupled thermal-hydraulic formulation of the CHF model  also expθ0∼0) but non-imbibing surfaces have only a slightly higher CHF than flat partially wetting or hydrophobic surfacesIn summary, we show the existence of maxima in CHF enhancement at intermediate texture density for pool boiling on thin (∼0.6 mm) micro- and nano-textured surfaces. Using liquid imbibition experiments and quantitative infrared temperature measurements of dry spots on the boiling surface, we show direct evidence that the boiling crisis is dictated by characteristic dry spot heating and rewetting timescales. Based on these observations, we develop a coupled thermal-hydraulic model for CHF, given by 2 thin-film heater was patterned on its backside by depositing a 150-nm Ti layer (heater) followed by a 300-nm Ag layer (contact pads) using e-beam evaporation and shadow masking. The wafers were diced into 5 × 5 cm2 samples using photoresist to protect the top surface from contamination. Photoresist was stripped off and samples were cleaned in acetone and isopropanol. Just before the boiling experiments, the samples were again cleaned in acetone, isopropanol and DI water to remove any organic contaminants.Using deep reactive ion etching and employing patterned photoresist as an etch mask, square micropillars were etched on the top surface of a 650-μm-thick double-sided polished <100> silicon wafer. After removing the photoresist and cleaning the wafer in Piranha solution, nanograss was etched on top of the micropillars (for the nano-textured samples only) using a custom reactive ion etch recipe. The wafer was then cleaned again in Piranha solution and a 1 × 2-cmrng=3.43 was determined using atomic force microscopy measurements conducted on nanograss grown on a flat silicon substrate  on the boiling samples, we need to account for the presence of a native oxide layer formed due to both oxidation in airθ∼36°) and Piranha cleaningθ∼22°). The contact angle of DI water on a flat silicon wafer with native oxide, and cleaned in the same manner as the boiling samples, was measured to be θ∼30° .Supplementary Figures 1-5, Supplementary Notes 1-5 and Supplementary ReferencesRaw infrared videos illustrating a CHF event on the nano-textured surface with 2 μm-spaced micropillars. Playback speed is 1/50 x real-time.Imbibition of 10 cSt silicone oil into the micro-textured surfaces with different micropillar spacing. Playback speed is 1x real-time.


FNDC5 on type 2 diabetes and clinical traits related to glucose metabolism in a large Chinese population sample.To investigate the impact of common variants of FNDC5 gene were selected and genotyped in 6822 participants. Detailed clinical investigations and biochemistry measurements were carried out in all of the participants. Subjects without diabetes were classified into normal weight and overweight/obese subgroups according to body mass index (BMI).Three tagging single nucleotide polymorphisms within the region of the 2), the rs16835198 major allele G was significantly associated with fasting insulin levels, and that each additional copy of the allele resulted in a 0.0178 mU/L increment of the values (p = 0.046). Moreover, after adjusting for confounding variables, there were trends towards correlation of rs16835198 with HOMA-insulin resistance (HOMA-IR) (p = 0.057) and low-density lipoprotein cholesterol (LDL-C) levels (p = 0.083). In overweight/obese subjects (BMI ≥25 Kg/m2), we noted rs16835198 showed trends towards association with fasting insulin (p = 0.057) and HOMA-IR levels (p = 0.091), both of which declined with additional copies of the major allele G. Moreover, rs16835198 was significantly associated with high-density lipoprotein cholesterol (HDL-C) levels (p = 0.013), and HOMA-β cell function (p = 0.028) in the overweight/obese subjects. Finally, we observed a significant interaction between BMI-rs16835198 and fasting insulin levels in the control group (p = 0.003).None of the SNPs were associated with either the risk of type 2 diabetes in all of the participants or with any of the clinical quantitative traits in the controls with normal glucose regulation. Subgroup analysis showed that in controls with normal weight (BMI <25 kg/mFNDC5 SNP rs16835198 on fasting insulin was significantly modified by BMI in the Chinese Han population.Our data indicate that the effect of the common  Diabetes is a major public health concern, and changes in diet and lifestyle have accelerated the rise in the incidence of this disease to pandemic proportions in many countries worldwide, including China. According to the national epidemiological survey conducted in 2008, China has the largest number of diabetics in any single country FNDC5  gene in both mice and humans Ucp1 and Cidea genes, implying its potential as therapeutic for combat obesity, diabetes and other disease could be ameliorate by exercise FNDC5 with insulin sensitivity and glucose metabolism FNDC5 variants in human metabolic disease, we set out to examine the association between FNDC5 variants and a range of metabolic parameters in a large Chinese Han population.Physical exercise is recognized not only as an indispensable component of lifestyle intervention but also, increasingly, as a cornerstone for non-pharmacological therapy in ameliorating obesity and insulin resistance The study was approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and in accordance with the Declaration of Helsinki. Written informed consent was obtained from each participant.2), and overweight/obese (BMI≥25 Kg/m2). The clinical characteristics of all participants are shown in The overall study group totally comprised 6822 participants of Han Chinese ancestry residing in Shanghai. Detailed information regarding this study population was described in detail previously et al.10 mean insulin.All participants underwent a detailed clinical investigation as described previously 2≥0.8, we selected three tagging SNPs in a region encompassing the FNDC5 gene and its 10 kb up- and downstream sequences. These three tagging SNPs tagged 100% of SNPs (5/5 SNPs in the HapMap Chinese Han sample) with a minor allele frequency (MAF) of >0.05. All SNPs were genotyped by primer extension of multiplex products with detection by matrix-assisted laser desorption ionization–time of flight mass spectroscopy, using a MassARRAY Compact Analyzer  with an overall call rate of 99.6%.Using the HapMap Phase III (release 27) Han Chinese database and a threshold of rp<0.05 in the controls) were excluded. Pairwise linkage disequilibrium including |D’| and r2 was estimated using Haploview (version 4.2). Allele and genotype distributions between the cases and control subjects were compared with χ2 test or logistic regression For each variant, the Hardy-Weinberg equilibrium test was performed separately in the cases and controls prior to association analysis. SNPs failing this test . Linkage disequilibrium analysis for these SNPs was shown in p value of 0.265 for rs3480. Similarly, logistic regression analysis found no association between any SNP and type 2 diabetes , rs16835198 major allele G was significantly associated with a 0.0178 mU/L increment in fasting insulin levels for each additional allele copy (p = 0.046). Moreover, after adjustment for confounding variables, this SNP exhibited trends towards association with HOMA-IR (p = 0.057) and serum LDL-C levels (p = 0.083) in the same subgroup (2) subgroup, with each additional copy of major allele (G) exhibiting lower values than TT homozygotes . Moreover, rs16835198 was also significantly associated with HDL-C and HOMA-β cell function levels in the overweight/obese subgroup both before  and after  adjusting for these covariates (p = 0.003). Results regarding the association between the other two SNPs and clinical traits in BMI-specific subgroups are shown in Supporting Information Click here for additional data file.


