Environmental safety has become increasingly important with respect to hydrogen use in society. Monitoring techniques for explosive gaseous hydrogen are essential to ensure safety in sustainable hydrogen utilization. Here, we reveal molecular hydrogen detection mechanisms with monolithic three-dimensional nanoporous reduced graphene oxide under gaseous hydrogen flow and at room temperature. Nanoporous reduced graphene oxide significantly increased molecular hydrogen physisorption without the need to employ catalytic metals or heating. This can be explained by the significantly increased surface area in comparison to two-dimensional graphene sheets and conventional reduced graphene oxide flakes. Using this large surface area, molecular hydrogen adsorption behaviors were accurately observed. In particular, we found that the electrical resistance firstly decreased and then gradually increased with higher gaseous hydrogen concentrations. The resistance decrease was due to charge transfer from the molecular hydrogen to the reduced graphene oxide at adsorbed molecular hydrogen concentrations lower than 2.8 ppm; conversely, the resistance increase was a result of Coulomb scattering effects at adsorbed molecular hydrogen concentrations exceeding 5.0 ppm, as supported by density functional theory. These findings not only provide the detailed adsorption mechanisms of molecular hydrogen, but also advance the development of catalyst-free non-heated physisorption-type molecular detection devices. Detection of molecular hydrogen in ambient atmospheres has become increasingly important because renewable energy, especially hydrogen-based energy, is a focus for the development of sustainable societies ,2. Users2O, CO, CO2, NO, NO2, O2, SO2, and NH3 at parts per billion (ppb) and parts per million (ppm) levels [2) using either graphene or rGO flakes is not precisely discussed in the literature because the weak physisorption strength and insufficient molecular detection area of the graphene surface mask the details of the electrical resistance behaviors. Since catalytic H2 detection with Pt [2 [2, the significant challenge of realizing physisorption of non-polar H2 and revealing the detection mechanisms in detail remains in the development of non-catalytic H2 detection devices.Conventional molecules are detected by catalytic combustion, resistance change, and thermal conductivity change ,4,5,6,7.) levels ,11,12,13) levels ,17,18,19 with Pt ,21, Pd [ with Pt ,23,24,25 with Pt , ZnO [27 with Pt , and SnOth Pt [2 ,30 at hi2 as compared to pristine nanoporous unoxidized graphene and rGO flakes. Such physisorption detection mechanisms based on a large surface area provide a new class of design direction for non-heated, flow-type, and physisorption-type graphene-based detection systems for non-polar gas molecules without metal catalysts.Here, we synthesized a single and monolithic nanoporous reduced graphene oxide (np-rGO) sheet. The synthesized sheet had an edge-free bicontinuous and open porous three-dimensional (3D) structure and was treated by a modified Hummer method. The np-rGO sheet maintained a projected surface area 500 times larger than that of a standard two-dimensional (2D) graphene sheet, performing significantly accurate detection via molecular physisorption of H30Mn70 ingots were prepared by melting pure Ni and Mn (purity >99.9 at.%) in an Ar-protected arc melting furnace. After annealing at 900 °C for 24 h for microstructure and composition homogenization, the ingots were cold-rolled into thin sheets (50 μm) at room temperature. Nanoporous Ni was prepared by chemical dealloying in a 1.0 M (NH4)2SO4 aqueous solution at 50 °C overnight. After dealloying, the samples were rinsed thoroughly with distilled water and then rinsed with ethanol before vacuum drying.Ni2) for 3 min [2 (100 sccm), Ar (200 sccm), and benzene  was introduced for graphene growth at 800 °C for 2 min. Subsequently, the furnace was opened for the rapid cooling of the inner quartz tube. The nanoporous Ni substrate was etched in a 1.0 M HCl solution and then the remaining nanoporous graphene was transferred into a 2.0 M HCl solution to completely remove residual Ni. The samples were repeatedly washed with water and subsequently kept in water.The nanoporous Ni substrates were loaded in a quartz tube (Ø26 × Ø22 × 250 mm), placed in a quartz tube furnace (Ø30 × Ø27 × 1000 mm), and annealed at 800 °C under a mixed gas flow , concentrated H2SO4 (3.45 mL), and NaNO2 (0.075 g) mixture without stirring. After 10 min, the mixture was diluted in an aqueous H2SO4 (5%) solution. Then, an aqueous H2O2 (30%) solution diluted in aqueous H2SO4 (5%) was slowly added to the mixture. The obtained transparent sheet was then washed several times with a mixture of aqueous H2SO4 (3%) and H2O2 (0.5%). The nanoporous graphene oxide (GO) sheet was then repeatedly washed with water and kept in 2-propanol. The samples were dried by a conventional supercritical CO2 drying method. The dried GO sample was transferred onto a SiO2 substrate and then connected to fine Cu lines (twisted) using silver paste in a standard pseudo four-probe configuration. The reduced graphene oxide (rGO) sample was prepared by annealing the GO sample on the substrate at 150 °C using ribbon heaters under vacuum in a self-made atmosphere-controlled glass cell connected to Swagelok vacuum components.The nanoporous graphene dispersed in water was oxidized via a modified Hummer method . A singl2 (scCO2) and thus 2-propanol was substituted for CO2 without the action of capillary forces. To this end, the nanoporous graphene sample was firstly transferred to a vial (volume: 5 mL) filled with 2-propanol (400 μL), and then the bottle was placed in an 80-mL pressure-resistant container (TAIATSU techno Corp). After removing the air inside the container via CO2 purging, the pressure of the container was gradually increased to 15 MPa by introducing liquid CO2  at a flow rate of 20 mL min−1 (~19 g min−1) using a high-pressure plunger pump . The scCO2 drying process was performed at 70 °C and at a constant CO2 flow rate of 5 mL min−1 (~4.8 g min−1) by forming a homogeneous phase of 2-propanol and scCO2, thus minimizing the capillary force. The pressure was maintained at 15 MPa for 5 h during the drying process. After drying the sample completely, the temperature was set at 40 °C and the container was gradually depressurized over 43 h from 15 MPa to atmospheric pressure by gradually pumping CO2 from the system. To prevent the collapse of the fragile nanoporous structures caused by water capillary forces during drying, the nanoporous graphene and rGO samples immersed in 2-propanol were dried by using supercritical COKa X-ray monochromator. Raman spectra were obtained using a micro-Raman spectrometer (Renishaw InVia Reflex 532) with an incident wavelength of 532.5 nm. The laser power was set at 0.1 mW to avoid possible damage or unexpected reduction by laser irradiation. The graphene samples were placed on a background-free glass slide. The accumulation time of each spectrum was 100 s. The microstructures of the nanoporous GO and nanoporous rGO were characterized using a scanning electron microscope . Chemical analyses on the graphene samples were performed via X-ray photoelectron spectroscopy  with an Al 2/Si (SiO2: 300 nm) substrate. The electrode was fabricated using Ag epoxy . The electrical resistance measurements were performed using the Physical Properties Measurement System .The temperature and magnetic field dependences of the electrical resistance of reduced nanoporous graphene oxide were measured using the four-probe method under a magnetic field at 300 K. The reduced nanoporous graphene sheets were placed on a SiO® to avoid leakage. The detection element of the nanoporous GO sheet (7.2 mm × 2.5 mm) was attached on a SiO2 substrate through fine twisted Cu lines fixed with silver paste. Then, the substrate was transferred to the glass cell and the fine Cu lines were electrically connected to the Cu wires (1.5 mm) inside the cell. The temperature inside the cell was controlled by an external ribbon heater for thermal reduction of nanoporous GO in the cell. The whole system and the connected vacuum components were heated to 150 °C, and the GO  through the fine Cu wires) was thermally reduced to rGO under vacuum (5 Pa). Briefly, the temperature for thermal reduction was fixed at 150 °C and the heating time was set at 4 h for oxygen contents of 20.0 at.%, 6 h for oxygen contents of 18.2 at.%, and 12 h for oxygen contents of 10.8 at.%. After cooling down to the room temperature (23 °C), the thermally reduced GO samples were sequentially measured in the cell by a standard pseudo four-probe method using a semiconductor parameter analyzer (Keysight B1500A). The applied voltage was set at −9 V for the nanoporous rGO samples  and to −10 mV for the pristine nanoporous graphene (without oxidation and reduction treatments) under a mixture of gaseous argon . The ultrapure gaseous Ar continuously flowed until the resistance value stabilized to exclude unexpected influences of impurities such as H2O, O2, CH4, CO2, CO, and H2. Thus, any changes in resistance values under the mixture of H2/Ar gas flowing could be attributed to the introduction of ultrapure H2 gas. After the resistance was stable, the mixture of gaseous Ar and gaseous hydrogen  was introduced in the cell. The total flow rate of the mixed gas was set at 30 sccm. To obtain the 1 ppm H2 gas in the Ar flow case, gaseous hydrogen was mixed with gaseous argon to a H2 concentration of 0.0001 vol.%, and then the mixed gas was supplied by the mass flow controllers at a flow rate of 30 sccm . Note that flow rates exceeding 30 sccm deformed the atomically thin porous structures, and the resistance values became unstable. Before all measurements, the whole system was vacuumed for 12 h at room temperature to avoid further thermal reduction of the rGO. A molecular detection glass cell was fabricated using an atmosphere-controlled separable three-neck glass cell with an O-ring. Swagelok vacuum components were inserted into two rubber stoppers, and the stoppers were installed in two of the necks. The Swagelok vacuum components were connected to mass flow controllers and a rotary pump. Four Cu wires (1.5 mm) and a thermocouple (type K) were inserted in a rubber stopper, and the stopper was installed in the third neck of the cell to communicate the inside and outside of the cell and the device; all connections were completely sealed with Torr Seal54H18, C54H18-2OH, C54H18-2O, C53H18-COOH, and C53H18-2O) were optimized using the density functional theory (DFT) method with the B3LYP functional [2 adsorption. All calculations were performed using Gaussian 16 quantum chemistry software [The geometries of the surface models  of 2.5 and of the D and G bands (ID/IG) of 0.08, corresponding to high-quality 1–2 layer graphene without detectable amorphous carbon features [ID/IG of 1.6 and I2D/IG of 0.91). The np-rGO partially restored graphene features (ID/IG of 0.98 and an I2D/IG of 1.7). The full width at half maximum (FWHM) values of the D, G, and 2D bands of the np-rGO were significantly increased in comparison to the pristine nanoporous graphene. Chemical binding was further investigated by XPS. The C1s X-ray photoelectron spectrum (sp2 and sp3 [ID/IG from the Tuinstra–Koenig relation: ID/IG = C(λ)/LD, where C(λ) is a proportionality constant at the excitation laser wavelength λ and LD is the average distance between the defects [LD value of np-rGO with an oxygen content of 20 at.