The nanoparticles are synthesized by solvent-induced nanoprecipitation of [Tb2(TCAn)2] complexes  with the use of polystyrenesulfonate (PSS) as stabilizer. The temperature responsive luminescence behavior of PSS-[Tb2(TCAn)2] within 293–333 K range in water is modulated by reversible changes derived from the back energy transfer from metal to ligand (M* → T1) correlating with the energy gap between the triplet levels of ligands and resonant 5D4 level of Tb3+ ion. The lowering of the triplet level (T1) energies going from TCA1 and TCA2 to their brominated counterparts TCA3 and TCA4 facilitates the back energy transfer. The highest ever reported temperature sensitivity for intracellular temperature nanosensors is obtained for PSS-[Tb2(TCA4)2] (SI = 5.25% K−1), while PSS-[Tb2(TCA3)2] is characterized by a moderate one (SI = 2.96% K−1). The insignificant release of toxic Tb3+ ions from PSS-[Tb2(TCAn)2] within heating/cooling cycle and the low cytotoxicity of the colloids point to their applicability in intracellular temperature monitoring. The cell internalization of PSS-[Tb2(TCAn)2]  marks the cell cytoplasm by green Tb3+-luminescence, which exhibits detectable quenching when the cell samples are heated from 303 to 313 K. The colloids hold unprecedented potential for in vivo intracellular monitoring of temperature changes induced by hyperthermia or pathological processes in narrow range of physiological temperatures.The work introduces hydrophilic PSS-[Tb Narrow intensive emission bands and long lifetimes of excited states provide a higher signal-to-noise ratio than that for the organic NPs and quantum dots. Versatility of lanthanide emitters derives from the variation of lanthanide ions, while their ligand environment enables to modify both intensity and response ability of lanthanide-centered luminescence. The temperature dependence of lanthanide-centered luminescence is the well documented basis for development of noninvasive submicrometer luminescent thermometric mapping19. Real-time in vivo and in vitro monitoring of temperature in living cells is of great impact to record temperature fluctuations during magnetic hyperthermia (MHT) or photothermal therapy (PTT), two emergent modalities for thermal treatment of cancer. The inorganic NPs doped by lanthanide ions are widely documented as nanothermometers based on up-conversion luminescence21. Mostly, they utilize the luminescence intensity ratio (LIR) approach19. The presence of the thermally coupled f-levels is the prerequisite for the temperature sensitivity of the inorganic NPs doped by Er22, Tm18, Nd23 and Ho24 ions, where the population distribution of thermally coupled energy levels can be re-distributed with temperature change due to Boltzmann statistics. Temperature responsive behavior of the lanthanide-based NPs is greatly affected by the energy gap between the thermally coupled f-levels of lanthanide ions. Thus, both the nature of the doped or co-doped lanthanide ions and the architecture of the lanthanide-doped NPs are the tools for tuning their temperature sensitivity19. However, the temperature sensitivities (S) defined as the rate of change of the thermo-sensitive parameter with temperature are typically low, usually being below 1% K−1 at the temperatures about 300 K for the major part of the reported lanthanide-doped inorganic NPs28.Lanthanide-based nanoparticles (NPs) have gained growing attention during recent decades due to their divergent application in cellular mapping and sensing. Specific photophysical characteristics of lanthanide compounds constituting the NPs significantly differentiate the latter from polymeric organic nanoparticles and quantum dots29. Greater temperature sensitivities of the molecular complexes, reported as up to 4.9% K−1, make them very promising basis for development of the lanthanide-based thermometers34. It is worth noting that both encapsulation of lanthanide complexes and their deposition onto polymeric nanomaterial  induce some changes in the inner-sphere environment of lanthanide ions, which, in turn, affect the thermal behavior of the complexes. However, the effects of the nanoparticulate hosts on the temperature responsive behavior of the lanthanide complexes are insufficiently studied. Moreover, the ligand environment of the ions commonly applied for these purposes (Tb3+ and Eu3+) is restricted to 1,3-diketonates. This mainly derives from both good luminescence and convenient thermal behavior of the lanthanide diketonates. However, the low solubility of the complexes in specific solvents  affects the efficiency of the doping procedure restricting their use as molecular blocks of silica nanoparticles36.Lanthanide complexes where lanthanide ions are coordinated by organic ligands of different structure represent promising basis for thermo-responsive nanomaterial because of the presence of different mechanisms, with respect to those regulating the thermo-responsive behavior of lanthanide ions in the inorganic matrices6. The main advantage of this morphology is the facile and easy way of conversion of the molecular lanthanide complexes into the aqueous colloids. Moreover, any complexes, which are soluble in any water-miscible organic solvent, but insoluble in water, provide a basis for the transition from molecular to nanoparticulate form. However, this molecule-to-nano transformation can affect both inner- and outer-sphere environment of the terbium ions, which, in turn, changes the Tb3+-centered luminescence and its thermo-sensitivity.The luminescent nanoparticles with core–shell morphology, where the core results from the solvent-induced nanoprecipitation of lanthanide complexes, while the hydrophilic shell derives from the deposition of the polyelectrolytes, represent very promising alternative to the polymeric nanoparticles doped by the lanthanide complexes3+ ions for both efficient Tb3+-centered luminescence and facile conversion into the aqueous luminescent colloids with high colloid stability37. The documented colloids were revealed as efficient fluorescent contrast agents with good cellular uptake behavior4. Moreover, some thiacalix[4]arene derivatives has been recently reported39 as the appropriate ligand environment of Tb3+ ions to generate the Tb3+-centered luminescence exhibiting the temperature responsive behavior in physiological temperature range. In particular, the dimeric 2:2 terbium complexes with the bromo-substituted thiacalix[4]arenes provide a promising basis for design of a temperature-sensitive nanomaterial39. This is the reason for focusing on the optimal conditions of an efficient conversion of the complexes from molecular blocks in the organic solutions to the water dispersed hydrophilic nanoparticles. The identification of main factors, including the structure of the ligands, influencing the temperature responsivity of the Tb3+-centered luminescence of the aqueous colloids based on the terbium complexes with four thiacalix[4]arene derivatives, will be discussed. Thus, the four thiacalix[4]arene derivatives bearing at the upper rims different substituents, including bromines 3·6H2O, Alfa Aesar, 99.9%), gadolinium(III) nitrate hexahydrate (Gd(NO3)3·6H2O, Sigma-Aldrich, 99.9%), triethylamine , D2O  (PSS) , DAPI (Sigma) and sodium chloride (Sigma-Aldrich) were used as received. N,N-Dimethylformamide (DMF) (Acros Organics) was twice distilled over P2O5. Thiacalixarenes TCA140, TCA241, TCA339 and TCA442 were synthesized according to published procedures. Both 1H NMR spectra of the thiacalix[4]arenes and their elemental analysis data  nitrate hexahydrate (Tb(NO3)3·6H2O with TCAn in the 1:1 ratio, and the solution was basified by the excess amounts of triethylamine (TEA) taken in 1:8 (TCAn:TEA) ratio43. The resulted colloidal solution was sonicated for 30 min (sonication bath temperature was controlled to be 20 ± 2 °C). Excess amounts of PSS were separated from colloids via centrifugation  and supernatant drain. One layered PSS-coated aqueous colloids were obtained via their redispersion (using sonication) in bidistilled water .The synthetic procedure is based on the formation of nanosized cores derived from precipitation of water insoluble Tb(III) complexes with ligands TCAn (n = 1–4) with the following adsorption of polystyrenesulfonate (PSS) molecules on their surfaces. The precipitation is induced by the pouring of 0.5 ml of the complex DMF solution under intensive stirring (2200 rpm) 2.5 ml of PSS-containing aqueous solution . The complex formation in the DMF solution was achieved by mixing of the Tb(NO2(TCAn)2] (n = 1–4) was examined by measuring of Tb3+-centered luminescence in the aerated aqueous colloids at different temperatures. The temperature acquisition performed by the water circulation thermostat required 5 min.The thermo-behavior of PSS-[Tb2(TCAn)2]  at different temperatures was performed in the manner shown in Scheme The monitoring of the luminescence intensity and fluorescent microscopy imaging of M-Hela cells stained by PSS-arene derivative (TCA2) has been also introduced in the present work to vary hydrophobicity of calixarene derivatives upon addition of tert-butyl groups to facilitate the solvent-induced aggregation of their complexes and to compare with TCA1.Figure TCAn n = –4 form t3+ ions in the DMF solutions evidences great similarity with TCA2. The steady state and time-resolved Tb3+-centered luminescence of the terbium complex with TCA1 in DMF reveals the insignificant deviation from the 2:2 complex with TCA2  of TCA2 was evaluated by the analysis of the fluorescence and phosphorescence spectra of the isostructural Gd3+ complexes in DMF  are very close to the values recently reported for TCA1 39. This allowed to assume that the sandwich-like structure of [Tb2(TCAn)2] complexes shown in Fig. 2(TCAn)2] colloids.UV–Vis study of the complex formation of TCA1 with TbCA2 Fig. . The eneDMF Fig. . It is w3+ ions and the excess of TEA tend to stay in the aqueous DMF solution due to their better solubility in water. The aqueous colloids resulting from the aggregation can be easily separated from the supernatant by centrifugation. The facile phase separation of the produced colloids provides the way to wash and analyze the Tb3+ in the supernatants. This enables to dispose of any water-soluble admixtures and to evaluate the extent of the Tb3+ complexes conversion from the molecular to the nanoparticulate state. The conversion extent was determined by spectrophotometric analysis of the Tb3+ ions in the supernatants, produced by the first phase separation after the synthesis, and the second one performed within the washing procedure. The percentages of the Tb3+ losses during the synthesis and the washing step are collected in Table 3+ ions during the synthetic procedure are about 3% relative to the initial concentration of the Tb3+ ions in the synthetic mixture, which indicates that the major extent of the complexes is converted from the molecular to the nanoparticulate state. Moreover, the EDS spectrum of the dried PSS-[Tb2(TCA3)2] colloids . The copper constituting of the grid is manifested by the Cu band in the spectrum 2], being about 1.7% for TCA1, while less than 1% for TCA2-4.The Tb2(TCAn)2] (n = 1–3)43, the electrokinetic potential values measured in the aqueous colloids range from − 46 to − 68 mV (Table 2(TCAn)2]-based cores, which are smaller than those measured by the DLS technique according to corresponding size distribution diagrams 2] colloids and their aggregation behavior are the well-known reasons for the deviation in the size-values measured by the different techniques44.Similar to PSS-[Gdn)2] n = –343, then)2] n = –343, then)2] n = –343, the2(TCAn)2] colloids is manifested by four narrow bands peculiar for Tb3+-centered luminescence 2] complexes in the DMF solutions39, the luminescence intensity is the highest for the thiacalix[4]arenes TCA1 and TCA2 compared to the bromo-substituted ligands TCA3 and TCA4. The integral intensities of the four emission bands measured in PSS-[Tb2(TCAn)2] colloids in the same