Metal alloy catalystspermitted a greater inclusion of Sn in Ge nanowires compared with conventional Aucatalysts, when used during vapour–liquid–solid growth. Theaddition of an annealing step close to the Ge-Sn eutectic temperature(230 °C) during cool-down, further facilitated the excessivedissolution of Sn in the nanowires. Sn was distributed throughout the Ge nanowirelattice with no metallic Sn segregation or precipitation at the surface or withinthe bulk of the nanowires. The non-equilibrium incorporation of Sn into the Genanowires can be understood in terms of a kinetic trapping model for impurityincorporation at the triple-phase boundary during growth.The development of non-equilibrium group IV nanoscale alloys is critical to achievingnew functionalities, such as the formation of a direct bandgap in a conventionalindirect bandgap elemental semiconductor. Here, we describe the fabrication ofuniform diameter, direct bandgapGe Direct band gap nanostructures compatible with Si-based electronicsare actively investigated. Here, Biswas et al. incorporate unusually largeamounts of tin in germanium nanowires by non-equilibrium kinetic trapping, and opticalcharacterizations suggest that the nanowires exhibit a direct band gap. Direct bandgap semiconductor materials are needed for new device architectures such asband-to-band tunnelling tunnel FETs3k=0 drops in energy until, in grey tin, the material acquires adirect (and vanishing) bandgap at k=0  were recently reported onthe fabrication of suspended GeSn nanowires through competitive etching betweenGex1−Snx and Ge layers20In recent years, considerable effort has been used to growGeOn the basis of thermodynamic limitations, a non-equilibrium growth scenario influencedby the kinetics of the system is required to incorporate a sufficient amount, far fromequilibrium, of Sn into a 1D Ge lattice to achieve a direct bandgap transition. Thetriple-phase boundary at the catalyst-nanowire interface in a bottom-up growth processis known to be a feasible pathway for impurity incorporation in a 1D lattice and can actas a localized non-equilibrium centre for excessive impurity dissolution24x1−Snx nanowires withconsiderable (x>0.09) Sn incorporation; around 10 times the equilibriumsolubility. Third-party metal catalysts  were used to guide thenon-equilibrium incorporation of Sn adatoms into the precipitated Ge bi-layers, wherethe impurity Sn atoms become trapped with the deposition of successive layers, thusgiving an extraordinary Sn content in the alloy nanowires.Here, we report the application of a three-phase bottom-up growth protocol to fabricatehighly crystalline, uniform diameter, direct bandgapGex1−Snx nanowires, wehave used dodecanethiol-stabilized phase pure Au andAu0.90Ag0.10 alloy nanoparticlesx1−Snx nanowires at440 °C on the surface of Si(001) substrates. Diphenylgermanewas used as the Ge source, whereas allyltributylstannane was used as the tinprecursor. Similar decomposition kinetics and solubility of the tin andgermanium precursors provoked the choice of diphenylgermane andallyltributylstannane, where the Sn precursor has a slightly higher boilingpoint (360 °C at atm. pressure) than the Ge precursor(325 °C at atm. pressure). The choice of Au and AuAgcatalysts and the growth temperature was driven by the Au-Ge and Au-Ag-Ge phasediagramsParticipation of Au and AuAg alloy seeds in the bottom-up growth of Ge nanowireshas been well-documented by our group in previous reports2627Both Au and AuAg nanoparticles successfully catalysed the growth of Ge nanowiresafter a 2 h time period, as determined by scanning electronmicroscopy see . The abs27x1−Snx nanowires with ahigh Sn content, it is essential to explore the quantitative and qualitativeincorporation of Sn in the nanowire body. Energy-dispersive x-ray (EDX) analysisand high-resolution electron energy loss spectroscopy (EELS) provide the meansto investigate the chemical environment in the bulk of the alloy nanowire andalso at the atomic scale. The composition of the nanowires and the distributionof elements within them were estimated through EDX point measurements andelemental mapping by STEM. For nanowires grown from pure Au catalysts, theamount of Sn in the injecting solution was varied from10–20 at.%, resulting in a gradual increase inthe actual Sn concentration in the nanowires. Very low amounts of Sn (meanconcentration of 1.5 at.%) was determined in nanowiresusing an injecting solution of 10 at.% Sn. Increasing theSn concentration in the injecting solution to 15 and20 at.% resulted in the incorporation of Sn in thenanowires at levels of 6 and 9 at.%, respectively. Thesevalues of Sn concentrations are much higher  than theextrapolated bulk equilibrium solid solubility of Sn in Gex1−Snx nanowires withsubstantial Sn incorporation and with negligible secondary nucleation ofunwanted spherical particulates in the sample. With the aim of including more Sninto the 1D Ge lattice, Au0.90Ag0.10 alloy nanoparticlecatalysts were used as seeds rather than pure Au, as the alloy seeds have beenpreviously shown to favour faster growth kinetics for phase pure Ge nanowiregrowthx1−Snx nanowire grown fromAuAg seed is shown in x1−Snx nanowire alsoconfirmed uniform Sn distribution in the entire nanowire volume  was followed by an annealing stepfor 2 h at 230 °C during the cool-down. Motivationto introduce a step cooling at 230 °C was driven by tworeasons: a small window in the Sn-rich side of the Au-Sn phase diagram at230 °C and the bulk Ge-Sn eutectic temperature at around230 °C. This step cooling technique further forces a colossalamount of Sn at.%), as determined by EDX measurements were traced (error bars aredefined in the Methods section). Two EELS maps were acquired at two vastlydifferent heights along the wires, to make sure Sn was not only present atcertain areas of each nanowire, such as close to the catalyst. Maps werede-noised by principal components analysis and the background was removed byfitting a power law over a region immediately in front of the core loss edges.The signal was then integrated over a 120 eV window above the onsetof the Sn M mapping  with a shotspots .Hence Shotspots , withoutLS edges . The redLS edges  correspox1−Snx nanowire with a9.2 at.