% (ID/IG = 0.98) was approximately 8–10 nm, which was almost of the same order as the distance between the oxidized functional groups estimated by the atomic concentrations.Raman spectra were analyzed to estimate the quality of the graphene samples and their respective oxidation levels . The prifeatures ,44. Aftespectrum  demonstr and sp3 ,47. Ther defects ,49,50. U−1 and 5000–8000 cm2 V−1 s−1, respectively [−1 and 0.31–1.0 cm2 V−1 s−1, respectively [2 V−1 s−1) [The transport properties of nanoporous graphene and np-rGO were measured at 300 K by a standard four-probe method. The typical electrical conductances and electron mobilities of the nanoporous graphene samples were 500–1000 S cmectively ,51. The ectively . Note thV−1 s−1) . Therefo2, we measured the time dependence of resistance changes under the H2/Ar mixed gas in the atmosphere-controlled glass cell. Firstly, we confirmed the reversibility of molecular physisorption of H2 under Ar and H2/Ar flow conditions . The response was relatively slower than that of conventional catalytic metal-based gas detection systems, probably because the replacement of Ar by H2 requires time owing to the low flow rate and geometrically requires time to reach the surface due to the complicated porous structure [To evaluate the molecular physisorption ability of Htructure  in compatructure ,28,29,302 gas volume concentrations from 0.1 ppm to 10,000 ppm under H2/Ar gas flow  was investigated for the np-rGO with an oxygen content of 20.0 at.%  and plotted for the various H2 ppm values  showed a similar behavior but did not show large changes in resistance values for 0.1–10,000 ppm H2 under the same conditions. Thus, the large changes in np-rGO were attributed to the oxidized functional groups on the graphene. Moreover, both samples exhibited a local minimum at approximately 1 ppm H2 under H2/Ar gas flow. Note that these increases and decreases have not been reported for graphene and rGO detection systems [2 ppm values under H2/Ar gas flow in comparison to the np-rGO sample with an oxygen content of 20.0 at.%. The change in resistance at H0.0 at.%  and nano0.0 at.% c. We foum values d. The no systems ,17,18,192 on the surface of graphene and graphene oxide , and there were no significant differences in the functional groups. In the models, it was found that two kinds of charge transfer phenomena occurred: (1) from oxygen-containing functional groups to graphene oxide  and (2) from a single H2 molecule to graphene oxide with +0.020 e   and lowest unoccupied molecular orbital (LUMO) decreased after the adsorption  and a charge transfer (+0.074 e by four H2 molecules) from the additional H2 molecule on graphene oxide further occurred  . This susorption . In partoccurred .2 lower than 1 ppm showed negative resistance changes, while those higher than 5 ppm showed positive resistance changes. The high resistance changes are attributed to the high oxidation level of the samples . Below 1 ppm H2, contribution of Coulomb scattering by the adsorbed H2 was not dominant/apparent and the charge transfer from H2 to np-rGO was a major effect. In this case, further hole doping from the adsorbed H2 of the p-type np-rGO reduced the resistance  and gradually increased after 300 s (Coulomb scattering domination) with an increase in hydrogen concentration  showed the largest change of resistance values at higher H2 ppm values in comparison to the nanoporous graphene and np-rGO with oxygen contents of 10.8 at.% (hydroxyl/epoxide group: 2.5 at.%) and 18.2 at.% (hydroxyl/epoxide group: 6.3 at.%). Furthermore, open porous structures with large total surface areas whose surface was modified by the oxygen-containing functional groups significantly promoted mass transfer and increased the sensitivity of molecular physisorption of non-polar H2. The excellent transport properties resulting from such monolithic characteristics supported accurate detection of resistance changes.The concentrations of adsorbed H samples  and lowetrations . This sur effect , while hl groups . Combinisistance e. Conver centers e,f, in antration . Thus, iWe developed a novel 3D nanoporous rGO for molecular hydrogen detection at room temperature under gas flow conditions. The detection mechanisms of molecular hydrogen were revealed by using 3D nanoporous rGO specimens featuring a large surface area, a single bicontinuous monolithic sheet morphology, and unique transport properties (non-electron hopping conduction), and were found to be based on the charge transfer effect and Coulomb scattering effect. Moreover, DFT calculations revealed that hydroxyl, epoxide, ether, ketone, and carboxyl groups are preferable molecular physisorption sites, and that hydroxyl groups benefit from the sequential adsorption of molecular hydrogen. Nanoporous rGO with tunable oxygen-containing functional groups could provide a general platform for a room-temperature physisorption-type gas sensor and provide a new design direction for nanoporous material-based gas sensors for combustible gases.


Propionibacterium acnes-associated sarcoidosis complicated by acute bird-related HP.The number of reports on sarcoidosis complicated by hypersensitivity pneumonitis (HP) is limited, and most describe cases complicated by chronic bird-related HP. Here, we present for the first time a case with P. acnes lipoteichoic acid. Poorly formed non-caseating granulomas without positively stained substances were also detected.A 62-year-old man with a past medical history of sarcoidosis was admitted to our department, and chest computed tomography showed diffuse ground-glass opacities, which appeared as he rapidly increased the number of pigeons he kept for a competition. Random transbronchial lung biopsy revealed well-formed non-caseating epithelioid granulomas, which contained positively stained substances on immunohistochemistry using the PAB antibody, a specific monoclonal antibody against We describe the successful identification of this exceptionally rare case of sarcoidosis complicated by acute bird-related HP in which two morphologically and immunohistologically different types of granulomas were present in the same lung. Propionibacterium acnes, although infection with these organisms is only one of the possible suggested pathogenetic mechanism [P. acnes was recently renamed as Cutibacterium acnes, but we continue to use the earlier nomenclature to avoid confusion [P. acnes-associated sarcoidosis that have involved the detection of P. acnes in sarcoid granulomas [Granulomatous lung diseases are a heterogenous group of disorders that include both infectious and noninfectious conditions with a wide spectrum of pathologies and various clinical manifestations and outcomes. This makes differential diagnosis based on histopathological assessment challenging . Sarcoidechanism . Note thonfusion . Reporteanulomas –6. Sarcoanulomas .However, the number of reports on sarcoidosis complicated by HP is limited, and most describe cases of sarcoidosis complicated by chronic bird-related HP Table . SpecifiP. Acnes mAb (PAB antibody), a specific monoclonal antibody against P. acnes lipoteichoic acid [Here we report a case of sarcoidosis complicated by acute bird-related HP in which two distinct types of granuloma were successfully distinguished by immunohistochemistry (IHC) using anti-oic acid .A 62-year-old man who had cough and dyspnea on exertion for 3 months and weight loss of 3 kg in 6 months was admitted to our respiratory department in March 2017. He had a 28 pack-year smoking history and used a duvet for warmth in winter. Thirty years earlier, he had started breeding more than 300 pigeons for competition, and although the number had gradually decreased to about 30–40 birds in April 2016, he had then rapidly increased the number up to 130 in the year before admission  on chest X-ray Fig. a and comTrichosporon asahii were not detected. Using the ImmunoCAP assay , serum levels of IgG specific to pigeons, parrots, and budgerigars were determined to be ≥200 mgA/L , ≥ 200 mgA/L , and ≥ 200 mgA/L , respectively [CO) 15.33 mL/min/mmHg (%DLCO 75.8%).Chest X-ray Fig. c and CT ectively . The lymectively . Pulmona5/mL, lymphocytes 17%, and CD4/CD8 ratio 1.5. Random transbronchial lung biopsy revealed well-formed non-caseating epithelioid granulomas with clear margin, characteristic of sarcoidosis, in the right B4a bronchus. Additional IHC using PAB antibody [P. acnes-specific epitope of lipoteichoic acids, which contain a glycolipid moiety and are anchored in the cell membrane and penetrate through the cell wall. Immunostaining using this antibody can identify cell wall-deficient L-forms of P. acnes causing latent infection in granulomas [P. acnes was detected in granulomas in ≥80% of patients diagnosed with sarcoidosis, irrespective of the organs involved: both the sensitivity and specificity of PAB staining are high [In April 2017, BALF results were total cell count 17.0 × 10antibody  detectedanulomas . P. acneare high . At the Taken together with the fact that dyspnea, ground-glass opacities on chest CT, and elevated serum KL-6 level improved without medication during 2 weeks of hospitalization, he was diagnosed as having sarcoidosis complicated by acute bird-related HP. Pulmonary function test results improved to VC 4.93 L %VC 125.4%) and FEV1 3.22 L (%FEV1 101.3%). On discharge from the hospital, no medication was prescribed, but he immediately stopped keeping pigeons  gene, currently named PSMB, codes for subunits of the proteasome. This is a multimeric enzymatic complex that degrades proteins into peptides in order to be presented in the MHC class I pathway, and genetic polymorphism in LMP7 was reported to be associated with both sarcoidosis [Sarcoidosis and HP have, at least in part, common immunopathogenesis including T-helper type 1 (Th1)-type immune responses in which phagocytosed antigens by macrophages present with major histocompatibility complex (MHC) class II to induce the immune response . In the coidosis  and birdcoidosis . Howevercoidosis , the reaConcerning the first observation in this case that sarcoidosis was complicated by acute bird-related HP, the patient had an approximately 40-year history of keeping pigeons, and sarcoidosis with BHL was diagnosed 4 years before admission to our department. One year before admission, as the number of pigeons kept increased, he developed dyspnea on exertion with ground-glass opacities observed on chest CT, but these improved by avoiding exposure to culprit antigens during a hospital stay. Given that the patient tested positive for both serum IgG against bird-related antigens and LST using pigeon dropping extract, a diagnosis of acute bird-related HP complicated with sarcoidosis was made. Long-term bird-keepers become sensitized to bird-related antigens after long-term exposure, resulting in elevated levels of serum antibodies against these antigens. Such conditions pose risk of developing chronic bird-related HP, although not all individuals develop it. Similar to our case, there have also been cases reported of acute bird-related HP triggered by sudden increases in the number of birds kept . Also, bConcerning the second observation that two morphologically and immunohistologically different types of granulomas were present in the same lung, the limited number of reports on sarcoidosis complicated by HP may be attributed, at least in part, to difficulties in distinguishing their distinct granulomas. Sarcoid granulomas can be differentiated from HP granulomas by histopathology . Sarcoid granulomas are well-formed non-caseating epithelioid granulomas distributed along the lymphatic tracts  , 25, andIn conclusion, we have presented the first case of sarcoidosis complicated by acute bird-related HP in which two distinct types of granuloma were successfully distinguished by IHC using PAB antibody. Cases of sarcoidosis complicated by HP need to be accumulated with precise clinical evaluations reported to improve our understanding of this condition.