concentration and instrumental conditions presented in Table 5D4 → 7F6) and 543 nm (5D4 → 7F5) are about 0.7 for the PSS-[Tb2(TCAn)2] colloids, which is greater than the ratios 0.56–0.59 previously reported for [Tb2(TCAn)2] in the DMF solutions39. Since the ratio is commonly considered as the factor affected by changes in a ligand environment of terbium ions45, the observed difference can be explained by the hydration effect arisen from the solvent exchange on going from the DMF to the aqueous solutions.The steady state luminescence of PSS-[Tb3+ ions in [Tb2(TCAn)2] shown in Fig. 38. Thus, the measurements of hydration numbers (q) of the Tb3+ ions in PSS-[Tb2(TCAn)2] were performed for confirming the complex stoichiometry. For this reason, in accordance with the well-known procedure46 the excited state lifetimes were measured for PSS-[Tb2(TCAn)2] in both H2O and D2O and reported in Table 46 and colloidal particles44 in H2O and D2O solutions allows calculating the number of coordinated water molecules with the uncertainty in q (number of water molecules) of ± 0.5 by the use of the Horrocks equation  were calculated according to the equation presented in SI along with the hydration numbers (q) presented in Table avg values among the ligands TCAn (n = 1–3). For comparison, the average values of the complexes in DMF are reported in brackets along with those measured in the colloids. The comparison of the lifetime values measured in PSS-[Tb2(TCAn)2] when n = 2, 3 indicates that they are smaller than those measured for TCA1 or previously reported for TCAn (n = 2–4) in the DMF solutions of [Tb2(TCAn)2] 2] (n = 1–3). Thus, the sandwich-like coordination of Tb3+ ions leading to the 2:2 stoichiometry is predominant in these colloids, while the small extent of the monomeric complex forms with the greater hydration number of Tb3+ ions (q = 5)39 cannot be excluded because of the shorter τ values contributing to τavg 2] for TCA4 correlates with the hydration number equal to 3. The greater hydration number revealed in PSS-[Tb2(TCA4)2] versus PSS-[Tb2(TCAn)2] (n = 1–3) can be explained by the increased contribution of the more hydrated monomeric form.The lower average τ-value of PSS-[Tb3+-centered luminescence for PSS-[Tb2(TCAn)2] is worth preceding by discussion of the main factors responsible for the temperature dependent luminescence of [Tb2(TCAn)2] in the solutions. As it has been previously documented39 the triplet energy level of the ligands is of the greatest impact on the thermo-behavior of [Tb2(TCAn)2] in solution. The recently reported invariance of the Tb3+-centered luminescence of complex [Tb2(TCA2)2] in solution upon heating within the range 293–323 K39 results from the higher energy of the triplet levels relative to the resonant 5D4 level of Tb3+ ion . The difference between the energies of these levels in accordance with the Latva rule is enough to provide efficient ligand-to-metal energy transfer and insignificant energy back transfer48. The similarity in the triplet levels for TCA1 and TCA2 in the terbium complexes is the reason for the insignificant change in the luminescence of [Tb2(TCAn)2]  in the solutions within the temperature range 293–323 K. However, the relative intensity of the main band at 545 nm of PSS-[Tb2(TCAn)2]  has a thermal behavior, manifested by the quenching under the heating to 333 K, which is most pronounced for TCA2 2] .The degradation of the colloids can be monitored by measuring the Tbds Table . This va50 can facilitate the degradation of the complexes during the luminescence measurements at different temperatures, where the samples are exposed to the irradiation for a long time (about twenty-thirty minutes). However, PSS-[Tb2(TCAn)2]  should be irradiated within two hours at least for the degradation of the luminescence on 20–30% relative to the initial value, which is much longer than the irradiation time for the heating–cooling cycle. The DLS measurements of the colloids after the heating–cooling cycle confirm the insignificant degradation of the colloids 2] when n = 3, 4 results in more pronounced quenching under the heating from 293 to 333 K 2]  being quenched by the heating to 333 K tends to recover after the cooling to 293 K, although the recovery is incomplete. It is worth noting that the previously reported energies of the triplet levels for ligands TCA3 (440 nm) and TCA4 (458 nm) reveal them as much worse antenna of Tb3+-centered luminescence than TCA1 and TCA2 due to the greater back energy transfer from the excited level of Tb3+ to the triplet level of the ligand39. The lifetime values of PSS-[Tb2(TCAn)2]  tend to decrease by 20% relative to the initial value under the same heating. The thermo-induced decrease in the lifetime values 2]  colloids than for the complexes in DMF solutions. However, the data in Fig. 2(TCAn)2] between n = 3 and 4.The luminescence of PSS-[Tbn)2] n = , 4 beingn)2] n = , 4 beingn)2] n = , 4 being3+-losses after the heating–cooling cycle, being no more than 3.5% relative to the initial concentration of PSS-[Tb2(TCAn)2] , indicates rather poor degradation of the complexes under their treatment within the cycle (Table 2(TCAn)2] becomes greater going from ligands TCA1 and TCA2 to the bromo-substituted ligands TCA3 and TCA4 2] tends to be larger with the increase of the number of the bromo-substituents of the ligand. The photobleaching explains the degradation of Tb3+-centered luminescence after the heating–cooling cycle 2] and PSS-[Tb2(TCA4)2], respectively. Indeed, the aforesaid percentages of the degradation agree with the real time duration (about 20–25 min for more details see the Exp. Section) required for the luminescence measurements during the heating–cooling cycle within 293–333 K. It is worth noting that both nanoparticulate state and aqueous environment are the factors enhancing the photobleaching, since the degradation of [Tb2(TCA4)2] in the DMF solutions after continuous irradiation for a half of an hour is no more than 10% of the initial luminescence intensity39. The literature data on the photo-oxidation of the phenols enhanced by their chloro-substitution50 can be assumed as an explanation of the bromo-substitution effect on the photobleaching. Thus, the upper-rim bromination of the thiacalix[4]arene ligands is the reason for the partially-reversible temperature-induced quenching of PSS-[Tb2(TCA3)2] and PSS-[Tb2(TCA4)2] through the back energy transfer mechanism, although the increased photobleaching contributes to the quenching of the colloids reducing the reversibility of their thermal behavior.The Tble Table . HoweverCA4 Fig. . MoreoveSI, defined by Eq. 2]  colloids and plotted versus temperature in Fig. 2(TCA3)2], and between 4.8–6.2 (heating) and 2.2–4.6 (cooling) for PSS-[Tb2(TCA4)2]. These numbers for SI are higher than the ones reported for inorganic lanthanide-based nanoparticles, being similar to the best ever reported examples and the highest ever reported temperature sensitivity for intracellular temperature nanosensors31. It is worth noting that the SI-values presented in Fig. 2(TCA4)2] results from the contribution of the photo-induced complex transformations. It is worth assuming that high activity of the complexes at the nanoparticle/water interface is the reason for transformations of the complexes under their heating and prolonged irradiation. Nevertheless, these transformations result in the insignificant release of toxic for living cells Tb3+ ions, which can be regarded as a prerequisite of the use of PSS-[Tb2(TCAn)2] as intracellular detectors of temperature changes. Moreover, the extent of the luminescence reversibility after the heating–cooling cycle for PSS-[Tb2(TCA3)2] seems to be promising for the reproducibility of the thermally-driven luminescence changes. The latter is important for monitoring of the intracellular temperature changes within several heating/cooling cycles under a cell treatment by hyperthermia. Taking into account that the temperature range can be reduced to 293–318 K to fit into the physiological temperature interval, the reusability of PSS-[Tb2(TCA3)2] was evaluated in the narrower temperature range 2] n = , 4 collon)2] n = , 4 collo3+-luminescence under the heating and cooling of the aqueous dispersions performed within five heating/cooling cycles. The graph in Fig. n/I0 and In/I(n−1) values versus the number of cycles  designate the luminescence intensities of the initial colloids, and those at the beginning of n- and (n−1)-cycles, respectively) illustrates smaller degradation of the luminescence after the first cycle. This reveals the possibility to run five heating–cooling cycles at least. Moreover, the SI-values calculated for the same temperature conditions (308 K) remain practically unchanged under the running of the cycles 2] colloids can be considered as cellular nanosensors convenient for recurrent monitoring of the temperature changes, while PSS-[Tb2(TCA4)2] nanoparticles with the greater thermo-sensitivity than PSS-[Tb2(TCA3)2] can be applied for single intracellular measurement. Both cytotoxicity and cell internalization of the colloids should be measured before their intracellular application.The data plotted in Fig. les Fig. c. It is 2(TCAn)2] is worth preceding by the discussion of the previously reported data on the PSS-coated colloids based on different lanthanide complexes37. It has been, in particular, already estimated that the PSS-based exterior layer of the lanthanide complexes deposited onto the hard cores is a reason for greater cell internalization versus the same hard cores deposited by PSS-polyethyleneimine (PEI) bilayer, where the PEI-layer is the exterior37. Moreover, the previously published colloids exhibit rather low cytotoxicity, which makes them efficient cellular contrast agents in confocal microscopy imaging of cell samples37. Thus, cell viability measurements of the three cell lines incubated by the colloids has been performed. The results are presented in Fig. The presentation of the cellular uptake and cytotoxicity of PSS-[Tb2(TCA3)2] and PSS-[Tb2(TCA4)2] is observed. In particular, the IC50 values of PSS-[Tb2(TCA3)2] evaluated for M-Hela, Chang liver and HSF cell lines are 380, 300 and 340 µM respectively, while the same values for PSS-[Tb2(TCA4)2] are above 380 µM.It is worth noting that both colloids exhibit rather low cytotoxicity, although some difference in the cytotoxic effect of PSS-[Tb2(TCAn)2]  colloids under various incubation time indicate the cell internalization of the colloids. Moreover, the data reveal the gradual growth of the cell internalization (derivative of measured luminescence intensity) under the increased incubation time from one to eight hours. The fluorescence microscopy images 2] ) into M-Hela cells.The flow cytometry measurements Fig. d,e perfoes Figs. ,S6 illus2(TCA3)2] and PSS-[Tb2(TCA4)2] into M-Hela cells was visualized by both fluorescence microscopy and confocal images of the cell samples incubated by the colloids. Figure 2(TCA4)2] and confocal microscopy for the cell samples incubated for 24 h. No detectable difference between the colloids is revealed in their cell internalization. It is worth noting that the confocal image with the staining of the cell nuclei by the dye  details the location of PSS-[Tb2(TCA4)2] within cell cytoplasm. Moreover, Fig. 5c indicates that the nanoparticles are in close proximity to the cell nuclei, but do not enter them. The efficient cell internalization of the PSS-coated colloids seems to disagree with their high negative surface charge manifested by the ζ-values in Table 51. The protein-corona, in turn, is the well-known factor modifying the cell internalization of different types of nanoparticles52, which has been previously exemplified for the silica nanoparticles bearing sulfonate-groups at their surface53. Thus, efficient cell internalization of the PSS-coated colloids manifested by the marking of the cell cytoplasm provides a basis for their use as intracellular transducers of the temperature changes.The internalization of PSS-arenes with