% Sn incorporation and with a2–3 nm amorphous oxide coating. Fast Fourier transform(FFT) analysis showed a pseudo hexagonal symmetry and the reflections can beassigned to the high-order Laue zone diffraction of {111} and {002} planes ingroup IV crystalsd) between {111} planes in the nanowire to be0.323 nm, which is very close to the d value for bulk diamondGe crystal (JCPDS 04–0545). An increase in the d value frombulk Ge is expected with the incorporation of large amounts of Sn in Ge lattice.However this discrepancy may arise from the fact that this particular nanowirecould only be aligned to a relatively minor zone axis. x1−Snx alloynanowires, with various Sn incorporations, exhibited a bulk diamond cubiccrystal structure with a 3C lattice arrangement without any stacking faults andtwin boundaries, with <111> being the dominant growth direction.Although there is a large lattice mismatch between the components (Ge and Sn) ofthe alloyd value, wehave calculated the inter planner spacing of 50 successive lattice planes (over>15 nm length) and determined the average d value to be0.331 nm, which is slightly above the bulk 3C-Ge value  in nanowires can induce structural defects, suchas twins and stacking faults and these defects can act as preferential sites forsubsequent impurity accumulationGe value . Bright-Ge value  of the i−1 in bulk Ge is attributed to theGe-Ge LO mode. The Ge-Ge Raman peak progressively shifts to a lower energy withincreasing Sn concentration (as determined by EDX measurements). A red shift of1.2 to 5.9 cm−1 of the Ge-Ge LO mode wasobserved for a variation in the Sn concentration from 1.5 to9.2 at.%, compared with bulk Ge. We could not compare theRaman shift with a sample of pure Ge nanowires, as the similar growth conditionswithout any Sn yielded Ge nanowires of entirely different dimensions andmorphology−1, as shown in x1−Snx alloys,where an increase in the intensity ratio between Ge-Sn and Ge-Ge LO modes withincreasing Sn content, implies a larger substitution of Sn in the Ge lattice forGex1−Snx nanowires. Theorigin of the Ge-Ge frequency shift in the Raman spectra of theGex1−Snx alloys is due tocompositional variations and strain effects. Participation of compressive andtensile strain towards the Raman shift is not justified for nanowire samples, asdue to the large surface area, strain can be effectively released for thesenanostructures. Compositional variations can originate from two factors: (i)mass disorder and (ii) bond distortion. The Ge-Ge LO mode progressively shiftstowards a lower frequency with an increasing Sn concentration as displayed inthe inset of ω) against Sn composition (x), asdetermined through EDX analysis, with a linear expression,ω(x)=ω0+Δωx,and the obtained value of Δω was found to be−(64.3±0.1) cm−1. This value isconsistent with the value of—(68±5)cm−1 reported by Li et al., who assumed thattheir alloy films were completely strain freex1−Snx nanowires.Compared with a few other recent reports, a discrepancy in theΔω value and Raman shift is observed for the9.2 at.% Sn containing nanowire sample with a relativelysmaller shift in the Ge-Ge LO peak21Raman scattering is an effective tool to estimate the structural and chemicalenvironment in the core of a nanowire. Raman spectroscopy was used to accuratelyprobe the local chemical bonding environment and also to estimate the amount ofSn in the alloy nanowire samples. x1−Snx thin films andbulk alloy to obtain a direct bandgap, it is essential to investigate theemission characteristics of strain free bottom-up grownGex1−Snx alloy nanowires.Bandgap information on Gex1−Snxalloys was extracted through photoluminescence measurements at low temperature.A photoluminescence study to probe the bandgap characteristics ofGex1−Snx nanowire sampleswas conducted on samples cooled to 7 K, using a He cryostat.Photoluminescence spectra of two nanowire samples (with an average Snconcentration of 6 and 9.2 at.%) recorded at7 K is shown in x1−Snx alloy nanowiresresulted in the reduction of the bandgap energy difference between the directand indirect transition, which was approximately 0.14 eV in bulk Ge.Nanowire samples containing a high Sn content (9.2 at.%)exhibited a single photoluminescence emission peak centred at2,233 nm (band gap (Eg) around 0.55 eV), with arelatively narrow line-width (202 nm) compared with thephotoluminescence plot from the low Sn contentGex1−Snx nanowire sample.The relatively narrower line-width of the photoluminescence emission confirmsthe single energy emission at the Γ pointx1−Snx nanowires, with9.2 at.% Sn incorporation, the direct band-to-bandtransition resulted in relatively narrow photoluminescence emission, comparedwith the broad emission fromGex1−Snx alloy nanowiresincorporating 6 at.% Sn. A single peak with a relativelynarrow line-width could signify emission from only the direct bandgap transitionrather than unification of both direct and indirect transitions. The lowphotoluminescence emission intensity observed from the nanowire samples could bedue to the luminescence quenching from metallic Sn impurities, which are presentin the catalysts at the tip of the nanowires and also in negligible amounts asspherical particles in samples. Also the high surface-to-volume ratio ofnanowires compared with thin films can account for the lower luminescenceintensity. Relatively broad photoluminescence spectra for both nanowire samples,compared with previous reports, could result from the random distribution of Snin the alloys. Sn incorporation in the nanowire samples also showed a standarddeviation (around 1%) which may also account for the broadness of thephotoluminescence spectra obtained.As there are contradictions regarding the amount of Sn needed inGex1−Snx nanowires matchedwell with the reported emission from alloy thin films and disks with similar Snincorporation (8–10 at.%)112140x1−Snx nanowires at lowtemperature also complements the low temperature photoluminescence observationfrom GeSn thin films11x1−Snx nanowires(9.2 at.% Sn) show a massive red shift in the emissionwavelength with the inclusion of Sn   the photoluminescence peak position was around2,150 nm. With increasing excitation (up to 16 P0)spectral broadening was observed, coupled with a blue-shift of thephotoluminescence peak position. We attribute this change to carrier filling ofclosely-spaced Γ and L energy bands. This shift was not observed inthe case of Ge nanowires within the scope of available excitation powers between350 and 700 mW. In addition, in order to achieve similarphotoluminescence intensity from both Ge andGex1−Snx nanostructures  depicts ructures, GenanoR2)close to unity, that is, for a good fit, indicates a single channel ofrecombination, while poor fit indicates competitive transition channels. For6 at.% Sn containing nanowire samples the value ofR2 was calculated to be 0.935 28462↔diamondA4+AuSn4 mediated by the formation of AuSn intermetallicphases. We did not observe the presence of these intermetallic phases within thenanowire  Sn diffusion in Ge at the growthconditions is negligible, (ii) the epitaxial mismatch between Sn and Ge results inelastic strains at and near the catalyst-nanowire interface and (iii) the lack oftruncating side facets at the catalyst-particle interface. Assuming that growth ofthe nanowire is layer by layer, the step flow kinetics can result in solute trappingof Sn from the Sn-rich droplet. The deviation of chemical equilibrium at theinterface is influenced by the kinetic parameter, that is, interfacial diffusionspeed in this case. For bulk metal-semiconductor systems impurity trapping at theliquid–solid interface is highly probable at a very high interfacevelocity in the order of m sec−1 than the growth velocity required for kinetic driven solute trapping. However, forour particular system a much higher Sn concentration in the catalyst seed(>90 at.