Chitosan nanofibrous membranes are prepared via an electrospinning technique and explored as potential wound healing patches. In particular, the effect of a physical or chemical crosslinking treatment on the mat morphological, mechanical, water-related, and biological properties is deeply evaluated. The use of phosphate ions  allows us to obtain smooth and highly homogenous nanofibers with an average size of 190 nm, whereas the use of ethylene glycol diglycidyl ether  leads to rougher, partially coalesced, and bigger nanofibers with an average dimension of 270 nm. Additionally, the physically crosslinked mats show enhanced mechanical performances, as well as greater water vapour permeability and hydrophilicity, with respect to the chemically crosslinked ones. Above all, cell adhesion and cytotoxicity experiments demonstrate that the use of phosphate ions as crosslinkers significantly improves the capability of chitosan mats to promote cell viability owing to their higher biocompatibility. Moreover, tuneable drug delivery properties are achieved for the physically crosslinked mats by a simple post-processing impregnation methodology, thereby indicating the possibility to enrich the prepared membranes with unique features. The results prove that the proposed approach may lead to the preparation of cheap, biocompatible, and efficient chitosan-based nanofibers for biomedical and pharmaceutical applications. Tissue reparation, which is a natural process, is often diminished by a broad number of factors with the employment of wound healing patches currently assuming a highly important role . Nowadayellulose . The exiellulose ,29,30. Hellulose ,32. Addiellulose . In thisellulose ,35.With these premises, in a previous work, the electrospinning of chitosan solutions was optimized by using poly(ethylene oxide) as a co-spinning agent, which was subsequently completely removed, and a physical or a chemical crosslinking treatment was specifically applied to enhance the fiber stability . Here, tv = 900 kDa, TritonTM X-100 laboratory-grade, glacial acetic acid, absolute ethanol (EtOH), ammonium hydroxide (NH4OH), sodium phosphate dibasic (Na2HPO4), and ethylene glycol diglycidyl ether (EGDE) ), thiazolyl blue tetrazolium bromide (MTT), and dimethyl-sulfoxide (DMSO) were purchased from Sigma Aldrich . Saos-2 human osteoblast cell line and mouse fibroblast L929 cell line were obtained from the American Type Culture Collection . Human keratinocyte HaCaT cell line  was obtained by the Cell Lines Service . DMEM cell medium, glutamine, Fetal Bovine Serum (FBS), penicillin/streptomycin antibiotic solution and ready-to-use trypsin solution were purchased from Microstech Srl . Tissue culture flasks and 96-well plates were purchased from VWR International .Low molecular weight chitosan was obtained from Sigma-Aldrich and used without further purification. A degree of deacetylation (DD) of 78% was estimated via a conductometric titration method . Poly, respectively. Then, the system was added with 1 wt% of Triton and maintained under gentle stirring for 24 h at room temperature and finally conserved at T = 4 °C.CS-PEO batch solution to be electrospun was prepared as previously reported . Firstly®  Professional Electrospinning Machine equipped with an aluminium flat collector was employed to electrospin the prepared chitosan-based solution. A detailed description of the fabrication procedure optimization is reported in a previously published work [4OH/H2O 7/2/1 at pH = 7.5, to neutralize the acetic acid and prevent the dissolution of chitosan in aqueous environments. The samples were then dried in an oven at T = 50 °C under vacuum overnight and subsequently immersed for 3 h in a crosslinking solution. Physically and chemically crosslinked membranes were prepared by using a 10 w/v% Na2HPO4 aqueous solution or a 2.5 v/v% EGDE aqueous solution heated up at T = 60 °C, respectively. Finally, chitosan-based mats were washed several times with deionized water and dried in an oven at T = 50 °C under vacuum overnight before being stored in a desiccator and characterized. It is noteworthy that chitosan chemical crosslinking was carried out at a temperature of 60 °C in order to accelerate the reaction hence reducing the loss of the nanofibrous structure.Doxa Microfluidicshed work . In a tyMembrane morphology was evaluated via field-emission scanning electron microscopy (FESEM) through ZEISS  SUPRA 40 VP operating at 10 kV in direct detector configuration (InLens). Good conductivity of the samples was assessed with a thin layer of sputter-coated carbon using a Polaron  E5100. ImageJ  open-source software was employed to analyse the nanofiber dimension. At least 200 diameter measurements were taken for each of the high-magnification micrographs.b), and the elongation at break (εb) were calculated from the obtained stress-deformation curves.Uniaxial tensile test was performed on the crosslinked mats by using a displacement-controlled dynamometer Instron 5565 . At least five rectangular specimens (40 × 10 mm) were tested for each sample. The thickness of the membranes was evaluated via a precision digital micrometer. A pre-load of 0.1 N and an elongation rate of 25 mm/min were employed for all the experiments. The Young modulus E, the tensile strength (σWater contact angle (WCA) of the crosslinked membranes was measured via an Attention Theta Lite optical tensiometer . A small drop of water  was placed on the sample surface and both right and left WCA were calculated via the instrument software.2) is the surface of the sample that permits the vapour diffusion, and ∆p (Pa) is the partial water vapour pressure difference across the two sides of the sample.Water vapour permeability (WVP) of the crosslinked membranes was assessed by means of a gravimetrical method according to ASTM E96-95. Briefly, circular samples were mounted on measuring cups with a diameter of 9.5 mm which were filled with distilled water up to 2 cm underneath the film. The cups were placed in an environmental chamber at 37 °C and 50% RH and weighted every hour for a period of 8 h. WVP was calculated as follows:i and Mf are the weights of the sample before and after the drying, respectively.The moisture content (MC) of the crosslinked membranes was determined by placing a small piece of each sample at T = 110 °C under vacuum for 24 h and evaluating the weight loss after the drying process. MC percentage on a wet-basis was expressed as:2 atmosphere in high glucose Dulbecco’s modified Eagle’s medium (DMEM), with glutamine supplemented wi th 10% FBS using penicillin/streptomycin as antibiotics. Experiments were performed in triplicate on 96-well plates. Crosslinked chitosan membranes were cut to obtain circular discs of 6 mm Ø and autoclaved before being positioned at the bottom of the wells.All cell lines were cultured at T = 37 °C in a humidified, 5% COTo evaluate the eventual cytotoxicity, crosslinked chitosan mats were soaked in complete cell medium and incubated in the 96-well plates with 100 μL of medium per well for 6 h at T = 37 °C. After the incubation with the membranes, the cell medium from each well was collected and added to 96-well plates containing the cell cultures  with a final dilution of the extracts of 1:1. After 24 h, cell viability was measured by the MTT test as already described .To evaluate cell adhesion on chitosan-based nanofibers, the cells were seeded at 10000 cells/well onto the membranes at T = 37 °C and incubated for 16 h. For each cell line, a standard curve was seeded in quadruplicate in the 96-wells in the 1250–10,000 cells/well range to obtain a linear regression curve to fit the experimental results and extrapolate the number of cells attached to the different membranes. At the end of the experiments, cell viability was assayed by MTT test as previously described . BrieflyData are the means ± S.D. of three independent experiments.Adsorption and desorption properties of the physically crosslinked electrospun mats were investigated by using methylene blue (MB) and methyl orange (MO) as model drug molecules of opposite charge. UV-vis absorption spectra were acquired utilizing a UV-1800 spectrophotometer  at room temperature.In a typical kinetic adsorption study, 5 mg of sample were placed within a fused silica cuvette with 1 cm pathlength and soaked with 3.5 mL of dye aqueous solution at a given concentration . The system was gently shaken for 6 h at room temperature and the solution dye concentration was periodically monitored.In a typical isotherm adsorption experiment, 5 mg of the membrane were immersed in 50 mL of dye solutions with increasing concentration  under gentle shaking for a week at room temperature and then the supernatant dye concentration was analysed.In a typical desorption experiment, a dye-loaded membrane was rapidly washed with EtOH and placed in a 1 cm pathlength fused silica cuvette. As the release medium, 3.5 mL of PBS at pH = 7.4 and T = 37 °C were used to simulate physiological conditions. The system was kept under gently stirring and the concentration of the supernatant was periodically monitored for 6 h.t), at equilibrium (qe), and the cumulative release percentage (R%) were determined as:0, Ct, Ce (mg/L) are the dye concentration at the initial time to, at a given time t and equilibrium, respectively, V (L) is the solution volume, M (g) is the weight of the membrane, mi (mg) is the dye load in the membrane at the time t0, and mr (mg) is the dye amount released at time t.The dye absorption capacity at a given time t  and smoother nanofibers that completely maintained their individuality, thereby leading to the formation of a high number of interconnected pores. On the contrary, the chemical crosslinking treatment promoted the partial coalescence of the nanofibers , which