four tert-buthyl-, two or four bromo-substituents. The synthesis was based on solvent-induced nanoprecipitation of the Tb3+ complexes, while a hydrophilic coating of the cores by polystyrenesulfonate (PSS) molecules provides their colloid stability. The hydration number equal to 2 was evaluated by the time-resolved luminescence measurements for the nanoprecipitated complexes with the unsubstituted, tert-buthyl-substituted and dibromo-substituted ligands. This indicates that specific dimeric complexes with sandwich-like coordination mode of Tb3+ ions, which are predominant in the DMF solutions, remain unchanged after the nanoprecipitation. The greater (close to 3) hydration number obtained for the complexes with tetra-brominated thiacalix[4]arene can be explained by a contribution of the monomeric complexes with the greater hydration number of Tb3+ ions. The bromo-substitution of thiacalix[4]arene causes the quenching of the Tb3+-centered luminescence due to the energy back transfer, which increases with the heating in the temperature range 293–333 K. The deviation of the luminescence intensities measured under the heating and cooling of the colloids indicates that the temperature-dependent luminescence of the colloids has a contribution coming from the irreversible transformation of the complexes. Thus, the greatest irreversible contribution to the heating-induced quenching of the luminescence makes the nanoparticles based on the complexes with tetra-brominated thiacalix[4]arene the most sensitive to the heating, but restricts their applicability to the single use only. However, the di-brominated ligands provide the optimal ligand environment of the Tb3+ ions in the nanoparticles for the recurrent temperature measurements for five heating/cooling cycles at least. The heating/cooling cycle results in the insignificant release of toxic Tb3+ ions from the nanoprecipitated complexes with the di-brominated thiacalix[4]arenes. This along with the low cytotoxicity of the PSS-coated colloids estimated for the three cell lines points to their applicability as intracellular sensors of temperature changes. The quick cell internalization of the colloids results in the efficient marking of the cell cytoplasm by green luminescence. The latter exhibit detectable quenching under the heating of the cell samples in the temperature range within 303–313 K. This study demonstrates the successful application of complex colloidal structures for monitoring the intracellular temperature changes with unprecedented sensitivity, and may represent a significant step forward for accurate 2D temperature mapping in processes like hyperthermia or pathological processes.The work introduces synthesis of hydrophilic core–shell nanoparticles exhibiting thermo-responsive TbSupplementary Information.


Cell proliferation was significantly accelerated by knockdown of CLIC3 in MKN7 cells. On the other hand, the proliferation was attenuated by exogenous CLIC3 expression in human gastric cancer cells (KATOIII and NUGC-4) in which endogenous CLIC3 expression is negligible. Our results suggest that CLIC3 functions as a Cl− channel in the plasma membrane of gastric cancer cells and that decreased expression of CLIC3 results in unfavorable prognosis of gastric cancer patients.Pathophysiological functions of chloride intracellular channel protein 3 (CLIC3) in human gastric cancer have been unclear. In the tissue microarray analysis using 107 gastric cancer specimens, CLIC3 expression was negatively correlated with pathological tumor depth, and the patients with lower expression of CLIC3 exhibited poorer prognosis. CLIC3 was expressed in the plasma membrane of cancer cells in the tissue. CLIC3 expression was also found in a human gastric cancer cell line (MKN7). In whole-cell patch-clamp recordings of the cells expressing CLIC3, NPPB-sensitive outwardly rectifying Cl Gastric cancer is one of the most common malignant tumors in the abdominal region , 2. Vari2+-activated Cl− channel, also contributes to tumor invasion and poor prognosis of human gastric cancer i and reduced cell proliferation with G0/G1 arrest [−]i affects the anti-cancer activity of paclitaxel, a microtubule-targeted chemotherapeutic drug, in MKN28 cells [−]i also attenuates cell proliferation of prostate cancer PC3 cells [− homeostasis in gastric cancer cells, resulting in the enhancement of cancer cell growth. Thus, the plasma membrane expression of gastric CLIC3 may explain why CLIC3 function in gastric cancers is different from other cancers. Future study is necessary to clarify the mechanism of regulation of CLIC3 expression in gastric cancer cells.The activities of Cl1 arrest . Interes28 cells . A decreC3 cells . ChangesOur study clarifies that CLIC3 has a channel activity in the plasma membrane of gastric cancer cells, and that decreased expression of CLIC3 results in unfavorable prognosis of gastric cancer patients via stimulation of the cancer cell proliferation. These findings suggest that pathophysiological role of CLIC3 in plasma membrane is different from that in cytosol and organellar membrane of the cancer cells.Additional file 1: Table S1. Expression of CLIC3 and clinicopathological characteristics patients with gastric cancer.Additional file 2: Fig. S1. Enlarged image of tissue microarray analysis  from − 100 mV to + 100 mV in the CLIC3-expressing HEK293T cells exposed to standard bathing solution (control: red) and the low Cl− bathing solution (blue).


Plasmodium falciparum parasite population diversity from two sites with low (pre-elimination) and high transmission in Senegal and Nigeria, respectively.Characterizing the genetic diversity of malaria parasite populations in different endemic settings (from low to high) could be helpful in determining the effectiveness of malaria interventions. This study compared P. falciparum malaria infected patients in Senegal (94) and Nigeria (93). Allelic polymorphism at merozoite surface protein 1 (msp1) and merozoite surface protein- 2 (msp2) genes were assessed by nested PCR.Parasite genomic DNA was extracted from 187 dried blood spot collected from confirmed uncomplicated msp1 and msp2 allelic families are the K1 and IC3D7 allelotypes in both Senegal and Nigeria. Multiplicity of infection (MOI) of greater that 1 and thus complex infections was common in both study sites in Senegal  than in Nigeria . The heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal  than isolates from Nigeria . In Senegal, K1 alleles was associated with heavy than with moderate parasite density. Meanwhile, equal proportions of K1 were observed in both heavy and moderate infection types in Nigeria. The IC3D7 subtype allele of the msp2 family was the most frequent in heavily parasitaemic individuals from both countries than in the moderately infected participants.The most frequent The unexpectedly low genetic diversity of infections high endemic Nigerian setting compared to the low endemic settings in Senegal is suggestive of possible epidemic outbreak in Nigeria. Plasmodium falciparum continues to be a significant public health havoc in many endemic countries in tropical and sub-tropical parts of the world  [(1 − ΣPi2)], where n = sample size, Pi = allele frequency as described by . A p valA total of 187 malaria infected participants were recruited for the study with almost equal proportion from the two countries . The mean age was not different from each country and locality within the countries. Although, participants were randomly enrolled into the study, however, more males (108) partook in the study than females (79)  infections, K1/RO33 combination was the most frequent in both countries but higher in Senegal (17%), and Nigeria (7%). However, the K1/MAD20/RO33 trimorphic allelic infections were least frequent; Senegal (3%) and Nigeria (2%).For P. falciparum isolates from Nigeria (190-300 bp). Similar pattern was also observed with the MAD20 alleles, where sizes in Senegal ranged from 100-300 bp while those from Nigeria ranged from 200 to 300 bp. In addition, a different pattern was observed in RO33 where only one clone type was seen in Nigeria (160 bp) as against multiple clones in Senegal (160–240 bp)  was different from that seen in msp2 gene, there was no difference in the frequency of isolates with IC3D7 allele in Senegal (57%) and Nigeria (58%), while the FC27 frequency was more prevalent in Senegal (26%) than in Nigeria (16%). Similar pattern was noticed with the IC3D7/FC27 dimorphic allelic family in both countries. Allele sizes of the IC3D7 variant of msp2 ranged from 300 to 700 bp in Senegal and 300–800 bp in Nigerian P. falciparum isolates, while FC27 alleles varied in size from 200 to 600 bp in Senegal and 290–550 bp in Nigeria  than the sites in Nigeria . Consequently, the heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal  than isolates from Nigeria . However, the heterozygosity of msp2 gene was not different for the two countries  from both countries. The K1 allele was more prevalent in individuals with higher parasitaemia infections (28) than those with moderate infections (18) from Senegal. However, in Nigeria, equal proportions of K1 were present in both moderate and high parasitaemic infections. Individuals with heavy infections from Senegal showed the highest proportion of K1/MAD20 (14) allele combination and same pattern was observed for the K1/RO33 mixture (13).The distribution of various allele types in both msp2 family showed high occurrence in high parasitaemic individuals from both countries  than in the moderately infected participants. Similar pattern was noticed with the FC27 allele  index which is related to the number of clones per infection and usually associated with the level of malaria transmission [msp1 and 2 genes were observed for both study locations in Senegal, but moderate MOI values were noted in Nigeria. Similar results for Senegal were reported by Niang et al. in 2017 [Control interventions targeting malaria parasite continues to face multiples hurdles from development of drug resistance in the parasite, insecticide resistance by the vectors, and unavailability of a reliable vaccine to confer the necessary protection against the parasite. Hence, there is need for continuous monitoring and evaluation of the effectiveness of control measures. Here, the diversity of smission –29 was amsp1 was more diverse in infections from Senegal than those from Nigeria. However, similar levels of diversity for msp2 was observed in both countries. This is in line with the differences in MOI probably due to differences in urbanization levels and recombination between genetically different clones. Indeed, there was a difference in the clonal structures of all allelic families  from both countries with Senegal showing the more sub-structured msp1 and msp2 populations. Nevertheless, similar trends has been observed in the Kingdom of Eswatini where a high genetic diversity was obtained in a low transmission area [Based on heterozygosity, which measures the level of genetic diversity at polymorphic loci, ion area . Circulamsp1 K1 allele in high and low parasite infections from both countries has been previously observed by various studies [The high occurrence of  studies , with th studies , 32, wes studies  and Iran studies .msp2 gene was also observed with both grades of parasite density, while no particular pattern was noticed with the FC27 allelotype. This finding is in agreement with the study of Hamid et al. in 2013 [Similarly, high frequency of IC3D7 allele type of  in 2013  where ICP. falciparum individuals should be evaluated in a bidirectional manner and a more holistic approach should be employed in determining the true epidemiological situation of any malaria endemic country as this will help in a more targeted control measure.Taken together, it can be concluded that evidence driving selection of these observed allelotypes in moderate and heavy Additional file 1: Table S1. Sequences of the primers used to amplify the msp 1 and msp 2 genes of P. falciparum isolates.