%) than the impurity concentration in a typicalbulk solidification process and a continuous Sn flux throughout nanowire growthcould account for the high Sn incorporationx1−Snx nanowires. The highSn concentration around the growing steps is further aided by low Sn diffusivitywithin the nanowire and along its sidewalls, and the kinetic pathway favors Snincorporation into the newly forming layer at the expense of elastic strainsrelative to the pristine Ge crystal. The elastic strains can be effectivelyaccommodated by the Sn-rich droplet. Finally, at equilibrium the catalyst-nanowireinterface also involves truncating side facetsx1−Snxnanowires and the interface is fully faceted which is indirect confirmation of theelastic strains due to non-equilibrium Sn incorporation. As a result, the Snincorporation is uniform through the nanowire, as opposed to being localized at thecore or within a surface shell.Non-equilibrium induction of Sn impurity in the Ge host is justified throughdiffusionless solute trapping at a finite growth velocity of the crystals. Solutetrapping is a process of solute redistribution at the interface resulting in anincrease of chemical potential and deviation of partition coefficientc−1 ref. . HoweverInduction of Sn in the alloy nanowires is further encouraged with a 2 hannealing at 230 °C, during the cooling down of nanowires . The chox1−Snx nanosystem, usuallymetastable under thermodynamic equilibrium, compatible with existing Si electronicsplatforms. Kinetic driven solute trapping model in a catalytic bottom-up growth withnoble metal catalysts facilitates the dissolution of high amounts of Sn into thealloy nanowires. The sparse spatial distribution of Sn in the nanowires and theformation of GeSn alloys in the nanowire cores were confirmed through atomicresolution EELS mapping and Raman spectroscopy. The use of innovative AuAg alloycatalysts, which enhances nanowire growth kinetics, facilitated larger incorporationof Sn into the Gex1−Snx lattice thanpure Au catalyst. Calcination of the nanowires near Ge-Sn eutectic temperatureduring the cool-down further aided Sn precipitation and dissolution to achievedirect bandgap Gex1−Snx alloy(x=0.092) nanowires. Further manipulation of growthtemperature, choice of precursors and catalysts could lead towardsGex1−Snx nanowires witheven a higher Sn content. Three-phase bottom-up growth is a feasible way toincorporate large amount of impurities and dopants in semiconductor nanowires.Demonstration of colossal incorporation of foreign atoms in the host semiconductorlattice allows new or added functionalities  in the existing semiconductorarchitecture. The demonstrated VLS process with its innovative catalyst andprecursors contributes towards the ongoing research on dopant/impurity incorporationin nanowires by directly demonstrating kinetics-dependent tin incorporation in Genanowire. The protocol demonstrated here is general and could be applied to otherdoped or alloy nanowire systems  to create new and innovative nanomaterials fornovel physics and devices. This paper also addresses towards the ongoing demand fora nanoscale material for group IV photonics. The demonstration of direct bandgapsilicon compatible nanowires will instigate a lot of initiative on fundamentalresearch on band-structure engineering of binary and ternary group IV alloynanowires and implementation of these nanoscale materials in photonic and electronicdevices. The great success of III–V quaternary semiconductors indecoupling strain and band-structure effects suggests that ternary compounds shouldhave a similar impact in the group IV arena. Direct bandgapGex1−Snx nanowires fabricated througha cheap and feasible bottom-up technique opens up unlimited possibilities in groupIV photonics, nanoelectronics and optoelectronics.In summary, we have successfully fabricated a stable direct bandgap 1DGe−3.Solutions of diphenylgermane and allyltributylstannane in anhydrous toluene wereprepared in an N2 glove box with a typical Ge precursor concentrationof 10 μM ml−1 andvarying Sn concentrations. The concentration of diphenylgermane in toluene wasfixed at 10 μM ml−1,whereas tin precursor concentrations were varied from1–2 μM ml−1for the incorporation of different amounts of Sn in theGex1−Snx nanowires. Aprecursor solution was loaded into a Hamilton sample-lock syringe inside anitrogen-filled glovebox. Before injection, the coated Si substrate was annealedfor 15 min at 440 °C under a flowingH2/Ar atmosphere inside a tube furnace. The precursor solution wasthen injected into the metal reaction cell using a high-pressure syringe pump ata rate of 0.025 ml min−1. AH2/Ar flow rate of0.5 ml min−1 was maintainedduring the entire growth period. A typical nanowire growth time was2 h. An additional annealing step was also introduced during thecool-down process where the substrate was kept at 230 °C for2 h under a H2/Ar flowing atmosphere. The reaction cellwas allowed to cool to room temperature and disassembled to access the growthsubstrate. Nanowires were washed with dry toluene and dried under N2flow for further characterization.Continuous-flow reactions for nanowire growth were carried out in a toluenemedium using a liquid-injection CVD technique. Metal nanoparticles werespin-coated onto a Si (001) substrate and loaded into a stainless steel microreactor cell, connected to metal tubing. The catalyst nanoparticle concentrationin each case was fixed at40 μM cmx1−Snx nanowireswere imaged on an FEI Helios NanoLab 600i scanning electron microscope. All EDXmeasurements were recorded in high-angle annular dark-field mode in the FEIHelios NanoLab 600i operating at 20 kV and 1.4 nA with anattached Oxford X-Max 80 detector. Error in the EDX measurements indicatesstandard deviation in EDX measurements measured over 50 nanowires. TEM analysiswas done in a JEOL JEM-2100 operating at 200 kV in bright-fieldcondition for imaging. High-resolution STEM imaging and EELS mapping was doneusing Nion UltraSTEM100 microscope, operated at 100 kV. Probe-formingoptics were adjusted to deliver a 0.9 Å probe, with120 pA beam current and 31 mrad convergence semi-angle.EELS data was acquired on a Gatan Enfina spectrometer, at 1 eV perchannel to capture both the Sn and Ge edges simultaneously. As a result, theeffective energy resolution was limited to 2.5 eV by the detectorpoint spread function (∼3 