in turn led to the establishment of a denser nanostructure presenting an average low porosity. Such findings are, to some extent, unexpected but may be most likely ascribable to the dissimilar reticulation mechanism and kinetic. Indeed, chemical crosslinking occurs via the formation of strong, stable, and not reversible covalent bonds between chitosan chains, whereas HPOch other . In any ch other ,42.Mechanical and water-related properties play a fundamental role in most biomedical devices where the existence of applied stresses and the existence of interfaces with native tissues are not negligible. In particular, wound healing patches should present features similar to those of human tissues, at the same time providing integrity and stability to offer a sufficient protective capability towards the external environment ,44,45. Ib, and tensile strength σb) are summarized in Stress-deformation curves obtained for both physically and chemically crosslinked mats via uniaxial tensile test are reported in Unexpectedly, the physical crosslinking led to more mechanically performing mats despite its weaker crosslinking efficiency. Specifically, the Young modulus and tensile strength were almost twice those of the chemically crosslinked membranes, whereas a slighter difference was observed for the elongation at break. However, rather than directly to the mechanism involved in the chitosan reticulation, the obtained disparities are most likely ascribable to the sample morphology. Indeed, the greater nanofiber homogeneity achieved via physical crosslinking led to the development of a much more ordered three-dimensional macroscopic structure able to provide for the superior mechanical performances. Conversely, the coalescence of the nanofibers occurring during the chemical crosslinking induces the establishment of a defect-rich network characterized by an inferior response ,49.Another key factor concerning wound healing patches is represented by their water-related behaviour ,51. As aThe water contact angle, the water vapour permeability, and the moisture content values of both physically and chemically crosslinked chitosan-based mats are reported in First of all, a marked discrepancy between the hydrophilicity of the samples can be observed with the physically crosslinked mats showing a much lower WCA with respect to the chemically crosslinked ones. Such a result is related to both the crosslinking mechanism and nanofiber morphology. Chitosan, owing to the presence of residual acetyl groups, is characterized by a poor water solubility that is even lowered after the crosslinking approach. Therefore, the stronger the links between the macromolecular chains , the smaller is the capability of chitosan to interact with water . Additionally, the thinner and defect-free nanofibers achieved via physical crosslinking present a higher surface area, thereby endowing the mats with a higher porosity, which, in turn, leads to capillarity effects that can promote the water-material compatibility. Further evidence of this phenomenon can be derived from the water vapour permeability value, which is nearly an order of magnitude smaller for the chemically crosslinked mats. In this regard, the presence of a great number of interconnected pores and the marked hydrophilicity of mats obtained via physical crosslinking seem to provide a much higher water vapour permeability. Finally, both samples presented low moisture content, hence being suitable to be stored over quite long periods without occurring in degradation issues and/or physical-chemical modifications.Considering the above-discussed results, the physically crosslinked chitosan-based mats seem to be much more promising compared to the chemically crosslinked ones for the development of potential wound healing patches. Indeed, the achieved mechanical properties indicate that such systems could be able to endure considerable stresses and deformations before showing mechanical failure, thereby being suitable to provide a highly stable environment to foster cell viability in different types of wounds . In addition, the marked hydrophilicity coupled with the great water vapour permeability may promote the exudate removal and gas exchange, thereby maintaining the tissues dried at the same time providing appropriate breathability, which are both important factors in the regeneration process.Chitosan is a well-known biocompatible material  even if p < 0.05). The L929 fibroblasts (p < 0.05). This latter result might be related to the fact that these cells preferentially adhere to fibrin or collagen substrates of protein origin. Such molecules, indeed, mimic more closely the basal membrane to which keratinocytes naturally adhere at the interface between epidermis and dermis [All cell types showed a clear preference for the physically crosslinked membranes with respect to the chemical ones. More in detail, the osteoblast cell line, Saos-2 f, showedroblasts b showed roblasts d showed d dermis ; therefop < 0.0001 and p < 0.005, respectively). Conversely, only one cell line—namely, HaCaT keratinocytes reported in p < 0.0005). In contrast, no toxicity was observed in the L929 fibroblasts  are summarized in In detail, MB was scarcely adsorbed by the mats with the process being almost arrested after only 2 h for the studied experimental conditions. Conversely, MO was rapidly and much more efficiently entrapped within the electrospun membranes with the phenomenon evolving even after 6 h, especially for the highest dye concentration . To better understand the kinetics of adsorption, pseudo-first-order and pseudo-second-order models were employed to fit the experimental data ,56. Intee values, it can be supposed that strong repulsive interactions arise between chitosan chains and the colorant molecules. Therefore, the adsorption is most likely driven by diffusive phenomena but remains highly hindered due to the electrostatic repulsions. In this sense, since oxygen-containing groups were proved to be frequently involved in the adsorbate-adsorbent mechanisms, the hydroxyl substituents of chitosan are likewise supposed to provide functional sites for the uptake through hydrogen bonding [e values. Nevertheless, since the adsorption process continues over a certain period of time, it is most likely that both electrostatic and diffusive phenomena play here an important role.The results suggest that a different adsorption mechanism occurs for MB and MO dyes, which is in agreement with their different charge. Specifically, MB being positively charged and taking into account the low q bonding . ConversIn this sense, further confirmation was obtained by studying the isotherms of adsorption for both MB and MO dyes with the related results shown in Independently on the phenomena driving the dye upload, a much more interesting aspect is related to the membrane release capabilities that are of topical importance in the development of controlled drug delivery systems. Calculated data are reported in In addition, future studies will be required, the achieved results represent a promising starting point in the development of simple, cheap, and efficient drug delivery systems with modulable release kinetics. Indeed, despite negatively charged molecules presenting a higher affinity for chitosan-based nanofibers and positively charged ones showing a better-controlled release, depending on both the drug nature and loading concentration, it could be possible to obtain DDSs able to provide a burst or a slow-release to satisfy the specific requirements.In the present work, electrospun chitosan-based nanofibers were prepared and subsequently subjected to a physical or chemical crosslinking treatment by using phosphate ions or ethylene glycol diglycidyl ether, respectively. Remarkably, physically crosslinked membranes were proved to show a broad number of advantages with respect to the chemically crosslinked ones. In particular, the use of phosphate ions led to much thinner and homogenous nanofibers , which, in turn, provided a greater global porosity and an enhanced mechanical response, as well as more marked hydrophilicity and water vapour permeability. Above all, significant differences were depicted between the mats in terms of cell adhesion and cytotoxicity. In particular, the nanofibers treated with phosphate ions showed no toxicity towards the different tested cell lines along with the capability to strongly promote their adhesion and proliferation, especially for osteoblasts. Conversely, the chemically crosslinked mats were found to be toxic for keratinocyte and osteoblast cell lines, thereby being able to marginally foster cell viability most likely due to the release of some residual chemicals. Owing to the aforementioned findings, the physically crosslinked mats were then loaded with two dyes, which were used as model drug molecules, and their delivery properties were intensively evaluated. Interestingly, by simply changing the charge of the dyes, the mats were proved able to show a burst or a slow sustained release, hence being suitable for different routes of administration. In conclusion, the proposed physically crosslinked chitosan-based mats represent a potential class of innovative, cheap, and highly efficient materials to be used as advanced wound healing patches with tuneable drug delivery properties. However, it is noteworthy that further in vitro and in vivo experiments are required to open the way to their effective use in biomedical and pharmaceutical products. Among others, some critical points that must be soon addressed include the improvement of the fabrication procedure , a better understanding of the product biological response  and drug delivery properties, as well as the evaluation of the scaffold degradation kinetics in physiological conditions.