Research found that post-selenization treatments are essential to produce stoichiometric thin films with desired crystallinity and orientation for the sputtered Sb2Se3. However, the influence of the sputtering process on Sb2Se3 device performance has rarely been explored. In this work, the working pressure effect was thoroughly studied for the sputtered Sb2Se3 thin film solar cells. High-quality Sb2Se3 thin film was obtained when a bilayer structure was applied by sputtering the film at a high (1.5 Pa) and a low working pressure (1.0 Pa) subsequently. Such bilayer structure was found to be beneficial for both crystallization and preferred orientation of the Sb2Se3 thin film. Lastly, an interesting power conversion efficiency (PCE) of 5.5% was obtained for the champion device.Magnetron sputtering has become an effective method in Sb It can be seen that although other devices presented decent OCV values, the final PCEs of these devices were restricted by the low FF and especially the poor SCJ, which were far inferior to that of the champion mixed-Sb2Se3 device. We attribute the unsatisfied SCJ of the devices sputtered at low working pressures (below 1 Pa) to the shunt paths caused by the pinholes within the films, whilst the low SCJ of the devices sputtered at elevated working pressures (above 1.5 Pa) could be attributed to the insufficiently thick absorber layer at such high working pressures, as shown in SEM images. In addition, the undesired grain orientations of other films  prepared under improper working pressures would increase the series resistance of the devices and thus lead to poor FF and SCJ [2Se3 device was much higher than that of the other samples in the wavelength region from 500 nm to 850 nm, suggesting less recombination losses of photogenerated carriers not only within the bulk film but also at the Sb2Se3/CdS heterojunction [The current density–voltage  and a low working pressure (1.0 Pa) subsequently. Such bilayer structure was found to be beneficial for both crystallization and preferred orientation of the Sb2Se3 thin film. Further, shunt leakages induced by pinholes within the bulk film could be effectively hindered by using the bilayer structure. Finally, an interesting PCE of 5.5% was obtained for the champion device. In summary, the influence of working pressure on the device performance of sputtered Sb


After this article  was publSpecifically:○37 and 39 degree panels for HepG2○37 and 39 degree panels for Huh7The following panels in Fig 1A appear to report overlapping data:Similarities were noted between Tubulin data reported in Fig 1B  and Fig 6C (lanes 1–4). The similar lanes represent samples exposed to different experimental conditions.The corresponding authors commented that the invasion results for HepG2 and Huh7 cells were highly similar in the 37 and 39 degree groups. The original data underlying the results reported in Fig 1A are no longer available.The authors provided available image data to support the results reported in Figs 1B and 6C, but these data did not resolve the concerns. The MMP-2, MMP-9, and Tubulin data reported in Fig 6C appear to match image data provided in support of Fig 1B, albeit with different blot and lane labels, suggesting that all three panels of Fig 6 may have reported the wrong data. None of the image data provided appear to match the results reported in Fig 1B. The authors commented that no additional western blot data are available currently.PLOS ONE Editors retract this article.In light of the unresolved concerns, the LY, JZ and WL agreed with retraction and apologize for the issues with the published article. The other authors either could not be reached or did not respond directly.


KCNQ1 gene. In rare cases, LOM of IC2 has been reported in families with KCNQ1 germline variants which additionally cause long-QT syndrome (LQTS). Thus, a functional link between disrupted KCNQ1 transcripts and altered IC2 methylation has been suggested, resulting in the co-occurrence of LQTS and BWS in case of maternal inheritance. Whereas these cases were identified by chance or in patients with abnormal electrocardiograms, a systematic screen for KCNQ1 variants in IC2 LOM carriers has not yet been performed.Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by disturbances of the chromosomal region 11p15.5. The most frequent molecular finding in BWS is loss of methylation (LOM) of the Imprinting Centre 2 (IC2) region on the maternal allele, which is localised in intron 10 of the KCNQ1 by MLPA and an amplicon-based next generation sequencing approach. We identified one patient with a splice site variant causing premature transcription termination of KCNQ1.We analysed 52 BWS patients with IC2 LOM to determine the frequency of germline variants in KCNQ1 transcription is required for the establishment of IC2 methylation, but that KCNQ1 variants cause IC2 LOM only in a small number of BWS patients.Our study strengthens the hypothesis that proper  Beckwith-Wiedemann syndrome  is a congenital imprinting disorder which was first described by Beckwith and Wiedemann in 1963 . Clinicainsulin-like growth factor 2 (IGF2) and H19 genes. The Imprinting Centre 2  controls the expression of the genes KCNQ1, KCNQ1OT1 and CDKN1C. In contrast to the IC1, the IC2 is maternally methylated leading to an expression of KCNQ1 and CDKN1C and a repression of KCNQ1OT1 on the maternal allele, while it is unmethylated on the paternal allele resulting in silencing of KCNQ1 and CDKN1C and expression of KCNQ1OT1  but more common in familial cases of BWS (20–40%) [The most frequent molecular cause of BWS is loss of methylation (LOM) of the IC2 (50–60% of patients) causing a biallelic expression of ed genes . Furthered genes ). Point (20–40%) . An epig(20–40%) .CDKN1C [KCNQ1 gene [KCNQ1, either by CNVs or single nucleotide variants (SNVs), causes a LOM of the IC2 [KCNQ1 and its regulatory elements, but not the IC2 itself. In mice, a maternally inherited poly(A) cassette, inserted into Kcnq1, terminates the transcription of Kcnq1 upstream of IC2 and results in LOM of the IC2, biallelic expression of Kcnq1ot1 and silencing of Cdkn1c and Kcnq1 [BWS mostly occurs sporadically, but in addition to inherited variants in CDKN1C , familiaCDKN1C , among tNQ1 gene –13. Some the IC2 –16. Remand Kcnq1 .KCNQ1 are the most frequent causes for congenital long-QT syndrome (LQTS) [KCNQ1 variant have a prolonged QT interval. Vice versa, 10% of the genetically affected patients who do not have a prolonged QT interval can be affected by a cardiac event until the age of 40 years [KCNQ1 variants but remained undetected so far due to the reduced penetrance of LQTS variants. Up to now, six LQTS families have been described in which altered KCNQ1 transcripts lead to BWS in combination with LQTS when inherited from the mother or to isolated LQTS when transmitted from the father [KCNQ1 variant and can therefore be explained by the biallelic expression of KCNQ1 in the heart but monoallelic expression in other tissues [KCNQ1 variants in BWS patients associated with IC2 LOM is missing.Loss-of-function mutations in e (LQTS) , a herede (LQTS) ). The pe40 years . It is ce father , 21–23. KCNQ1 among BWS patients with IC2 LOM.In our study, we therefore aimed to determine the frequency of pathogenic germline variants in KCNQ1 by multiplex ligation-dependent probe amplification (MLPA) did not reveal any deletion or duplication in the cohort of 52 BWS patients with IC2 LOM.Copy number analysis of the coding sequence of KCNQ1 was detected in one patient. The variant was located in the donor splice site of intron 1 of KCNQ1 . The change was also detected in the mother but not in the father. The variant affects the canonical donor splice site of intron 1   approach, heterozygosity for a splice variant in The boy was born at 26 weeks of gestation by caesarean section due to premature placental separation and preeclampsia with a birthweight of 950 g (50th percentile), a length of 33 cm (30th percentile) and a head circumference of 23 cm (15th percentile). He showed marked macroglossia, cleft palate, umbilical and inguinal hernias, hypospadias, ear lobe creases and a nevus flammeus in his face. He suffered from chronic respiratory insufficiency due to bronchopulmonary dysplasia as a consequence of his prematurity. He underwent multiple surgeries during the first months of life, including reduction of macroglossia. He showed a severe feeding disorder and required tube feeding for several years. His psychomotor development was significantly delayed putatively due to his premature birth. At the age of 3 years, he was able to walk without support, and he started speaking a few words at the age of 7 years. On the last examination at the age of eight and a half years, he was attending a school for special needs. His vision was restricted because of retinopathy of prematurity, and he needed regular controls for a nephrocalcinosis °I–II.Clinical diagnosis of BWS was confirmed by molecular detection of IC2 LOM at the age of 2 months. Molecular karyotyping did not reveal any CNVs (resolution of 50 kb). He had one healthy brother. His mother was healthy as well and did not show any BWS features. MLPA of the mother showed a normal methylation pattern.KCNQ1 and revealed a borderline prolonged QTc interval of 440–450 ms. The long-term ECG showed normal findings but sporadic extrasystoles. The mother’s ECG revealed no pathologic findings.An electrocardiography (ECG) of the patient was performed after the results of the molecular analysis of KCNQ1 germline variants, we identified one case with a maternally inherited splice site variant (c.386+1G>T) affecting the splice donor site of intron 1 of KCNQ1. A variant at the same position with another base pair substitution (c.386 +1G>C) has been described by Valente et al. [KCNQ1 transcript. The resulting monoallelic expression of KCNQ1 could explain the LQTS phenotype as KCNQ1 is expressed biallelically in the heart [By analysing 52 BWS patients with IC2 LOM for e et al.  in a BWSe et al. . The authe heart . In tissKCNQ1 upstream of IC2 results in LOM. A deletion in KCNQ1 has been reported that does not include the IC2 but causes LOM, suggesting that transcription of the entire KCNQ1 gene is required for the establishment of the imprinting mark at the IC2 locus [KCNQ1 upstream of IC2 which leads to an IC2 LOM and BWS symptoms when inherited from the mother but to a normal phenotype in case of paternal inheritance [KCNQ1 results in a LOM of the IC2 and BWS when maternally inherited [KCNQ1 and one with a 160-kb KCNQ1 duplication. Both patients were affected by LQTS and BWS due to an IC2 LOM [Five further cases corroborate the hypothesis that interrupted transcription of C2 locus . Demars eritance . In a thnherited . Valente IC2 LOM . In our  IC2 LOM . The borIn fact, functional analyses have not been conducted but the effect of the splice site variant can be delineated from findings published recently by Valente et al. .KCNQ1 variants in BWS patients with IC2 LOM have not yet been performed. Demars et al. screened 78 BWS patients with IC2 LOM for CNVs and discovered one patient with a duplication including exon 2 and parts of intron 1 and 2 of KCNQ1. However, they did not perform ECG examinations [KCNQ1 mutation carriers without a QT prolongation [KCNQ1 germline variants and multiple miscarriages [KCNQ1 [KCNQ1 gene. However, our screening report has some technical limitations. The used MLPA kit does not cover exon 5 of KCNQ1 . In case of therapy refractoriness, left cardiac sympathetic denervation or an implantable cardioverter defibrillator (ICD) is necessary. Also, the patients should avoid competitive sports (e.g. swimming) and the intake of QT interval prolonging drugs (for review: ). The prKCNQ1 variants. The study was approved by the ethical committee of the Medical Faculty of the RWTH Aachen University (EK303-18).We investigated 52 BWS patients carrying an IC2 LOM. The molecular diagnosis had been confirmed by methylation-specific multiplex ligation-dependent probe amplification  according to the manufacturer’s instructions. Multilocus imprinting disturbances (MLID) were excluded by screening with a MS-MLPA-based multilocus assay . Patients with MLID were excluded as they are probably caused by other (genomic) alterations than by KCNQ1 gene by MLPA  following the manufacturer’s instructions.The patient’s DNA samples were analysed for deletions or duplications of the KCNQ1 by an amplicon-based next generation sequencing approach. First, the exons of the whole coding region of KCNQ1 were amplified by polymerase chain reaction (PCR); primers and PCR conditions are listed in All patients were screened for SNVs in Additional file 1: Supplementary tables. Primer sequences and PCR conditions for the amplicon-based NGS analysis of KCNQ1.