pixels), even though the cold field emissiongun of the instrument had a native energy width of 0.35 eV in theoperating conditions. Raman scattering measurements were performed in abackscattering geometry using a micro-Raman setup consisting of a spectrometer and a Peltier-cooled charge-coupled devicedetector. An air cooled He-Ne laser of wavelength 633 nm was used asan excitation source. The photoluminescence measurements were performed using aconfocal configuration. Samples were cooled to 7 K using a Heliumcryostat. A pulsed titanium-sapphire 800 nm laser was used as anexcitation source. Laser frequency was 76 MHz and pulse width was300 fs. The laser beam was focused down to a50 μm spot and the power was measured to be500 mW. For temperature- and power-dependent experiments, thestructures were encased in a liquid He and liquid nitrogen cryostat,respectively equipped with KBr window and cooled to desired temperature. Thephotoluminescence emission was collected by a monochromator and then sent to athermoelectrically cooled, photoconductive, extended-range InGaAs detector,sensitive in the mid-IR spectral range from 1.2 to 2.6 μmand facilitated by CaF2 optical components. Lock-in and chopper was used as astandard noise-cancellation tool.Bottom-up grown GeHow to cite this article: Biswas, S. et al. Non-equilibrium inductionof tin in germanium: towards direct bandgapGex1−Snx nanowires. Nat.Commun. 7:11405 doi: 10.1038/ncomms11405 (2016).Supplementary Figures 1-15


TNF G-308A, in sudden infant death syndrome (SIDS) infants, SIDS and control parents, and ethnic groups with different incidences of SIDS; (2) the effects of two risk factors for SIDS, cigarette smoke and virus infection, on TNF-α responses; and (3) to assess effects of genotype, cigarette smoke, and gender on TNF-α responses to bacterial toxins identified in SIDS infants. TNF G-308A genotypes were determined by real-time polymerase chain reaction for SIDS infants from Australia, Germany, and Hungary; parents of SIDS infants and their controls; and populations with high , medium (Caucasian), and low (Bangladeshi) SIDS incidences. Leukocytes from Caucasian donors were stimulated in vitro with endotoxin or toxic shock syndrome toxin-1 (TSST-1). TNF-α responses were measured by L929 bioassay (IU/ml) and assessed in relation to genotype, smoking status, and gender. There was a significantly higher proportion of the minor allele AA genotype among Australian SIDS infants  compared to 3/62 (4.8%) controls (p = 0.03). There were no significant differences in TNF-α responses by TNF G-308A genotypes when assessed in relation to smoking status or gender. Given the rarity of the TNF G-308A A allele in Caucasian populations, the finding that 24% of the Australian SIDS infants tested had this genotype requires further investigation and cautious interpretation. Although non-smokers with the AA genotype had higher TNFα responses to both TSST-1 and endotoxin, there were too few subjects with this rare allele to obtain statistically valid results. No effects of genotype, smoking, or gender were observed for TNF-α responses to these toxins.Dysregulation of the inflammatory responses has been suggested to contribute to the events leading to sudden infant deaths. Our objectives were (1) to analyze a single nucleotide polymorphism (SNP) associated with high levels of tumor necrosis factor-α (TNF-α) responses,  TNF G-308A, has been associated with severe responses to infection. Cytokine gene polymorphisms can significantly affect the level of these substances produced in response to infection  is a key pro-inflammatory cytokine which could play a role in several of the mechanisms proposed to explain sudden infant death syndrome (SIDS). In relation to the anaphylaxis hypothesis, TNF-α is stored in mast cell granules and is released on antigen stimulation. TNF-α also induces fever and affects respiration  and can nfection . In vitresponses  and indiesponses . The molesponses .TNF G-308A SNP indicate the AA genotype carried an increased risk of death from cerebral malaria , moderate (Caucasian), or high  incidences of SIDS.Reports on the  malaria , and dea malaria . Two stu malaria , 12. BecStaphylococcus aureus  to assess effects of virus infection and cigarette smoke on TNF-α responses; and (3) to assess the effect of gender, TNF G-308A, and cigarette smoke on responses to bacterial antigens (LPS and TSST-1) identified in SIDS infants.The objectives of the study were (1) to analyze the distribution of Approval for the study was obtained from the Lothian Health Ethics Committee (UK), Hunter Area Research Ethics Committee  and the University of Newcastle Human Research Ethics Committee .n = 34) and Australia (n = 60) and a comparison group with no family history of sudden infant deaths . Paraffin-fixed samples of tissue from SIDS infants were obtained from Australia (n = 25), Hungary (n = 18), and Germany (n = 46). Stored frozen blood samples from Aboriginal Australians (n = 117) and buccal epithelial cells from Bangladeshi donors (n = 32) were used as sources of DNA for comparisons among ethnic groups. The methods for extraction of DNA from the samples have been described previously  genotype was determined by a commercial allelic discrimination polymerase chain reaction (PCR) assay (Assay ID: C___7514879_10) (PE Applied Biosystems). Primers and probes were provided in a 20× assay mix, sequences and concentrations of which are unknown.Each PCR reaction contained 10 ng of sample DNA, 1× Assay mix, and 1× TaqMan Universal PCR Master Mix (PE Applied Biosystems) made up to a final volume of 5 μl with sterilized MilliQ water. PCR was performed using the ABI PRISM 7900HT sequence detection system (PE Applied Biosystems) at the following thermal cycling conditions: 50°C for 2 min; 95°C for 10 min; 92°C for 15 s; and 60°C for 1 min, for 40 cycles.2) test or Fisher’s Exact test, if appropriate, was used to assess the distribution of TNF G-308A in SIDS infants, parents of SIDS infants, and between ethnic groups. Genotype distribution from the Hardy–Weinberg equilibrium (HWE) was assessed using the χ2 test.Data were analyzed using the statistical software package Statistics/Data Analysis™ (STATA) Version 8.0 . The Chi-square (χTumor necrosis factor-α responses of the THP-1 human monocytic cell line were measured as previously described in the model system to assess the effects of surrogates for infection (IFN-γ) and exposure to CSE on cells with a uniform genetic background .in vitro cytokine stimulation assays and plasma was collected for the assessment of cotinine for evidence of exposure to cigarette smoke, a confounding variable for altered cytokine responses. TNF G-308A genotype was determined as described above. Donors with detectable levels of cotinine were excluded from the analysis. Only ARCBS