First of all, we would like to thank all the authors for their contribution and the editorial staff who enabled the achievement of this “Management of Sarcoidosis: Challenges and Solutions” Special Issue. This issue covers a selection of hot questions on sarcoidosis pathogenesis and on everyday clinical practice in sarcoidosis. This issue is more an impressionist view of sarcoidosis than an exhaustive one, as significant progress was made both in the understanding of the pathogenesis and in diagnostic and therapeutic approaches—particularly in terms of changes in new concepts concerning genetics and auto-immunity, and a more comprehensive approach to the care. Therefore, the issue contains very didactic and practical general reviews, as well as original articles focused on little-known or mutating topics.Sarcoidosis is a disease with a large phenotypic variability due to the complex combination of genetic susceptibility, immune networks and still unknown infectious and/or environmental causal agents. Mycobacterium tuberculosis (PPD)-coated polystyrene beads. This model makes it possible to have access to the initial cellular and molecular events in granuloma formation [One way to penetrate the still opaque world of this systemic disease is in the development of selected models with a high level of confidence, which is Besnard and Jeny’s review’s objective . As theyormation . The secormation . As a coormation ,6. The tormation . Consecuormation . If the ormation . Of the ormation .Thanks to the development and mastery of innovative tools, significant pieces of genetics in the sarcoidosis jigsaw puzzle are added on a regular basis, which is clearly and extensively described by Calender et al. . A recenMycobacteroides abscessus or other bacteria. This noteworthy contribution from Baerlecken et al. reinforces the discussion about the classification of sarcoidosis among the vast spectrum of immune or inflammatory systemic diseases, which has always been a puzzle—immune granulomatous disease , auto-inflammatory disease (inflammasome activation) or auto-immune disease  [Autoimmunity features observed in sarcoidosis have been observed for a long time, but they raise the hypothesis of an underlying autoimmune disease that co-occurs with sarcoidosis , or a rontigens) ? Or, clontigens) ,22? To end with the pathogenic domain of the issue, McClain Caldwell et al. contributed a research paper opening a new field in sarcoidosis treatment, focused on the potential benefits of stem-cell therapy . It shouIn the area of clinical knowledge of sarcoidosis, classical reviews are still not outdated ,28,29. HJain et al. underscored the difficulty of the diagnosis, with often a significant delay, and most encounter comorbidities . Concern18FFDG-PET is a very efficient biomarker for determining treatable inflammatory lesions in symptomatic patients, particularly those with normal serological inflammation, like serum angiotensin-converting enzyme and serum sIL2R [18FFDG-PET has been indicated in three clinical contexts concerning patients with confirmed sarcoidosis, as follows: (i) patients with pulmonary fibrosis, for whom the expected beneficial treatment effect on Forced Vital Capacity can be predicted thanks to lung inflammation assessed by lung FDG uptake; (ii) patients with cardiac sarcoidosis for whom, subject to the patients’ diet before 18FFDG-PET being correct, a left ventricular ejection fraction gain under treatment might be expected thanks to cardiac FDG uptake; and (iii) patients with persistent disabling symptoms. Otherwise, for patients with suspected extrapulmonary sarcoidosis difficult to diagnose, such as isolated uveitis of unknown cause, central nervous system or cardiac manifestations with normal thorax CT, the evidence of 18FFDG uptake in both hilar and mediastinal lymph-nodes can usefully guide the diagnosis. As underscored by Keijsers, um sIL2R . 18FFDG-Advanced pulmonary sarcoidosis is the main cause of dangerous sarcoidosis. In their paper, Sawahata et al. had the original idea of comparing the thorax CT in patients with supplemental oxygen need with a previous one done several years before for a better understanding of the radiological findings’ significance . They prPediatric sarcoidosis is an extremely rare disease with a 0.6–1.02/100,000 estimated incidence, but in the absence of international registers, the disease is probably under-reported ,44. ChauEvidencing non-caseating granulomas on histopathology is very important for diagnosing sarcoidosis. Despite many original articles and reviews focusing on endobronchial ultrasound-guided transbronchial needle aspiration (EBUS), the particularly well-written paper by Pedro et al. offers a means of getting very clear and accurate data concerning the advantages and the pitfalls of every endoscopic technique in sarcoidosis, and the impacts of many technical factors . EBUS haIn conclusion, while all the numerous questions raised by sarcoidosis could not have been included in the register of this Special Issue, such as for example the potential role of the microbiota in sarcoidosis , or a ne


The results from our analyses indicate that at least for the given systems, force field and simulation protocol, the two methods diverge in their predictions. The possibility of a force field-dependent deficiency is examined as a possible source of the observed discrepancy.Both molecular mechanical and quantum mechanical calculations play an important role in describing the behavior and structure of molecules. In this work, we compare for the same peptide systems the results obtained from folding molecular dynamics simulations with previously reported results from quantum mechanical calculations. More specifically, three molecular dynamics simulations of 5 μs each in explicit water solvent were carried out for three Asn-Gly-containing heptapeptides, in order to study their folding and dynamics. Previous data, based on quantum mechanical calculations within the DFT framework have shown that these peptides adopt  Type I and II β-turns are the most common types of β-turns found in proteins, with the ideal torsion angles  in each of these categories being  and , respectively, whereas, the corresponding values for their “mirror-image” type I’ and II’ β-turns are  and  [esidues) –5. They s i to i+ with the[β-turns , the almβ-turns can be considered structurally as no well-behaved model systems, as they occasionally adopt random conformations, it is both the nature and position of specific residues that favors the propensity towards β-turn formation usually by stabilizing the formation of β-hairpin motifs [β-turns [β-turns which are common among observed protein structures [φ,ψ angles of this turn type are typically constrained in a small region of the Ramachandran plot for L-amino acids [i+1 and i+2 of type I’ β-turns indicate that, among others, Asn is generally favored in position i+1 and Gly in position i+2 [β-turn and β-hairpin conformations, though not as effectively as the D-Pro-Xaa loop-forming segment [β-hairpin promoter sequence D-Pro-Gly is balanced to some extent by an enthalpic advantage of the Asn-Gly sequence, as the rigid nature of the D-Pro-Gly segment may prevent energetically favored contacts between side-chains [β-turns, no extensive computational investigation has been made on the folding of short peptides containing this segment. A recent study by Kang and Yoo [β-turn structures using ab initio quantum mechanical (QM) calculations within the DFT framework, indicated that Asn-Gly type I’ β-turn was indeed the preferred structural motif for all the three heptapeptides. The optimized torsion angles for every β-turn type in each heptapeptide as obtained from their work [ψGly value that despite residing within the ±30° deviation limit, has a slightly increased flexibility compared to the rest of the angles.Although short peptides that appear in n motifs –12. Anal[β-turns , 10–12. ructures , 7. The no acids . Studiestion i+2 . Analysi segment –23. Intee-chains . However and Yoo , who exaeir work  at the Sβ-hairpin-like [β-turn motif [β-turn conformations in water, and examined to what extent the AMBER99SB*-ILDN force field, combined with the TIP3P [Folding molecular dynamics (MD) simulations using empirical force fields have matured to the point of becoming a powerful and useful analytical tool for studying the structure and dynamics of a wide variety of peptide systems. Our perspective on the current literature is that among all extensively tested and validated force fields , the AMBpin-like , 37–39. pin-like . So far,rn motif —possiblyrn motif , 42. Thehe TIP3P  water moβ-turn structures we conducted three MD simulations for the same three capped heptapeptides that Kang and Yoo used in their work: Ac-Ala-Ala-Asn-Gly-Ala-Ala-NHMe (hpNG-1), Ac-Leu-Val-Asn-Gly-Gln-Tyr-NHMe  [Asn-Gly-Leu-Phe-NHMe  [In order to study the propensities of the Asn-Gly segment to form ry 1EST)  and Ac-P β-turn) . The sys β-turn) –38 and i β-turn) , 47 for  β-turn)  and the  β-turn) , 29, whi β-turn)  was applΤ step of 20 K) over a period of 32 ps. Subsequently, the system was equilibrated for 10 ps under NpT conditions without any restraints, until the volume equilibrated. This was followed by the production NpT runs with the temperature and pressure controlled using the Nosé-Hoover Langevin dynamics and Langevin piston barostat control method as implemented by the NAMD program, with adaptive tempering applied through the Langevin thermostat, while the pressure was maintained at 1 atm. The application of the adaptive tempering in combination with the choice of the NpT ensemble guarantees the absence of significant variations of the system’s mean density . The Langevin damping coefficient was set to 1 ps-1 and the piston oscillation period set to 200 fs with a decay time of 100 fs. The production run was performed with the impulse Verlet-I multiple time-step integration algorithm [−6. A cutoff for the van der Waals interactions was applied through a switching function, acting between 7 and 9 Å. The SHAKE algorithm [−8 was used to restrain all bonds involving hydrogen atoms. Trajectories were obtained by saving the atomic coordinates of the whole system every 1 ps.The simulation protocol for all the peptides was the following: the system was first energy minimized for 1000 conjugate gradient steps followed by a heating-up phase to a final temperature of 320 K  between the DFT-derived φ,ψ torsion angles (from ref 24) and their corresponding torsion angles from each frame of the trajectory (or from each frame of a dPCA-derived cluster). In the second step, the distributions of these angle deviations were calculated and the resulting histograms plotted. In the final step, we treated these distributions as a sum of independent Gaussian distributions, and we obtained numerical values for the means (μ) and standard deviations (σ) of those peaks residing closest to the reference DFT angle values  parameters was performed with the nlsLM function of the R package) [A significant part of this work focused on analyzing the extent to which the ulations  and the ulations  agree wipackage) . These vnot been observed in a given trajectory. These probabilistic estimates are presented in the form of graphs that depict the probability of observing a new/different structure, as a function of the minimal RMSD—of this (thus far unobserved) structure—from all those structures already observed. The results from the application of Good-Turing statistics are shown in The heptapeptides—as will be discussed in the next section—demonstrate a highly dynamic behavior throughout the molecular dynamics simulations. Their structural flexibility/disorder leads to a rugged folding landscape and, consequently, to the absence of a well-defined gradient towards a would-be ‘native’ structure. The implication of this finding is that the whole configurational space available to the peptides is