PSD95, DLGA, ZO-1) domains, to an extraordinarily large number of other proteins besides NUMB. Many of these interactions suggest additional roles for LNX1/2 proteins in the nervous system in areas such as synapse formation, neurotransmission and regulating neuroglial function. Twenty years on from their initial discovery, I discuss here the putative neuronal functions of LNX1/2 proteins in light of the anxiety-related phenotype of double knockout mice lacking LNX1 and LNX2 in the central nervous system (CNS). I also review what is known about non-neuronal roles of LNX1/2 proteins, including their roles in embryonic patterning and pancreas development in zebrafish and their possible involvement in colorectal cancer (CRC), osteoclast differentiation and immune function in mammals. The emerging picture places LNX1/2 proteins as potential regulators of multiple cellular signalling processes, but in many cases the physiological significance of such roles remains only partly validated and needs to be considered in the context of the tight control of LNX1/2 protein levels in vivo.Ligand of NUMB Protein X1 and X2 (LNX1 and LNX2) are E3 ubiquitin ligases, named for their ability to interact with and promote the degradation of the cell fate determinant protein NUMB. On this basis they are thought to play a role in modulating NUMB/NOTCH signalling during processes such as cortical neurogenesis. However, LNX1/2 proteins can bind, via their four PDZ ( Ligand of NUMB Protein-X) protein family evolved from a common ancestral protein that contained a RING  domain and four PDZ  domains [PDZ and RING). Mammalian LNX1 and LNX2 were named for their ability to interact with NUMB and have retained the ancestral RING plus four PDZ domains structure [Lnx1 gene by McGlade et al., this focus allows me to comprehensively summarize the published literature on these intriguing but incompletely understood proteins [The LNX , the NUMB-binding NPAY/F motif, four PDZ domains and a carboxyl terminal PDZ binding motif  Figure . A multiLNX1/2-like gene is present in some, but not all, invertebrate lineages [LNX1/2 orthologues, whereas those in Nematoda and Arthropoda do not. This indicates that LNX1/2 proteins are not essential in all invertebrate lineages. In the vertebrate lineage, duplication of an ancestral LNX1/2-like gene gave rise to LNX1, LNX2 and LNX2b. These three genes seem to be conserved in all vertebrate lineages with the exception of the loss of LNX2b in eutherian mammals. This loss of LNX2b is quite interesting in that the LNX2b pseudogene contributed several exons to the non-coding Xist RNA that control X-chromosome inactivation in eutherian mammals [A  mammals .LNX1/2-like genes in all vertebrate lineages suggests the acquisition of essential vertebrate-specific functions. Intriguingly, vertebrate LNX1/2 proteins have either an NPAY (LNX1) or NPAF  NUMB-binding motif, whereas the invertebrate proteins lack this motif [LNX1/2 orthologues in all vertebrates. Conversely however, this same observation tells us that the ancestral LNX1/2 protein probably evolved to perform cellular function(s) not related to an interaction with NUMB. It seems likely that vertebrate LNX1/2 proteins would also have retained some of these functions. Thus an important lesson from examining the evolutionary history of LNX1/2 proteins is that, despite their name, they should not be considered a ‘one-trick pony’ and are likely to have significant functions independent of their regulation of NOTCH signalling via NUMB.The maintenance of at least two The X-ray crystallographic structure of the LNX2 RING domain was determined by Nayak and Sivaraman in 2015  and provin vitro ubiquitination assays. They also showed for the first time that LNX2 could indeed ubiquitinate NUMB, something that had previously only been shown for LNX1. LNX2 autoubiquitination diminished but did not abolish this activity. Another finding from their study was that the ZnF-RING-ZnF LNX2 construct was present as a dimer in both the crystal structure and in solution. However, dimerization was not required for LNX2 autoubiquitination activity.Using LNX2 autoubiquitination as a readout of E3 ubiquitin ligase activity Nayak and Sivaraman  saw thatHaving determined the structure of the LNX2 RING domain with its flanking ZnF motifs, Nayak and Sivaraman  have rechttp://www.rcsb.org). Specifically, structures of mouse LNX1 PDZ2 , human LNX1 PDZ3 in complex with a peptide ligand from the coxsackievirus and adenovirus receptor (CAR) (PDB ID: 3B76) and human LNX2 PDZ2  have been submitted to the PDB. Most of these structures are not associated with any journal publication, though interestingly LNX2 PDZ2 was used in a recent, highly innovative study in which the application of strong electric field pulses to protein crystals was combined with time-resolved X-ray crystallography in order to probe the internal protein mechanics of a PDZ domain [Apart from the RING domain, structures of a number of individual PDZ domains are available in the Protein Data Bank  . This brThe fact that yeast two-hybrid screens failed to identify any peptides that bind to PDZ4 from either LNX1 or LNX2  is notewWhile we have learned a lot from the study of individual LNX1/2 domains, a structure of a full-length LNX1/2 protein would now be extremely informative. Such a structure would resolve the nature of PDZ1-mediated intra- and/or intermolecular interactions of LNX1/2 proteins. It would hopefully also reveal how the 3D arrangement of PDZ domains in a full-length LNX1/2 protein might orient ligands of specific PDZ domains as potentials substrates for ubiquitination by the ZnF-RING-ZnF domain.LNX1 and LNX2, the expression of lnx2b in zebrafish is discussed separately below. Northern blot analysis revealed expression of murine Lnx1 mRNA in adult heart, brain, lung, skeletal muscle and kidney with little or no message detected in spleen, liver and testis [LNX1 mRNA was likewise detected in adult heart, brain and kidney as well as the placenta and pancreas, whereas much lower levels were detected in lung, liver and skeletal muscle [Lnx1 mRNA seems to be expressed in many adult tissues. Notably, both these and subsequent studies [Lnx1 transcripts are expressed in the brain and spinal cord compared with other tissues. These transcripts are predicted to give rise to 70 and 62 kDa proteins that lack the ZnF-RING-ZnF domain present in the 80 kDa protein that is expressed in other tissues  that lack the catalytic ZnF-RING-ZnF region is intriguing. The p70 isoform is found across diverse vertebrate species and the short amino terminal sequence unique to this isoform is highly conserved . In addiLNX1/2 mRNA and protein levels in vivo have significant implications for interpreting reports on the role of LNX1/2 proteins in disease, and the physiological relevance of the many interactions of LNX1/2 proteins that have been identified to date. The seemingly tight control of LNX1/2 protein levels suggest that LNX1/2 proteins (i) may have general functions in the many cell types, but that they are only required at extremely low levels or (ii) have very specific roles in certain cell types in which they are expressed at higher levels but in a temporally or spatially restricted manner. The fact that LNX1/2 proteins are normally present in vivo at relatively low levels must be kept in mind when interpreting experiments that rely heavily on LNX1/2 overexpression.Overall the low expression levels of LNX1 and LNX2 proteins, and the possible lack of correlation between in vivo is not known, so the physiological relevance of a LNX1–SHC interaction is unclear.LNX1 was first identified in a yeast two-hybrid screen to identify proteins that bind to the PTB domain of murine NUMB . The intThe ubiquitin ligase activity of LNX1 was confirmed and NUMB was shown to be a substrate for LNX1-mediated ubiquitination . UbiquitNUMB interacts with the NOTCH receptor and can negatively regulate NOTCH signalling in several contexts. NUMB is thought to achieve this inhibition of NOTCH signalling by modulating either the endocytosis of NOTCH or the trafficking of NOTCH post-endocytosis, however the exact mechanism is a matter of debate . Given NHes1 [Hes5 is unaffected and rather point to other functions of NUMB to explain the phenotypes observed in these mice [Importantly, some evidence that a loss of LNX1/2 function can lead to altered NOTCH signalling has emerged more recently. In zebrafish embryos expressing a dominant negative form of LNX2 it was found that NUMB levels were increased and the number of cells exhibiting NOTCH signalling was decreased, as assessed using a transgenic fluorescent protein-based reporter . In a diHes1 . Howeverese mice . In anotese mice .These observations make it clear that relating increased or decreased LNX1/2 protein expression to alterations in NOTCH signalling in a given context is not straightforward. Firstly, LNX1/2 may or may not affect NUMB levels depending on the particular isoform(s) of NUMB present; secondly, redundancy of function between LNX1/2 isoforms and between NUMB and NUMB-like need to be considered and thirdly, even if NUMB levels are altered this could affect pathways other than NOTCH signalling. Finally, it is possible that LNX1/2 may modulate NUMB and/or NUMB-like function independently of triggering their degradation, for example by altering NUMB or NUMB-like subcellular localization. Nevertheless, LNX1/2 proteins certainly can act as activators of NOTCH signalling through the ubiquitination of NUMB, but this mechanism must be carefully examined in each cellular context for the reasons outlined above.NUMB functions as a cell fate determinant in many types of neural progenitor cells, as well as in non-neural cells. During neurogenesis asymmetric localization of NUMB in dividing neural progenitors causes it to partition predominantly into one of the daughter cells—specifying a distinct fate for this cell by antagonizing NOTCH signalling . For exa−/− mice defects in the specification of ependymal cells were observed. These mice exhibit dramatically decreased levels of NUMB protein, which may at least in part explain the phenotype observed. Intriguingly, Gli3−/− mice have increased LNX2 levels, particularly in the SVZ, leading the authors to speculate that LNX2 overexpression in Gli3−/− mice may target NUMB for proteolytic degradation. While this mechanism seems plausible, a causal relationship between LNX2 and NUMB levels was not definitively demonstrated. Nevertheless, this is an interesting finding that begs the question of whether LNX2 plays a role in normal SVZ development beyond this proposed role in Gli3−/− animals.While examining knockout mice lacking the Sonic hedgehog signalling component GLI3, Wang et al.  uncovereThe interaction of contactin-associated protein-4 (CASPR4) with LNX2 has also been proposed to play a role in neuronal differentiation . CASPR4 A distinct role for LNX1/2 proteins in cortical neurogenesis has been proposed based on their interaction with SHOOTIN1 . SHOOTINin vivo during normal brain development. Notably, gross brain morphology was found to be normal and no changes in NUMB levels were observed in mice lacking both LNX1 and LNX2 in the nervous system [The examples above indicate several potential roles for LNX1/2 proteins in regulating neurogenesis and neuronal differentiation and fit with the well-established role of NUMB in such processes. However, none could be considered definitive proof of such a role s system . FurtherLnx1 and Lnx2 mRNAs are significantly up-regulated during osteoclast differentiation in vitro. To probe the requirement for LNX2 the authors employed lentiviral transduction of shRNAs to knockdown LNX2 expression in bone marrow derived macrophages. This drastically decreased osteoclast formation in vitro and increased apoptosis of pre-osteoclasts. LNX2 knockdown also dampened the activation of the signalling pathways that promote osteoclast formation, namely M-CSF induced ARK and AKT activation, as well as RANKL induced NF-κB and JNK activity. Notably LNX2 knockdown caused an increase in NUMB