donor samples that were cleared for infectious agents were received. Leukocytes were collected and stored as described previously  have been described previously. Buffy coats from 14 male and 14 female donors, aged 20–55 years, were purchased from the Australian red cross blood service (ARCBS) . Ethical permission was obtained from University of Newcastle Human Research Ethics Committee (H-229–0606) and ARCBS Ethics Committee (07-11NSW-07) for the purchase and use of human buffy coats for the purposes of the study. PBMC were collected from each donor for eviously .n = 34) and a comparison group, individuals unrelated to the families and who had no family history of SIDS (n = 59). DNA was extracted from the leukocytes and screened for the TNF G-308A alleles as described above. The leukocytes were assessed for inflammatory responses to TSST-1 and LPS as described previously. All samples were coded and tested without knowing the sex or smoking status of the donors. Leukocytes (1 × 106 cells ml−1) were stimulated in vitro with 0.01 μg ml−1 or 1 μg ml−1Escherichia coli LPS or 0.5 μg ml−1 TSST-1  for 24 h. Cell culture conditions have been described previously  collected from British parents of SIDS infants  and German  (p < 0.01) SIDS infants.The majority of each SIDS group possessed the GG genotype; however, Australian SIDS infants had a significant increase in proportion with the AA genotype  compared to the Hungarian  (p = 0.02). Only 3/62 (5%) of controls had the AA genotype compared with 24% of SIDS infants. No significant differences were detected between the distribution of TNF G-308A genotype for the combined SIDS infant group compared to the Caucasian controls (p = 0.41).The distribution of allele frequencies for the Australian control population differed significantly from that observed for Australian SIDS infants . Parents of SIDS infants recruited from Britain had a genotype distribution similar to their control population; the majority of individuals had the GA genotype (p = 0.89).The distribution of TNF G-308A varied significantly among individuals from different ethnic groups. The majority of both British and Australian control populations possessed the GG genotype with fewer than 5% of individuals possessing the AA genotype.The distribution of p = 0.01) and Aboriginal Australians (p < 0.01). More than 90% of the Bangladeshi and Aboriginal Australian populations possessed the GG genotype, and 0% had the AA genotype.As there were no differences between the allele frequencies for British and Australian control populations, these data were combined for further comparison with the Bangladeshi and Aboriginal Australian populations. The distribution of allele frequencies differed significantly between the Caucasian group and both Bangladeshis (−1 (low) or 10 ng ml−1 (high) concentrations. Priming of THP-1 cells with INF-γ resulted in dose-dependent enhancement of TNF-α responses to LPS (50 ng ml−1) . As observed with THP-1 cells, CSE did not elicit TNF-α from PBMC. In contrast to the results obtained with THP-1 cells, the high dose of CSE significantly reduced TNF-α responses to LPS (p < 0.0001) .  compared with responses from cells of male donors (4621 pg ml−1) if both INF-γ and CSE were used to pre-treat cells prior to exposure to endotoxin (p = 0.014). There were no significant differences in TNF-α responses associated with genotypes GG (n = 15), GA (n = 10) or AA (n = 3) under any of the conditions tested. For donors with the GG genotype, there were higher responses to LPS from cells of female donors (n = 9) (1674 pg ml−1) than from cells of male donors (n = 6) (989 pg ml−1) (p = 0.03). While the responses from cells of females with the GA genotype (n = 2) were approximately twice that of those from cells of male donors (n = 8), the differences were not significant (p > 0.05). There were only three donors with the AA genotype and all were female.When assessed by gender of the donor, there were no significant differences between: TNF-α responses to LPS, INF-γ, or CSE; or combinations of LPS with CSE or LPS with INF-γ. There was a significant difference in TNF-α responses of cells from female donors (6510 pg mlTNF G-308A for smokers (n = 38) or non-smokers (n = 60). For the GG and GA genotypes, the geometric mean TNF-α responses were below 10 IU ml−1. For the three donors with the AA genotype, there was one smoker and two non-smokers. The responses of the smoker donor with the AA genotype were similar to those of the GG and GA donors (<10 IU ml−1). The response of the two donors who were not smokers was nearly 20 IU ml−1 for responses to TSST-1 and over 50 IU ml−1 for responses to the higher dose of LPS. There were not enough donors with the AA genotype for statistical analyses.This experiment utilized cells from the UK donors. There were no significant differences in the median TNF-α response to TSST-1 or LPS associated with different genotypes of TNF G-308A genotype AA in Australian SIDS infants. Given the rarity of the TNF G-308A A allele in the Caucasian population, it was an unusual observation that 24% of the Australian SIDS infants tested in this study were homozygous. The number of infants assessed in this study was small; therefore, data should be interpreted with caution. In the Norwegian study, 3% of SIDS infants, 5% of borderline SIDS, and 3% of infants who died of infection had the AA genotype; while none of the controls had this SNP genotype  or low (UK Bangladeshi) incidences of SIDS had similar genetic profiles.While the distribution of If the finding of a significant increase in the AA genotype associated with high TNF-α reflects a risk factor for SIDS, the findings need to be assessed in relation to the various hypotheses proposed to explain the physiological events leading to SIDS.Long QT interval is reported to be an important risk factor for SIDS based on a prospective study of a large group of infants . The resDysregulation of glucose metabolism was proposed as one mechanism triggering the physiological events leading to SIDS . Acute hThe A allele has been associated with increased production of TNF-α , 28. TheTNF G-308A genotype on TNF-α responses to TSST-1 or LPS in the different populations. Although the responses of the non-smokers with the AA genotype were higher than those observed for the other genotypes, there were too few donors with this genotype for statistical analyses. These studies need to be repeated with more samples from individuals with the rare genotype.Despite finding interactions with smoking and cytokine gene polymorphisms in our previous studies , this stIn the experiments with cells from healthy Australian blood donors which were controlled for exposure to cigarette smoke and infection, females generally had higher TNF-α responses than males . When asTNF G-308A genotype