accessible during the simulations. This immediately raises the issue of convergence and statistical significance of our results. To tackle this problem, we apply a recently proposed probabilistic method  for estiβ-turn is rather dramatic: the curves fall quite fast to negligible punobserved values for RMSDs of the order of 1 Å. The apparent difference between these two cases may be attributed to the fact that the four-residue central parts are structurally more stable, possibly promoting the formation of secondary-structure-like patterns, whereas the peptides' termini are highly mobile and disordered.To clarify these graphs, the high probability of unobserved species for low RMSD values indicates that if we were to continue the simulation  it would be highly probable to observe structures, which although similar to some of the already observed ones, they would still be slightly different from them. As the RMSD from the already observed structures increases, the corresponding probability decreases. It is therefore the exact form of this graph and the inclination of each curve towards low probability values that inform us to what extent the trajectories have been sampled or, in other words, how significant is the structural variability that has been missed due to limited sampling. Focusing on the application of Good-Turing statistics in our trajectories, the results have been obtained from multiple calculations and are organised in two groups. To conclude, the results drawn from the analysis of our 5 μs trajectories indicate that the length of the simulations is probably sufficient and guarantees a reasonable sampling of the peptides’ configurational space for the given force field. A similar type of analysis has also been made but using only structures whose corresponding adaptive tempering temperature was ≤ 360 K, thus corresponding to more stable (from the simulation’s point of view) conformers. As shown in β-turn motifs for each heptapeptide can be seen in To place our observations on a structurally firm ground, we calculated the free energy landscapes of the heptapeptides using the dihedral angle Principal Component Analysis (dPCA) method to identify the prominent conformations. Various studies have already indicated that dPCA is a high resolution, powerful and more appropriate method for studying such highly flexible systems –56. We rhpNG-1, 34.7% of the total frames were assigned by the program CARMA to clusters, while for hpNG-2 and hpNG-3 the corresponding percentages were 36.7% and 31.8%, respectively. The most highly populated cluster recorded for each heptapeptide occupied only 11.9%, 20.1% and 20.7% of the clustered frames , clearly indicating that the peptides demonstrate a very dynamic behavior. β-turn motifs in each cluster. There is a structural variability concerning the preference among turn types and the positional preference of each amino acid residue in positions i+1 and i+2. Asn-Gly turns, not only failed to be the dominant structural motif, but the analysis also revealed the presence of a plethora of β-turn motifs as well. Examination of clusters’ representative structures reveals that structures can vary among different β-turn and unfolded conformations. β-turn, clearly demonstrating the large diversity of the turn types that have been sampled. Many representative structures corresponding to significantly populated clusters adopt a β-turn conformation, but the presence of unfolded random coil representatives is nonetheless considerable. Asn-Gly is not favored to be the turn segment among the representatives, as there is a variety in the positional preference of each residue for this site.As it can be seen in β-turn motifs, β-turns sampled in each trajectory taking into account both the different combinations of i+1 and i+2 positional preferences and the different types of β-turns. Of the turns located, βIV turns seem to be the most prominent turn motif in each trajectory, but this can be attributed to the fact that this miscellaneous type does not follow any of the stringent criteria being used for turns’ classification scheme [βVIII turns’ sampling on the other hand is limited, resulting only in a negligible amount of observations. As for the most common classical turn motifs βI, βI’, βII, βII’, we must also focus on the various analyses of turns derived from experimental data, apart from the DFT calculations, so that we examine the possibility that our simulations have accurately described the peptides’ folding behavior. X-1-Asn and Gly-X+1βI turns appear to be the most frequently sampled turn motifs in each trajectory, in agreement with the overall positional potentials which show a distinctive preference for Asn in positions i and i+2 [i forming a hydrogen bond with the main-chain nitrogen of residue i+2, stabilizing consequently type I β-turns [i to i+3 form another turn-like structure known as the “Asx-turn”, which is commonly observed in type I β-turns [φ = -90°, ψ = 0°) for position i+2 in βI turns [αL conformation in position i+3 of type I turns [-1-Asn and Gly-X+1βI turns in our trajectories. Regarding type II’ turns, although poorly sampled, especially in the case where Asn-Gly occupies the turn position, Gly-X+1 has a relatively increased preference as a turn segment for this turn type, in agreement with the crystallographic data where Gly shows a major preference for position i+1 in βII’ turns due to φ,ψ restrictions around +60°, -120° respectively [βII’ Gly-X turns in model peptides containing the Asn-Gly segment [βII’ turns (followed by βI turns) [+1 populations. Finally, the Asn-Gly segment is slightly to moderately favored as a turn segment depending on the heptapeptide, showing a rather increased preference towards βII and βI’ turns . Although Gly residue very often occupies the i+2 position in both of the aforementioned turn types (as it most readily adopts the αL and γL conformations) [i+1 of βII turns in protein structures [βI’ turns contrarily, the Asn residue occurs very frequently in this position, as several studies have already indicated [φ,ψ constraints around the γL region of the Ramachandran plot [βI’ turns is apparent in our results, contrary to the consistent intrinsic preference of this dipeptide for the turn segment in βI’ turns as shown by numerous above-mentioned studies.To have a more comprehensive understanding of the preferences among the different n scheme . βVIII t and i+2 , 7, 10.  β-turns . AdditioI turns) , justifyructures . In βI’ ndicated –20, 23, βI’ turn motif . In contrast, the present MD analysis shows that Asn-Gly turns are overall , these residues do not occupy proper pair-facing positions that strongly influence stabilization interactions [βI’ turns is not unequivocally established, as previous NMR and MD studies implied either the concomitant presence of other turn motifs [Although the general pre-existing conformational preferences of the amino acid residues are important for the turn formation, side-chain to side-chain or side-chain to backbone interactions also play a key role in the folding and stabilization of turns and by extent hairpin motifs, especially in the context of a three-dimensional protein structure , 19–23. ractions , 24. Thermations , 64. Thi studies . Though  studies , 64, 65,ractions . Finallyn motifs  or the in motifs .ab initio level might overshadow the structural distribution of these systems.Having said the above and seeing our results in a positive light, given that MD simulations are superior in sampling a large conformational phase space, they might adequately interpret at first sight the behavior of such short and flexible peptides isolated in water solvent. Despite arguing in favor of our MD results though, our aim at this point is not to dispute the validity of the QM calculations, but to note that generating single structures on the d = 0° indicate similarity with the DFT model , while values far from d = 0° indicate large deviations. To further quantify these observations, we treated the distributions of these deviations (green curves in d = 0°) and obtained numerical values for their means (μ) and standard deviations (σ) through a nonlinear regression fitting performed with the nlsLM function of the R package . The outliers are again ψAsn in βII’ hpNG-1, ψAsn in βII’ hpNG-2 and ψAsn in βII’ hpNG-3, which show a very significant deviation from the ideal β-turn values, far exceeding the ±30° limit set by Thornton and coworkers.To complete this set of comparisons, we also calculated the deviations (in degrees) between the mean Asn/Gly torsion angles from molecular dynamics and the corresponding ornton’s  definitiψAsn βII’ angles disputes the validity of our simulations. The fact that molecular dynamics utterly fail to sample energetically allowed—as indicated by QM calculations—angle values, along with the absence of a strong propensity towards the Asn-Gly βI’ conformation, might underlie a force field discrepancy. Indeed, previous studies on the Asn-Gly β-hairpin Trpzip-2 model peptide using the 99SB*-ILDN force field, showed the inability of this force field variant to sufficiently fold and stabilize the β-hairpin conformation [Although, as mentioned above, the results from our MD simulations, under the scope of the application regime they have been designed for, manifest a substantial convergence with data concerning the general amino acid frequencies in turns, such conspicuous disparity with the experimental/DFT results regarding ormation , 42. Thiφ,ψ angles in each cluster’s frame from the corresponding DFT-calculated value. β-turns and a representative structure for each cluster. A closer look at the histograms reveals a significant divergence of the Asn/Gly φ,ψ peaks from the d = 0° value for several clusters, showing that Asn-Gly turns have not been adequately sampled in the major dPCA clusters. The cases where this torsion angle analysis indicates convergence with the DFT data can also be verified through a comparison with the results shown in hpNG-1, for example, have significant populations of Asn-Gly type II and I β-turns respectively, as the sharp Asn/Gly φ,ψ peaks lying around d = 0° indicate, but the Asn-Gly βI’ motif has not been sampled at all  of Asn-Gly turns sampled in the clusters. The results are in good agreement with the previously observed populations of βII’ turns have been identified. As for the Asn-Gly βΙ’ turn, although it is present, it is definitely not a predominant structural motif of the dPCA clusters. A question that arises at this point is this: Can the clusters be characterized by a single structural motif? In other words, can a highly populated motif be representative of a cluster’s structural ensemble? If we look in β-turn type, they cannot be unequivocally identified by a single motif. As can be seen in To compare our previous observations with the results obtained from the dPCA we calculated, as described above, the deviations of the d at all . Clusterectively , whereasectively . S8 Fig β-turns in each cluster gives rise mainly to coil or non-classical βIV representative structures, suggesting that clusters are barely characterized by native-like conformations. While the tendency towards β-turn formation is in general present, the structural diversity observed in our trajectories (and clusters) precludes us from identifying adequately large populations of specific turn motifs that would be indicative of a propensity towards specific β-turn structures, such as the type I’ Asn-Gly turns. That being said, along with the incapability of the force field to sample certain torsion angles—and therefore conformations—identified as energetically favoured by the DFT calculations [To summarize, turn populations in clusters are indicative of what is mentioned above: the small fraction of stable, folded ulations , cast doβII’ turns that—although meagerly reported in previous statistical analyses of database [βI’ turns, as previously has been confirmed