levels and decreased levels of the NOTCH2 intracellular domain, as well as decreased expression of the NOTCH target gene Hes1. NOTCH signalling is known to play multiple roles in maintaining bone homoeostasis and controlling osteoclast differentiation [in vitro differentiation system can be confirmed in vivo, perhaps by examining osteoclast differentiation and homoeostasis in detail in Lnx2 knockout mice.A possible role for mammalian LNX1/2 proteins in the differentiation of osteoclasts that seems to involve the modulation of NOTCH signalling via NUMB has recently been described . This inntiation ,48. ThesSeveral hundred proteins have been described as potentially interacting with LNX1/2 proteins and the overwhelming majority of these interactions are mediated by the LNX1/2 PDZ domains ,13,18,20The cytoplasmic domains of several components of epithelial tight junctions including CLAUDINS ,51, juncCLAUDINs comprise a large family of transmembrane proteins involved in the formation of tight junctions that maintain epithelial and endothelial barrier functions. CLAUDIN-1 cytoplasmic domain was found to interact with LNX1 via its carboxyl terminal PDZ binding motif . LNX1 ovJAM4 is a member of the immunoglobulin superfamily of adhesion molecules that localizes to tight junctions. JAM4 was shown to interact via its carboxyl terminal PDZ binding motif with PDZ2 of LNX1 and LNX1 was seen to partly co-localize with JAM4 and other tight junction components in epithelial cells . LNX1p70In vivo both CAR and LNX2 proteins are present in several tissues in embryonic day 16.5 mice and in particular co-expression of both protein was noted in a subset of blood vessels [in vivo is a major strength of these studies but the functional consequence of the interaction of LNX1/2 proteins with CAR either in epithelial or sperm cells remains to be established. Notably mice lacking LNX2 appear to have normal fertility, suggesting that LNX2 is not absolutely required for sperm development and fertilization in any case [CAR like JAM4 is a member of the immunoglobulin superfamily that can interact with LNX1 via its cytoplasmic tail . CAR is  vessels . CAR and vessels . The demany case . Similarany case .These studies suggest multiple ways in which LNX1/2 proteins might be involved in the formation or remodelling of tight junctions in epithelial cells. This may be relevant in the context of EMTs that occur developmentally and that also contribute to tumour development and metastasis . InteresPotential functions of LNX1/2 proteins in presynaptic nerve terminals are suggested by the characterization of interactions of LNX1/2 proteins with several presynaptic proteins. The finding that both LNX1 and LNX2 are predominantly expressed in neurons  suggestsProteomic analyses of the LNX1 interactome revealed an interaction of LNX1 with LIPRIN-α proteins ,14. ThisA number of other proteins that play important roles in synapse formation and function have also been reported to interact with LNX1/2 proteins. These include MYCBP2 (PAM/HIGHWIRE/RPM-1), a well-known regulator of synapse formation  and CASPERBB2 is a member of the epidermal growth factor receptor family of receptor tyrosine kinases with important signalling functions in many contexts during embryonic development, including the differentiation of myelinating Schwann cells in the peripheral nervous system. ERBB2 was found to interact with the PDZ domain region of LNX1 and endogenous ERBB2 could be co-immunoprecipitated with LNX1 from mouse brain lysate . ImmunosLnx1 and Lnx2 mRNA was detected in purified T cells, the levels of LNX1 or LNX2 proteins were not assessed and are likely to be significantly lower than are achieved by heterologous expression. It will be important therefore to show that the endogenous levels of LNX1/2 proteins in T cells can affect CD8-α levels and /or subcellular localization, perhaps by examining T cells under conditions of LNX1/2 knockdown or loss of function.CD8 is a T-cell co-receptor that can stabilize the interaction of the T-cell receptor with the class I MHC on antigen presenting cells and recruit effector proteins to the T-cell receptor. The cytoplasmic tail of CD8-α was found to bind to both LNX1 and LNX2 . The intThe best characterized interactions of LNX1/2 proteins with transcriptional regulators are those of zebrafish LNX2b (described separately). However, a few interactions of mammalian LNX1 and LNX2 with nuclear proteins have been described. Several reports describe LNX1 as being partly localized in the nucleus ,58,59, aSki interacting protein (SKIP/SNW1) was identified in a yeast two-hybrid screen using PDZ1 of LNX1 as a bait . EmployiArmbruester et al.  describePDZ-binding kinase (PBK/TOPK) is another nuclear protein that has been shown to interact with LNX1 ,15,18. PThe non-receptor tyrosine kinase c-SRC can interact via its carboxyl terminus with both PDZ1 and PDZ3 of LNX1 . The twoZheng et al. . found ain vivo cellular contexts these interactions and potential modulatory influences of LNX1/2 proteins on cell signalling are relevant.It clear from the examples above that LNX1 has the ability to interact with, and in some cases ubiquitinate, key components of intracellular signalling pathways. Going forward, it will be interesting to determine in which LNX1 exon3 eliminates expression of the neuronally expressed LNX1p70 and p62 proteins, but is not expected to affect LNX1p80 expression in other tissues [https://www.mmrrc.org/catalog/sds.php?mmrrc_id=32436). However, only basic neurological and behavioural tests were performed, so subtle neural defects cannot be ruled out. The sole immunological abnormality reported is difficult to reconcile with the fact that only the neuronal LNX1 isoforms are eliminated in these mice.The first LNX1/2 loss-of-function mouse model was an isoform-specific LNX1 knockout generated by Lexicon Pharmaceuticals as part of a large-scale gene knockout programme. In this mouse deletion of  tissues . This moLNX2 knockout mouse was subsequently generated in my laboratory [LNX1exon3−/− line mentioned above to generate LNX1/LNX2 double knockout (DKO) mice in which LNX2 is eliminated globally in all tissues, while the loss of LNX1 is expected to be restricted to the exon 3-containing LNX1 p70 and p62 isoforms in the CNS. These mice thus represent a loss-of-function model to study neuronal functions of mammalian LNX1 and LNX2. LNX1/LNX2 DKO mice are viable, healthy and fertile and do not exhibit gross neuroanatomical defects [−/− mice was associated with decreased NUMB levels and abnormalities in SVZ formation [Lnx1 and Lnx2 mRNA expression.A boratory ,20. Sinc defects . Corticaormation   in zebrafish development have been extensively examined by Ro and Dawid  , making  markers . Lnx2b wectoderm . Taken tcdx4 during zebrafish embryogenesis has also been shown [Cdx4 expression is repressed by Tcf3 in the absence of Wnt signalling, whereas the transcriptional regulator E4f1 was shown to de-repress cdx4. Lnx2b can interact with E4f1, but promotes repression of cdx4 —counteracting the action of E4f1. While Lnx2b can ubiquitinate E4f1 in cell-based ubiquitination assays, this did not lead to E4f1 degradation and its E3 ubiquitin ligase activity was not required to antagonize the de-repression of cdx4 by E4f1. Thus the interaction of Lnx2b with E4f1 appears to be sufficient to modulate the Tcf3 repression complex on the cdx4 promoter, although interactions of Lnx2b with other components of the complex could play a role.A distinct role for Lnx2b in modulating the expression of the caudal homeobox gene en shown . Cdx/cauPtf1a transcription factor that is required for differentiation of these cells. Other exocrine markers such as trypsin were also drastically diminished, whereas endocrine cell differentiation seemed unaffected. The specificity of this phenotype tallied with the expression of lnx2 mRNA in the ventral pancreatic bud from which exocrine cells are derived, but not in the dorsal bud from which endocrine cells arise. The mechanism underlying this phenotype appears to involve LNX2/2b-mediated regulation of Notch signalling via Numb, based on the observations that Numb knockdown substantially rescued the phenotype and that increased levels of Numb and decreased Notch signalling were seen in pancreatic cells of embryos expressing dominant negative Lnx2. Destabilization of Numb by Lnx2 and Lnx2b is proposed to promote Notch signalling in ventral bud-derived cells which appears to be important in the specification and/or proliferation of the exocrine precursor cell population [A third function for Lnx2 and -2b proteins during zebrafish development has also been revealed by Dawid et al. . In thispulation .in vivo. A key question is whether these functions are shared in other lineages, particularly in eutherian mammals that lack LNX2b. The absence of a clear mammalian Bozozok orthologue suggests that this function of Lnx proteins in very early embryonic patterning may be specific to fish. By contrast, Ro and Dawid [Together these studies in the zebrafish model represent the best mechanistic descriptions of LNX1/2 protein function nd Dawid  demonstrLNX1 mRNA was noted in gliomas compared with healthy brain tissue [LNX1 and LNX2 mRNA are expressed predominantly in neurons [LNX1 gene in brain tumours and osteosarcoma xenografts [KIT, PDGFR-α and VEGFR2) and so amplification of LNX1 may be incidental. A few cases with specific amplification of LNX1 were reported [LNX1 missense mutations were found [LNX1 mRNA expression in HEK cells using siRNA led to cell cycle arrest and caused alterations in the expression levels of several cancer associated genes. Nie et al. [LNX1 mRNA and particularly LNX1 protein in tumours, the question of whether LNX1 actually promotes a cancer phenotype in vivo remains unclear and further evidence is required to demonstrate a compelling causative role for LNX1 in brain tumours.A number of reports have highlighted altered LNX1 expression in the context of brain tumours, although in most cases a causative role for LNX1 in driving tumorigenesis has not been demonstrated. Down-regulation of n tissue , though  neurons  and as snografts . It is nreported  and somere found , howeverre found  highlighe et al.  reportedLNX2 gene maps to chromosome 13q12.2—a region that is frequently amplified in CRC. Examining genes that are amplified in this chromosomal region, Camps et al. [LNX2 mRNA to be overexpressed in CRC compared with normal colon mucosa as well as in CRC cell lines. Furthermore, it was shown that LNX2 knockdown using siRNA reduced the viability of CRC cell lines and induced apoptosis. Using genome-wide expression profiling they observed dysregulation of 680 genes upon silencing of LNX2 in a CRC cell line (SW480) that exhibits chromosome 13 amplification. The expression of multiple target genes of both the NOTCH and WNT/β-catenin signalling pathways were prominently decreased and alterations of upstream components of these pathways were noted at both the mRNA and protein levels. In particular, TCF7L2, a transcription factor that is a major effector of WNT signalling, and many of its target genes were down-regulated in LNX2 knockdown cells. Similarly, levels of NOTCH and the NOTCH ligand JAG1 as well as several NOTCH target genes were significantly decreased. Thus, LNX2 has the ability to simultaneously regulate two signalling pathways known to play critical roles in CRC tumorigenesis. Surprisingly, decreased NUMB protein levels were observed upon LNX2 knockdown—the opposite of what one might expect for NUMB as a substrate for LNX2-mediated ubiquitination and an inhibitor of NOTCH signalling. Thus, the observed changes in NOTCH signalling cannot be explained simply by LNX2 modulating NUMB levels. Nevertheless, the findings suggest a credible model whereby overexpression of LNX2 as a consequence of LNX2 gene amplification promotes NOTCH and WNT/β-catenin signalling, which drives cell proliferation and prevents apoptosis in CRC cells [The most comprehensive studies linking LNX1/2 proteins to tumorigenesis are in relation to CRC ,35. The s et al.  found LNRC