results indicated that for the Australian population, infants with the AA genotype might be at increased risk of SIDS. The study needs to be expanded and investigations need to include thorough investigation for microorganisms and their toxins as well as investigation of the genetics and cytokine responses. While the effects of genotype on TNF-α responses remain unclear in the models tested, the effects of INF-γ are unequivocal for both THP-1 cells and PBMC. The results indicate that virus infection might enhance TNF-α responses to minor bacterial infection. While exposure to cigarette smoke is a risk factor for SIDS, it does not appear to enhance TNF-α responses to LPS. For the PBMC studies, CSE reduced significantly the enhanced responses of IFN-γ primed cells to LPS.In conclusion, the Each of the authors made substantial contributions to the conception, design, analyses, and interpretations of the work. They assisted in preparing the article, critically assessed the final version and agree to be accountable for the accuracy and integrity of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer’s disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME).Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression.The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.The online version of this article (doi:10.1186/s12868-016-0288-1) contains supplementary material, which is available to authorized users. PICALM (phosphatidylinositol binding clathrin assembly protein) [BIN1 (bridging integrator 1) and SORL1 (sortilin-related receptor) that reached genome-wide significance [In 2009 Genome Wide Association Studies (GWAS) were published describing polymorphisms in a number of genes associated with an increased risk of developing Alzheimer’s disease (AD) including protein) , 2. PICAprotein) . Subsequificance , 5. Pathificance  identifiificance .The amyloid cascade hypothesis is the leading theory to explain the aetiology of AD , suggestAltered CME has been described previously in AD. Early endocytic changes (an increase in number and size of Rab5-positive endosomes) have been seen in post-mortem AD brains , 21. InhPICALM and Clusterin show significantly poorer episodic memory, a marker for AD onset [PICALM homologues protected against Aβ-induced toxicity [PICALM protective alleles appeared to be associated with an increase in PICALM mRNA expression in human brain [PICALM is involved in the recruitment of clathrin and other proteins to the membrane and thus regulates the formation of clathrin-coated pits and vesicles in CME ; though AD onset . In yeastoxicity . In contan brain  and to pan brain . Decreasan brain . Thus PIWe used the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP as we wanted to look at the effects of PICALM on physiologically relevant levels of APP, more relevant to the situation found in AD patients. We found that these cells expressed PICALM isoforms 1 and 2. Having depleted levels of PICALM by siRNA targeting all four isoforms, we showed that the levels of intracellular APP, the intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 itself was not affected. Functional CME measured by transferrin uptake was significantly reduced after both depletion of PICALM and clathrin heavy chain (CHC). In contrast, depletion of clathrin did not affect APP levels but did reduce those of β-CTF. PICALM depletion altered the intracellular distribution of clathrin with less staining found in the perinuclear transgolgi region while clathrin depletion affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. In contrast to the findings for depletion of all PICALM isoforms, individual depletion of PICALM isoforms 1 and 2 by specific siRNAs did not affect APP levels. Clathrin depletion had a more complicated differential effect on the isoforms, increasing isoform 1 protein and mRNA expression while decreasing isoform 2 protein expression but not affecting mRNA levels.In H4 cells, we detected two bands for PICALM using Western blotting with molecular masses of 72.2 ± 0.4 and 65.8 ± 0.9 kDa Fig. a. Four mUsing an siRNA sequence directed against all the isoforms of PICALM (Seq. A), we significantly reduced the total levels of PICALM to 17.9 ± 6.2 % of levels in control cells incubated with siRNA to GFP where PICALM levels were unaffected  compared to mean cell numbers following treatment with siRNA targeting GFP (82.0 ± 8.2).Having demonstrated an extensive reduction in the levels of PICALM, we investigated whether other proteins involved in CME were affected by siRNA to PICALM. Figure siRNA to total PICALM significantly reduced the expression of APP to 73.3 ± 3.4 % of the GFP control levels in H4 cells Fig. a. We repPICALM rs3851179 [PICALM from IGAP rs10792832 [Using the Gibbs et al.  eQTL dats3851179 , and the10792832 , are botPICALM SNPs were associated with differential changes in the expression of the isoforms of PICALM, we examined the contribution of the isoforms of PICALM to its functions in endocytosis and potential involvement with the processing of APP. We designed siRNA to the two most abundant isoforms we identified in the H4 cells, isoforms 1 and 2. Figure Given that the In contrast to the siRNA to total PICALM which significantly reduced APP levels, neither siRNA to isoform 1 nor isoform 2 significantly affected the levels of APP Fig. a. SimilaHaving shown that depletion of PICALM affected functional endocytosis, clathrin localisation and APP processing, we examined whether knocking down clathrin itself would have any effect on PICALM or APP processing. Using siRNA to CHC and compared to GFP siRNA-treated control cells, we significantly reduced the expression of CHC protein to 14.9 ± 1.5 % Fig. a, b, CHCWe have demonstrated that reducing the expression of PICALM or clathrin inhibits CME to a similar extent and reduces β-secretase-mediated cleavage of APP but only PICALM siRNA affects the levels of APP. Thus PICALM can influence the processing of APP via more than one route possibly contributing to its role as a risk factor for AD.The gene for PICALM gives rise to a number of isoforms , 33–35 oReducing PICALM expression by siRNA significantly inhibited the uptake of transferrin by functional CME in the H4 cells suggesting that, in these cells, PICALM is essential for normal levels of CME to occur, probably due to its involvement in the formation of clathrin-coated vesicles , 35. TheThe consequent decrease in functional endocytosis following PICALM depletion does not involve dynamin or a change in the levels of clathrin since neither mRNA nor protein levels for CHC were altered by knockdown of PICALM. However, the localisation of clathrin in the transgolgi network was reduced by PICALM depletion possibly due to redistribution in the cell since overall protein expression was unaltered. A reduction in