by several experimental and statistical analyses [β-hairpin peptide, the Trpzip-2, giving no dominant cluster but a small fraction of folded structures under equilibrium conditions, possibly due to backbone torsion parameterization and problems in describing local conformational preferences [β-hairpin formation could not be stabilized, as well as that there is an increase in a-helix propensity due to an imbalance of the local conformational preferences [α-helical conformations, and therefore of an inadequate balance between various secondary structure elements, thus making the stabilization of the β-hairpin unachievable.As previously mentioned, the inability to sample Asn-Gly database –5, 7—havanalyses –20, 23, ferences , 42. In ferences , 42. Theferences , 42. ThihpNG-1 peptide, but this time using the 99SB-ILDN force field instead of the 99SB*-ILDN variant. Results from the aforementioned studies on the Trpzip-2 showed that although 99SB-ILDN underestimates ΔΗF, it achieves a relatively better folding of the peptide [β-turns was introduced in the force field during its optimization to better sample α-helical structures. Analysis of this 99SB-ILDN trajectory with the PROMOTIF program and comparison with the results shown in hpNG-1 entries in I’: 0.41→0.47%, 2.34→2.54%, 0.38→0.31%, II’: 0.17→0.08%, 0.04→0.03%, 7.35→6.75%, implying that there is no profound variation in the performance between the star-corrected force field and its uncorrected counterpart. This is not necessarily an unexpected outcome. Despite the success of the 99SB-ILDN variant  in folding Trpzip-2 in previous studies, the increased flexibility of our systems leads correspondingly to an increased difficulty of tracking these systems computationally. Note, however, that this remark should not be taken trivially. Constantly increasing computing power has concomitantly facilitated the application of high quality, longer simulations . At least for such short peptides, such long simulation times eliminate obscurities associated with insufficient sampling and allow these studies to be seen as an acid test for the ability of force fields to successfully interpret physical reality. In this spirit, the above reported discrepancies add one more piece of evidence to the existing literature by showing that two force fields give a totally different view contrary to QM and X-ray determined results. This, by extent, points towards the necessity of numerous independent case studies for such systematic inaccuracies to be detected, despite the long-term validation a force field might have undergone.Given the long and successful history of applications of the AMBER99SB family of force fields to peptide folding , 69, we  peptide . The aimζ basis sets, such as the 6-31G(d) used in ref 24, frequently suffer from inaccuracies due to basis-set superposition (BSSE) and basis-set incompleteness (BSIE) errors. These problems affect not only energies but also properties derived from them such as geometries, as previous studies on peptides and protein fragments have already indicated [βII’ turn in our case, may account for inconsistencies in the DFT calculations that were performed. That being the case, we could argue that our MD analysis might after all adequately provide a physically correct description of the structures and dynamics of the Asn-Gly heptapeptides.Having noted, though, the above conclusions and aiming to provide the full scenery of the complications accompanying this work, should we solely attribute such discrepancies to the parametrization of the force field? The comparison between DFT and MD rises and falls with the quality of the DFT calculations as well. The differences between these two methods, especially in terms of the potential energy surface calculation, are widely known to the scientific community, and disagreement is not unexpected. Despite the approximations used in MD simulations, the reliability of QM calculations is also not unquestionable. Concerning our case, although the hybrid meta-GGA M06-2X functional  coupled ndicated –78. Despβ-turn is the most preferred motif in aqueous solution for the three heptapeptides containing the Asn-Gly segment. The picture painted by our molecular dynamics analysis, however, is fundamentally different: according to the simulations, the peptides are highly flexible with multiple relatively shallow free energy minima that allow the peptides to quickly interconvert between structurally diverse conformations. The dPCA-based free energy landscapes in β-turn formation. As β-turns containing Asn/Gly as one of their central residues occupy the largest part of the respective MD trajectories with cumulative frequencies reaching ≈85% for two of the peptides (becoming ≈45% if the type IV turns are excluded). The residue-specific preferences for positions i+1 and i+2 on the β-turns vary significantly between the four central residues, with the Asn-Gly segment preferring the aforementioned positions almost equally with segments formed by neighboring residues (X-1-Asn and Gly-X+1). Type I β-turns appear to be the most prominent MD-derived motif in each trajectory, while Asn-Gly βI’ turns show relatively significant populations only for the hpNG-2 and hpNG-3 peptides. In agreement with the dPCA results, the torsion angle analysis also demonstrated the structural malleability of the peptides and their pronounced tendency to form various types of β-turns. But it was also this more detailed analysis that allowed us to convincingly demonstrate the inability of the AMBER99SB*-ILDN force field to sample the Asn-Gly βII’ motif. This apparent force field discrepancy was also confirmed through a comparison between MD and the ideal β-turn values obtained by the Thornton group. This comparison , and can thus be argued that we could be completely wrong in assuming that these specific peptides should actually sample, for example, the  studies , 23, 63  studies –5, 7 thaS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigResults from the application of Good-Turing statistics to the three trajectories, for both the full-length peptides and their four-residue central part, obtained using only structures associated with temperatures ≤ 360 K.(TIF)Click here for additional data file.S2 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-1 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S3 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-2 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S4 Figφ,ψ torsion angle values of residues i to i+3 for (A) βΙ, (Β) βI’, (C) βII and (D) βII’ DFT-obtained hpNG-3 turn structures. Areas in red, yellow, beige and white represent the core, the allowed, the generous and the disallowed regions respectively. Non-glycine residues are depicted here with black square signs and glycine residues with black triangles. Figures were generated using PROCHECK.Ramachandran plots showing the (TIF)Click here for additional data file.S5 FigNG-1 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Ala-Asn-Gly-Ala part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S6 FigNG-2 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Val-Asn-Gly-Gln part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S7 FigNG-3 for every turn type. Shown from left to right is the number of the cluster along with its population, the occupancy of β-turn motifs in each cluster, the histograms showing the distribution of deviations (in degrees) between the reference DFT φ,ψ values of the 4-residue central part and the respective φ,ψ values obtained from the simulation, and the corresponding backbone representative structures of each cluster. Representatives with random coil conformation are depicted here only with their Val-Asn-Gly-Leu part.Results from the torsion angles analysis of the five most prominent dPCA clusters of hp(TIFF)Click here for additional data file.S8 Figβ-turns among a set of 500 randomly selected structures from every dPCA cluster of the three heptapeptides. Clusters in which β-turns were not identified among the set of structures are omitted. Shown at the horizontal axis are clusters’ numbers and the different turn types. The vertical axis shows the % occupancy of the different turn types in each cluster. The structural assignment was performed using the PROMOTIF program.The above barcharts show the occupancy of Asn-Gly (TIFF)Click here for additional data file.


Once internalized, receptors reach the sorting endosome and are either targeted for degradation or recycled to the plasma membrane, a process mediated at least in part by tubular recycling endosomes (TREs). TREs may be efficient for sorting owing to the ratio of large surface membrane area to luminal volume; following receptor segregation, TRE fission likely releases receptor-laden tubules and vesicles for recycling. Despite the importance of TRE networks for recycling, these unique structures remain poorly understood, and unresolved questions relate to their lipid and protein composition and biogenesis. Our previous studies have depicted the endocytic protein MICAL-L1 as an essential TRE constituent, and newer studies show a similar localization for the GTP-binding protein Rab10. We demonstrate that TREs are enriched in both phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphosphate P2), supporting the idea of MICAL-L1 recruitment by PA and Rab10 recruitment via PIP2. Using siRNA knock-down, we demonstrate that Rab10-marked TREs remain prominent in cells upon MICAL-L1 or Syndapin2 depletion. However, depletion of Rab10 or its interaction partner, EHBP1, led to loss of MICAL-L1-marked TREs. We next used phospholipase D inhibitors to decrease PA synthesis, acutely disrupt TREs, and enable monitoring of TRE regeneration after inhibitor washout. Rab10 depletion prevented TRE regeneration, whereas MICAL-L1 knock-down did not. It is surprising that EHBP1 depletion did not affect TRE regeneration under these conditions. Overall, our study supports a primary role for Rab10 and the requirement for PA and PIP2 in TRE biogenesis and regeneration, with Rab10 likely linking the sorting endosome to motor proteins and the microtubule network. The internalization of receptors and lipids from the plasma membrane is an essential process in all mammalian cells . Once inOne of the mechanisms proposed for efficient endosomal sorting is the use of a large surface membrane area to segregate select molecules and target them to distinct pathways . Indeed,TREs containing MICAL-L1 have been implicated in the trafficking of receptors both from the SE toward the perinuclear endocytic recycling compartment (ERC) and from the ERC toward the plasma membrane . HoweverRecently, Rab10 has also been identified as a component of tubular endosomes . Indeed,A–F. By performing 3D surface volume analysis, we were able to avoid interference from non–membrane bound protein and focus almost exclusively on the surface volume of the endosomal proteins. In addition, the serial sections ensured that we measured true overlap and not merely proteins that overlapped in the X-Y axis (see G). The Rab10 overlap with MICAL-L1 was considerably lower, likely resulting from the more intense overall Rab10 staining pattern that includes nontubular endosomes in addition to the TREs. Even the total volume overlap, which measures the total surface volume of each protein and the percentage of overlap, was about 20%, further highlighting the degree of overlap on TREs. Moreover, overlap between MICAL-L1 and Rab10 was further validated using super-resolution Structured Illumination Microscopy . The PA marker also localized primarily to long, tubular structures, but unlike PLCδ was largely devoid of localization to the plasma membrane and to perinuclear spherical vesicles (B). The localization of endogenous Rab10 closely mirrored that of the PA marker (C) and similarly was absent on the plasma membrane and perinuclear spherical vesicles. Significantly, endogenous