cells .LNX1 mRNA and protein were down-regulated in a population of putative cancer stem cells (side population cells) isolated from the CRC cell line HT29. Cancer stem cells in tumours are proposed to give rise to cells with highly proliferative and invasive phenotypes. LNX1 knockdown increased the number of cancer stem cells and led to the formation of more colonospheres in extreme limited dilution assays, and more tumours in mice injected with LNX1 knockdown HT29 cells. This suggested that LNX1 is a negative regulator of cancer stemness in CRC. Seeking a mechanism for this effect the authors examined LNX1 interacting proteins and found that knockdown of CAR, a known LNX1 ligand [LNX1 knockdown leads to increased CAR protein levels, suggesting that CAR may be involved in mediating the effect of LNX1 on cancer stemness, although the mechanism by which LNX1 regulated CAR levels and whether it involved LNX1-mediated ubiquitination of CAR was not examined. Finally, this study showed that tamoxifen increased LNX1 gene expression and could inhibit colonosphere formation in HT29 cells. Overall these findings suggest that LNX1 may act as a tumour suppressor and that up-regulating LNX1 expression may represent a novel therapeutic strategy to negatively regulate cancer stemness in CRC.A more recent study has now also implicated LNX1 in CRC . Here th1 ligand , caused LNX1 mRNA to be up-regulated in CD133− compared with CD133+ stem cells, suggesting that an involvement of LNX1 in cancer stemness may not be specific to CRC [LNX2 promoter region that was linked to increased susceptibility to diffuse large B-cell lymphoma [LNX2 gene to differential responses to the chemotherapeutic drug cytosine arabinoside in lymphoblastoid cells [Interestingly, a previous study of cancer stem cells in glioblastoma reported c to CRC . Furtherlymphoma  and anotid cells . These sLNX1 that are associated with susceptibility to Kawasaki disease—an inflammatory paediatric condition that causes damage to the corony arteries and is thought to be triggered by an unidentified infection [LNX1 transcript levels in acute compared with convalescent Kawasaki disease. A different report found that blood levels of both LNX1 and LNX2 mRNAs were significantly elevated in chronic compared with acute Q fever [Coxiella burnetti infection and the acute form usually resolves by itself, whereas chronic Q fever develops as an endocarditis and is associated with an impaired immune response. The authors suggest that LNX1/2 expression could be used as a prognostic biomarker in Q fever patients. These reports, along with the previously discussed interaction of LNX1/2 proteins with CD8 [A couple of studies have found associations of LNX1 and LNX2 with infectious diseases. A GWAS found intronic SNPs in nfection . This st Q fever . Q feverwith CD8  suggest LNX2 mRNA expression in placental tissue was found to be associated with preeclampsia in pregnancy [LNX2 gene to be hypomethylated in smokers [Differential expression of regnancy , while a smokers . The funin vivo (with the notable exception of studies of zebrafish Lnx1/2 proteins). While our understanding of some basic aspects of LNX1/2 function is still incomplete, it is improving. It is now appreciated that LNX1/2 protein levels are tightly regulated. We are beginning to understand the differential functions of LNX1, LNX2 and LNX2b, as well as the neuronal and non-neuronal LNX1 isoforms. Fantastic structural insights into the mechanisms of LNX1/2 ubiquitin ligase activity have emerged in the past few years. The LNX1/2 interactome has been thoroughly explored using a variety of complementary approaches. Loss-of-function analyses of mammalian LNX1/2 proteins in both cellular and mouse models have been published. Many pieces of the puzzle are falling into place.Two decades of research have highlighted a multitude of potential functions for LNX1/2 proteins, based largely on their ability to interact with many binding partners . Howeverin vivo. The molecular basis for the subtle phenotypes already observed in mice lacking LNX1 and LNX2 proteins in the CNS can be dissected more precisely. The generation of mice lacking both LNX1 and LNX2 in all tissues will clarify proposed roles for LNX1/2 proteins in immune function and bone homeostasis and allow the role for LNX1/2 proteins in pancreas differentiation to be explored in mammals. Further structural studies will hopefully reveal structures of full-length LNX1/2 proteins and/or structures of LNX: substrate complexes. Translational studies will be also be needed to tease out the disease associations of LNX1/2 proteins, particularly in CRC, where both LNX1 and LNX2 have been implicated. These studies will determine whether LNX1/2 proteins are worth considering as potential drug targets, particularly as methods to inhibit both E3 ubiquitin ligases and PDZ domain interactions with small molecules develop [Going forward armed with this knowledge and the new research tools that are available, researchers in this field can be optimistic about putting more pieces of this puzzle together. The currently available and newly generated LNX1 and LNX2 knockout mice can be used to explore the significance of putative functions of LNX1/2 proteins  develop .Click here for additional data file.


Identifying immunogens that induce HIV-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict T-cell epitopes for various HLA types.We tested the performance of the NetMHCpan4.0 computational neural network in re-identifying 93 T-cell epitopes that had been previously independently mapped using the whole proteome IFN-γ ELISPOT assays in 6 HLA class I typed Ugandan individuals infected with HIV-1 subtypes A1 and D. To provide a benchmark we compared the predictions for NetMHCpan4.0 to MHCflurry1.2.0 and NetCTL1.2.p = 0.0000005). MHCflurry1.2.0 similarly predicted all but 2 of the peptides that NetMHCpan4.0 predicted and NetCTL1.2 predicted only 14 of the 93 experimental peptides.NetMHCpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched HLA class I alleles. Receiver Operator Characteristic (ROC) analysis gave an area under the curve (AUC) of 0.928. Setting NetMHCpan4.0 to predict 11-14mer length did not improve the prediction (37–79 of 93 peptides) with an inverse correlation between the number of predictions and length set. Late time point peptides were significantly stronger binders than early peptides (Wilcoxon signed rank test: NetMHCpan4.0 class I epitope predictions covered 95% of the epitope responses identified in six HIV-1 infected individuals, and would have reduced the number of experimental confirmatory tests by > 80%. Algorithmic epitope prediction in conjunction with HLA allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort. Computational algorithms were initially demonstrated as useful tools for predicting potential epitopes that might elicit quality T-cell responses , 2. Compag genes  in cynomag genes  necessarag genes . Computaag genes , therefoag genes –10.For HIV-1 vaccine design purposes an important consideration for the suitability of a computational algorithm is the breadth of discrete number of T-cell epitopes it generates that could reach particular levels of coverage  of circuin-silico approaches in epitope prediction and its application for vaccine design reported a meagre 22, 44%, and relatively higher 78% match for three computational tools namely YFPEITHI, CTLPRED and IEDB respectively [A reliable pan-HLA-specific algorithm NetMHCpan4.0 –20 that ectively . Using eet.al [The data used was from an independent study that did not include this analysis in its objectives. Experimental data of peptides previously mapped for HIV-1 epitope recognition of 6 individuals for a separate study . The web version of NetMHCpan4.0 [http://www.cbs.dtu.dk/services/NetMHCpan/) was configured to predict 9mer through 14mer epitopes for 22 HLA class I alleles  plot. The hit rate (sensitivity) and false hit rate (specificity) of binder predictions as determined by the NetMHCpan4.0 threshold of peptides within the top 2% (with a score of 2 or less) were calculated and the strength of the model was determined by calculating the area under the curve, AUC of the ROC plot –43. PearTo experimentally determine epitopes for 757 peptides spanning the whole HIV-1 proteome for clades A and D as well as both time points of the 6 individuals required a total of 4230 test assay wells. For each test subject these included 9 antigen proliferation wells, 384 culture ELISPOT wells and an average of 164 epitope mapping ELISPOT wells . Using the 22 HLA alleles represented in the study subjects we were able to computationally predict 95% of the experimentally mapped epitopes. This approach could have reduced the test assays by eliminating all the T-cell antigen proliferation and culture ELISPOT steps totalling to 3258 assay wells (77%) and leaving only 972 (23%) epitope mapping assays required. Applying a pooling strategy to the computational predictions similar to that used in the experimental pooling where each pool contained approximately 20 peptides with a coverage of 3 per peptide pool, the 923 potential peptides  eligible epitope mapping peptides) would make at most 46 pools. Consequently the computational prediction approach could have reduced the experimental assays by at least 80%.rs = 0.88  epitopes were experimentally mapped of which 12 were recognized at both baseline and later time points, 34 only at baseline and 54 only at the later time point. Comparison of the ranked computational score for Netmhcpan4.0 binders of early (n = 34) versus later peptides showed that the later time point predictions were stronger binders reaching statistical significance  (Fig. The experimental peptide mapping data was derived from a baseline time point corresponding to HIV-1 Fiebig stages IV, V and VI (Table In this analysis we showed that the computational method NetMHCpan4.0 predicted 95% of previously experimentally mapped HIV-1 epitopes in 6 HIV-1 infected individuals expressing a total of 22 different HLA class I alleles. In our IFN-γ ELISPOT assays we evaluated 757 17mer peptides overlapping by 11 amino acids and covering the whole HIV-1 subtype A1 and D consensus proteomes. Out of the 5 experimentally determined epitopes missed by the algorithm Table , 4 were As the participants had been enrolled in the acute/early phase of HIV-1 infection and we had observed intra-participant changes in epitope recognition between early and late time points after infection, we compared the binding scores of confirmed epitopes at these time points and found a statistically significant change towards recognition of higher binding peptides as the infection entered the chronic phase. This might represent better support of the T-cell response directed at more stable HLA/peptide complexes as the infection progresses into chronicity.The NetMHCpan4.0 algorithm, which is based on binding affinity and integrates data on eluted naturally processed ligands, reflected optimal HLA class I binding for 9-mers, producing a decreasing number of predictions when the peptide size was increased from 9 to 11 amino acids. With a single exception, predicted binders between 11 and 14 amino acids included at least one 9mer predicted to bind on its own, suggesting a destabilizing effect of the extra amino acids beyond the canonical HLA class I binding pockets at positions 2 and 9 could account for fewer predictions.Important limitations are the lack of predictions of HLA class II restricted epitopes, which might have contributed to a fraction of IFN-γ ELISPOT responses. Approximately 5% of the computational predictions may be false positives that only increase the size of planned wet experiments and approximately 1% of true positives may also be missed.In this analysis, using NetMHCpan4.0, MHCflurry and NetCTL to predict previously experimentally mapped epitopes, we demonstrate that the computational methods reliably predict an acceptable portion of binder epitopes. We recommend the use of such computational methods to reduce the size of experiments required cost associated.