clathrin expression in the transgolgi network has been described previously for both decreasing  and incrConsistent with previous findings showing that APP is internalised by endocytosis  and thatDepleting PICALM expression decreased β-secretase cleavage of APP as the levels of βCTF were reduced and a decrease in the sAPPβ/sAPPα ratio was seen. α-secretase activity was unaffected by depletion of PICALM. A role for PICALM in targeting CTF fragments of APP for degradation by autophagy has been suggested . Thus anPrevious work has shown that PICALM affects the endocytosis of γ-secretase and this was proposed to explain the effects of depletion of PICALM on Aβ generation . HoweverHaving examined the effect of depletion of total PICALM on APP metabolism and, given that the expression of isoform 1 was lower than isoform 2 in H4 cells, we investigated whether the isoforms had differential effects on APP endocytosis and processing. We depleted isoform 1 and 2 individually using specific siRNAs which did not significantly affect the other isoform thus confirming that the two bands we saw in the H4 cells were indeed isoform 1 and 2. Interestingly, depletion of either isoform alone had no significant effect on the levels of APP or βCTF so both PICALM isoforms must be involved in functional endocytosis in the H4 cells. However, the differential effect of clathrin depletion on the expression of the isoforms at both the mRNA and protein level does suggest that they may have different roles in CME. Clathrin depletion increased both the protein and mRNA for isoform 1 but decreased isoform 2 protein without altering the mRNA. The discrepancy for isoform 2 may be due to differences in mRNA stability between the two isoforms. The increase in one PICALM isoform and the decrease in the other may explain why clathrin depletion had no effect on APP levels. The intracellular localisation of total PICALM was altered by clathrin depletion which could reflect differential changes in the expression of isoforms 1 and 2. We could not investigate this finding further to understand the mechanism involved as there are no commercial antibodies available which can distinguish between isoforms 1 and 2. Our findings from the eQTL data sets, showing an association between a decrease in isoform 2 mRNA expression but no effects on isoform 1 mRNA for the Harold and co-workers  looked aIn conclusion, we have demonstrated that the depletion of PICALM in H4 cells has significant effects on the processing of APP, probably by reducing cleavage by β-secretase. The reduction in βCTF production is particularly interesting as this is now increasingly thought to have toxic effects on cells independent of Aβ. These effects are likely to be mediated by at least two processes, a reduction in CME and an alteration in the endocytosis of endocytic vesicles. Based on our data and on evidence from the literature, we hypothesise that polymorphisms of PICALM could alter the expression of the protein over the lifetime of an individual which would be associated with either an increased risk of developing AD or a protective effect. Further studies are required to elucidate the relative importance of these different roles of PICALM and the relevance of the isoforms to APP processing.All chemicals and reagents were purchased from Sigma-Aldrich, Poole, UK, Life Technologies (Invitrogen), Paisley, UK or Fisher Scientific, Leicester, UK and all reactions were performed at room temperature unless otherwise specified. All siRNA (sequences of 21–23 residues) were purchased from Eurofins Genomics, Ebersberg, Germany.Antibodies were purchased as follows: Anti-APP , anti-β-actin directly conjugated to horseradish peroxidase  or GAPDH , anti-dynamin II , anti-clathrin heavy chain , anti-PICALM , anti-BACE1  and anti-rabbit (PI-1000) and anti-mouse (PI-2000) antibodies conjugated to HRP .Neuroglioma H4 cells  were cultured in Opti-MEM media containing Glutamax (Invitrogen) supplemented with 4 % foetal bovine serum .The expression of PICALM and clathrin heavy chain (CHC) was depleted using siRNA as previously described . Briefly® First-Strand Synthesis System (Invitrogen) using Oligo(dT) and Superscript™ II reverse transcriptase. Real-time PCR was carried out on the LightCycler ® 2.0 Real-Time PCR System  using TaqMan gene expression assays (Table ® TaqMan® Master hot start reaction mix (Roche). Reactions were carried out according to the manufacturer’s protocols. Data were normalised either to polymerase (RNA) II (DNA directed) polypeptide A (POLR2A) or 18S RNA levels. Relative quantification was calculated using the 2−ΔΔCt method [After siRNA treatment cells were lysed in Trizol™ reagent (Invitrogen) then total RNA was extracted and purified using RQ1 RNase-Free DNase  followed by Qiagen RNeasy™ Mini kits . cDNAs were synthesized from 800 ng RNA with SuperScriptt method  using meAfter siRNA treatment, cells were lysed as previously described  and totaThis assay was performed according to Al-Soraj and co-workers  as descrAPP in the lysed cells samples was quantified using the APP DuoSet  following the manufacturer’s guidelines , 58. BriFor analysis of Aβ40, media was subjected to immunoprecipitation and ELISA as described previously , 58. Bri® MTS Aqueous One Solution Cell Proliferation Assay (Promega) following the manufacturer’s guidelines. The total number of cells was also counted using a Z™ Series Coulter Counter (Beckman Coulter (UK) Ltd, High Wycombe, UK).Viability studies were performed after incubation with siRNA to total PICALM for 48 and 72 h using the CellTiter 96® HardSet™ Mounting Medium (Vector Laboratories) with DAPI and visualized using a Leica SP5 confocal laser scanning microscope. GFP siRNA did not affect the localisation or expression of any of the proteins examined compared to media controls so the GFP siRNA images are shown for comparison with those obtained following siRNA for PICALM or clathrin.H4 cells were plated on collagen-coated coverslips as previously described  and trea® 5. Results were first normalised to total protein concentration, where relevant and expressed as % of media control values. Both sAPPβ and sAPPα were quantified in ng/ml and then expressed as the ratio of sAPPβ to sAPPα. Where there were only two factors, data were compared to GFP using unpaired Student’s t tests, at the two-tailed significance level. Elsewhere data were analysed with one-way ANOVA followed by the relevant post hoc test comparing all data to GFP.Western blots were quantified using Image J, analysed as detailed previously  and exprWhere necessary, data were transformed to fulfil the assumptions of normality and homoskedasticity and to therefore allow the use of parametric testing. If this proved impossible the equivalent non-parametric test was used. All data used to produce Figs.