Rab10 was found on TREs that were marked by both PIP2 and PA , indicating that these phospholipids are both enriched on TRE.Given the requirement for PA in recruiting MICAL-L1 to TREs , and thein vitro . As demoin vitro A. The PAvesicles B. The loA marker C and simA–C). When MICAL-L1 was depleted by siRNA , the apparent volume of Rab10 TREs was not decreased . Indeed, the apparent volume of Rab10 TREs displayed a small but significant increase in the absence of MICAL-L1 . Since Syndapin2 interacts with MICAL-L1 and colocalizes with it on TREs, we similarly knocked down Syndapin2 and measured the apparent Rab10 and MICAL-L1 TRE volume. As demonstrated, Syndapin2 depletion led to reduced overall expression of MICAL-L1 . As a result, significantly fewer MICAL-L1 TREs were measured , likely owing to the instability of MICAL-L1 on membranes and its degradation  upon MICAL-L1 or Syndapin2 knock-down. As anticipated, in Mock-treated cells, MICAL-L1 and Rab10 displayed a partial overlap on the same TREs , A–C. Whradation . Howeverradation .Figure 3J; quantified in Fig. 4K). Compared with Mock-treated cells (A–C), Rab10 knock-down cells (D–F) displayed a significantly decreased apparent volume of MICAL-L1-containing TRE . Since Rab10 functions with EHBP1, a protein that interacts with PIP2 and reportedly affiliates with TREs . When the localizations of Rab10 and MICAL-L1 were analyzed, significantly reduced Rab10- and MICAL-L1-TRE were observed, with Rab10 displaying an apparently more cytoplasmic localization . Indeed, membrane fractionation experiments demonstrated that significantly more Rab10 could be detected in the cytosolic fraction and less Rab10 was detected in the membrane fraction when EHBP1 expression was impaired by EHBP1 siRNA knock-down . These data suggest that both Rab10 and EHBP1 are required for TRE generation and/or maintenance.Given the surprisingly superfluous requirement for MICAL-L1 and Syndapin2 expression to maintain Rab10-containing TRE, we next asked whether Rab10 is required for the generation of MICAL-L1-containing TREs . To thisFig. 4ith TREs , 31, we A; quantified in Fig. 5I), significantly fewer TREs were observed in Rab10 KO MEF cells . Indeed, the total apparent tubular volume of MICAL-L1-marked TRE was typically two- to threefold higher in WT MEF cells than in their MEF Rab10 KO counterparts . Significantly, when the Rab10 KO MEF cells were “rescued” by transfection of WT Rab10 (+red fluorescent protein-Rab10) into these cells as opposed to a green fluorescent protein control, the apparent volume of MICAL-L1-marked TRE increased by ∼ three-fold . Overall, these data support the notion that Rab10 is a major protein involved in the generation of TRE.To further address the role of Rab10 in TRE biogenesis, we examined MICAL-L1-containing TRE in mouse embryonic fibroblast (MEF) cells derived from Rab10 knockout (KO) embryos . CompareFig. 5A–F). Similarly, TREs marked by Rab10 were significantly impaired upon PLD treatment but recovered rapidly upon PLD washout (G–L). These data further support the idea that MICAL-L1- and Rab10-TREs mostly mark the same tubular endosomes.We have previously demonstrated that phospholipase D (PLD) inhibitors may be used to acutely disrupt the TRE network within 30 min of treatment, likely by affecting levels of PA and potentially also PIP2 . It is i washout , G–L. ThM; quantified in Fig. 6N). We then calculated and compared the apparent MICAL-L1-TRE volume between Mock and Rab10 knock-down cells under control conditions (dimethyl sulfoxide [DMSO] treatment), PLD inhibitor treatment, and following inhibitor washout . As expected, the apparent MICAL-L1-TRE volume was reduced by over 50% in DMSO-treated cells upon Rab10 knock-down compared with Mock-treated cells. PLD inhibitor treatment further reduced the apparent TRE volume levels in both Mock-treated and Rab10 knock-down cells. Most significantly, whereas the Mock-treated cells displayed a massive recovery in TRE regeneration upon washout, the Rab10 knock-down cells did not, and the TRE levels remained as low as the DMSO treatment baseline . These data are consistent with the requirement of Rab10 for TRE biogenesis. We also tested the role of EHBP1 in TRE regeneration, since like Rab10, it appeared to be necessary for the biogenesis and/or maintenance of these endosomes. Accordingly, we used DMSO, PLD inhibitors, and washout of the inhibitors to compare apparent TRE volumes  in Mock-treated and EHBP1 knock-down cells. As expected, EHBP1 knock-down significantly reduced the apparent volumes of TREs marked by either MICAL-L1  or Rab10 . Also as anticipated, PLD inhibitor treatment further reduced TRE levels of both Mock-treated and EHBP1 knock-down cells. However, somewhat surprisingly, upon PLD inhibitor washout the EHBP1 knock-down cells (O-P) displayed TRE regeneration almost identical to that seen in Mock-treated cells (R–S). These data suggest that Rab10 is a key protein involved in the biogenesis, maintenance, and regeneration of TREs. However, although EHBP1 appears to be required for TRE biogenesis and/or maintenance, unlike Rab10, EHBP1 appears to be expendable for TRE regeneration.Using this system, we now addressed the role of Rab10 in TRE regeneration by knocking it down with siRNA. As demonstrated by immunoblot, Rab10 expression was significantly reduced with siRNA , only the presence of Rab10 is necessary for acute regeneration of TREs following PA depletion. EHBP1 has been implicated in fission of tubular endosomes , 41, a fMycoplasma infection.The HeLa cervical cancer cell line was obtained from the american type culture collection and grown in Dulbecco's modified Eagle's medium (high-glucose) containing 10% FBS, 1× penicillin–streptomycin (Invitrogen), and 2 mM glutamine. The MEFs derived from Rab10 knockout (KO) embryos were previously described  and growThe following antibodies were used: anti-MICAL-L1 , anti-Rab10 , anti-EHBP1 , anti-Syndapin2/Pacsin2 , anti-EEA1 , anti-pPKCα , anti-GAPDH-horseradish peroxidase (HRP) , mouse anti-rabbit IgG light chain–HRP , Alexa Fluor 568–conjugated goat anti-rabbit , Alexa Fluor 568–conjugated goat anti-mouse , and Alexa Fluor 488–conjugated donkey anti-mouse . The PLD inhibitors CAY 10593  and CAY 10594 were purchased from Cayman Chemical Co and were typically used at 50 μM for 30 min at 37 °C.Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and were then scraped off plates with a rubber policeman into ice-cold lysis buffer . Protein levels of postnuclear lysates were quantified using the Bradford assay (Bio-Rad) for equal protein level loading. For immunoblotting, 20 to 30 μg of protein per lysate was separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and blocked for 30 min at room temperature in PBS with 0.3% Tween (PBST) plus 5% nonfat dry milk. The membranes were then incubated overnight at 4 °C or for 1 h at room temperature with primary antibodies diluted in PBST. Membranes were then washed three times with PBST and incubated at room temperature with appropriate secondary antibodies in PBST for 30 min. The membranes were then washed again three times with PBST, before being subjected to enhanced chemiluminescence.g. Supernatants (cytosolic fractions) and pellets (membrane fractions) were boiled in 4× Laemmli sample buffer, and proteins samples were separated and visualized by SDS-PAGE and immunoblotting.HeLa cells plated on 10-cm plates were treated with EHBP1 siRNA or mock transfected (48 h) using Lipofectamine RNAiMAX according to the manufacturer's instructions. Cells were then resuspended with Cellstripper (Corning #25-056-CI) and centrifuged. Cell pellets were resuspended with Homogenization Buffer  containing protease inhibitors  and homogenized on ice. Homogenates were centrifuged for 1 h at 4 °C at 100,000https://imagej.nih.gov/ij/docs/menus/analyze.html#gels. Statistical significance was calculated using a Student's t-test with the Vassarstats program (http://www.vassarstats.net).The adjusted relative density of the immunoblots was measured in Fiji ImageJ based on the method in the following protocol: HeLa cells were grown either on coverslips or 35-mm culture dishes for 24 h in Dulbecco's modified Eagle's medium containing 10% FBS with 2 mM L-glutamine and 1× units/ml penicillin/streptomycin. The cells were then subjected to siRNA treatment with human MICAL-L1 , Syndapin-2 , Rab10 , or EHBP1  oligonucleotides from Dharmacon for 48 h at 37 °C using Dharmafect , following the manufacturer's protocol.MEFs were cultured on coverslips and transfected with either green fluorescent protein or red fluorescent protein-Rab10 using GeneExpresso Max transfection reagent (Excellgen) for 48 h.Red fluorescent protein-Rab10 was a gift from Dr Barth Grant. The PLCδ1 PH domain P2) and MICAL-L1 CC (marker for PA) have been described previously , 51.HeLa or MEF cells were treated as indicated in the text and then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then rinsed three times in PBS and incubated with primary antibody in PBS containing 0.5% bovine serum albumin and 0.2% saponin for 1 h at room temperature. Cells were then washed three times in PBS and incubated with the appropriate fluorochrome-conjugated secondary antibodies diluted in PBS containing 0.5% bovine serum albumin and 0.2% saponin for 30 min. Cells were then washed three times in PBS and mounted in Fluoromount. Z-Stack confocal imaging was performed using a Zeiss LSM 800 confocal microscope with a 63×/1.4 numerical aperture oil objective, and more than 10 cells from three independent experiments were processed using the IMARIS software.t-test with the Vassarstats program (http://www.vassarstats.net). Snapshots and/or videos were obtained from the IMARIS program and used as representative images.Z-Sections of images (8–12 slices) acquired from the confocal microscope were imported into IMARIS x64 9.1.2 software  coupled with custom MAT-LAB (2009 and 2014) programming for 3D surface rendering and quantitative analysis, as indicated. Briefly, the image display was adjusted for both of the channels , and the rendering quality was set to 100%. Surfaces were created by selecting the source channel and smooth surface detail set at 0.198 m. Background subtraction was set to 0.743 m, and the threshold was reduced for surfaces to fully cover all voxels. The surface area and volume of the surfaces generated were quantified by IMARIS for both of the channels, and the values were exported into Excel for graphical and statistical analysis. To quantify apparent surface overlap volume between two surfaces (MICAL-L1 and Rab10), the IMARIS XT bundle Kiss and Run was first integrated with MATLAB and launched in IMARIS. The 3D surface-reconstructed images were then processed for Kiss and Run analysis using the surface–surface overlap module, which uses a surface mask for the target and tracks the surface and determines the overlap for each surface independently. This particular Xtension program analyzes contact events between surfaces that are defined by having at least one overlapping voxel. The volume of overlap for each surface was then quantified and exported to Excel for further analysis. The mean and the SE of the mean were calculated from data obtained from three independent experiments with at least 10 images taken per treatment. Statistical significance was calculated using a Student's Structured illumination microscopy (SIM) images were collected with a Zeiss Elyra PS.1 illumination system (Carl Zeiss) using a 63× oil objective with numerical aperture of 1.4. Two laser lines were used in acquisition of images: 488 and 568 nm. Three orientation angles of the excitation grid were acquired for each Z-plane, with Z spacing of 110 nm between planes. SIM processing was performed with the SIM module of the Zen Black software (Carl Zeiss). The processed SIM images were then exported in TIF format.All data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.