Scientific Reports 10.1038/srep34626, published online 11 October 2016Correction to: This Article contains errors in the Supplementary Information file, where the size distributions of the nanoparticles given in Figure S12b and Figure S13c are incorrect. The correct Figures S12 and S13 are given below as Figs.


ObjectiveTo compare 5 French (Fr) and 6 Fr guiding catheters regarding the volume of contrast administered, fluoroscopy time, and total procedure time during transradial percutaneous coronary intervention (PCI).BackgroundPrevious studies had compared 5 Fr and 6 Fr catheters and deemed 5 Fr catheters safe and effective. In this study, we retrospectively compared the 5 Fr catheter to 6 Fr catheter with an attempt to eliminate the effect of inter-operator skill level variability.MethodsIn a single-center, retrospective cohort study, we randomly selected patients who had received PCI through transradial access using 5 Fr or 6 Fr catheters. The study involved two groups of 100 patients each. These groups were comprised of an equal number of cases from each operator. The primary endpoint was contrast medium volume. Secondary endpoints were fluoroscopy time and procedure time.ResultsLess contrast was used in the 5 Fr group vs. 6 Fr catheter group . PCI using 5 Fr catheters was associated with shorter fluoroscopy time  and shorter procedure time , but this was not statistically significant.ConclusionTransradial PCI using 5 Fr guiding catheters was associated with less contrast medium usage, but there was no advantage regarding procedure time or fluoroscopy time when compared to 6 Fr catheters. Similar to 6 Fr catheters, 5 Fr catheters achieved high PCI success rates through radial access when compared in the treatment of coronary lesions with the same level of complexity. Since its inception in 1978, the field of percutaneous coronary intervention (PCI) has been evolving to constantly better itself [Among the numerous medical devices manufactured, only a few make the cut and are deemed to be the standard of care after being studied and tested in the field. Before the 6 Fr catheter was determined to be the standard of care, it had also been compared and tested against 7 Fr and 8 Fr catheters; and it was crowned as the standard of care only after it was deemed to be effective -4. With Study design and patient populationOur study was a single-center, retrospective cohort study designed to compare procedural variables of 5 Fr versus 6 Fr catheters in patients who underwent PCI using radial access. A total of 200 patients were involved in the retrospective analysis in this study. The study population was randomly selected from a pool of 463 patients that included all the consecutive PCI cases performed at St. Mary’s Medical Center - Marshall University by four different interventional cardiologists with expertise in transradial procedures during May 2016-May 2017. All of the cases used radial access as initial access, and at least one stent was deployed using 5 Fr or 6 Fr as an initial catheter. Indications for the procedure were either elective for stable angina or emergent for non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI). The total study population was then divided into two groups: 5 Fr group and 6 Fr group. Of the total 463 cases that we considered, 265 were performed using 6 Fr catheter and 198 using 5 Fr catheter. Each of these two groups was then divided into four subgroups based on the operator  grade 3 anterograde flow without the occurrence of complications such as death from any cause, urgent coronary bypass surgery, or acute myocardial infarction.Data analysisBaseline clinical characteristics were analyzed for all patients included in the study. Results were expressed as proportions or mean with standard deviation (SD). The primary endpoint was the amount of contrast used in the procedure. Secondary endpoints included procedure time and fluoroscopy time. All were continuous variables and they were assessed by t-test using SPSS Statistics version 24.0 . A p-value of <0.05 was considered statistically significant.A total of 200 patients were included in the analysis, with 100 each from the two study groups. Both groups consisted of unselected randomized patients in routine clinical practice with a diverse clinical profile. The two groups were well balanced regarding baseline characteristics  in the 5 Fr group, while crossover from 6 Fr to 5 Fr occurred in two cases (2%) in the 6 Fr group. As defined in the methods section, a 99% success rate was achieved in the 5 Fr group while 98% success rate was achieved in the 6 Fr group. Compared with transradial PCI using 6 Fr catheters, the 5 Fr group had lower contrast use . PCI using 5 Fr catheters was associated with shorter fluoroscopy time . Again, procedure time was less with the 5 Fr catheters . The latter two findings were statistically insignificant.Initial prototypes of the 5 Fr and 6 Fr catheters did not have a lumen large enough for an adequate stent insertion as the lumen was less than 0.058 inches; hence, these catheters could only be employed for diagnostic purposes . It was The appeal of 5 Fr catheter came to light when several studies reported favorable prospects of utilization. In 2001, Schöbel et al. reported a success rate of up to 95% in 5 Fr catheters used in transfemoral access site depending on the type of the lesion. However, it was also reported that 5 Fr catheters had limitations in the application such as poor back-up support in long lesions as well as severe stenosis . Gobeil The 5 French catheter use has strongly been associated with decreased usage of contrast volume. Schussler et al. studied 5 Fr catheters with transradial PCI in preselected cases where they were shown to be effective. They concluded that the usage of 5 Fr catheters reduces morbidity and the cost of the procedure since it required less amount of contrast medium and resulted in minimal complications . Rakhit Gobeil et al. compared the 5 Fr and 6 Fr in transradial PCIs . Their sThe success of the procedure was defined as less than 50% residual stenosis with TIMI grade 3 anterograde flow without the occurrence of complications such as death from any cause, urgent coronary bypass surgery, or acute myocardial infarction. 5 Fr catheters were associated with a 99% success rate, which is higher than the 6 Fr group (98%). Previous studies have shown the superiority of the use of 5 Fr catheters in terms of success and complications rates; however, the success rate of 99% is significantly higher than the rate reported in older studies like Gobeil et al., which showed a success rate of 90% . In receDahm et al. conducted a randomized study of 5 Fr vs. 6 Fr transradial PCI aiming to ascertain whether the 5 Fr guiding catheter has equivalent procedural success and complication rates as the 6 Fr. Their study showed that transradial PCI of non-complex lesions can be successfully performed with either 5 Fr or 6 Fr guiding catheters, with 5 Fr showing higher procedural success rates and lower vascular access complications in comparison to 6 Fr guiding catheter . In thisOur findings revealed that transradial PCI using 5 Fr guiding catheters was associated with less contrast medium usage when compared to 6 Fr catheters. 5 Fr catheters would thus be the preferred choice for patients with reduced renal function or when minimizing the use of contrast medium is of concern. 5 Fr catheters did not have an advantage over 6 Fr catheters in terms of procedure time or fluoroscopy time. Similar to 6 Fr catheters, 5 Fr catheters can achieve a high PCI success rate through radial access when compared in the treatment of coronary lesions with the same level of complexity.


Small ubiquitin‐related modifier (SUMO)‐specific protease 2 (SENP2) is essential for the development of healthy placenta. The loss of SENP2 causes severe placental deficiencies and leads to embryonic death that is associated with heart and brain deformities. However, tissue‐specific disruption of SENP2 demonstrates its dispensable role in embryogenesis and the embryonic defects are secondary to placental insufficiency. SENP2 regulates SUMO1 modification of Mdm2, which controls p53 activities critical for trophoblast cell proliferation and differentiation. Here we use genetic analyses to examine the involvement of SUMO2 and SUMO3 for SENP2‐mediated placentation. The results indicate that hyper‐SUMOylation caused by SENP2 deficiency can be compensated by reducing the level of SUMO modifiers. The placental deficiencies caused by the loss of SENP2 can be alleviated by the inactivation of gene encoding SUMO2 or SUMO3. Our findings demonstrate that SENP2 genetically interacts with SUMO2 and SUMO3 pivotal for the development of three major trophoblast layers. The alleviation of placental defects in the SENP2 knockouts further leads to the proper formation of the heart structures, including atrioventricular cushion and myocardium. SUMO2 and SUMO3 modifications regulate placentation and organogenesis mediated by SENP2. Genetic analyses reveal that hyper sumoylation caused by SENP2 deficiency can be compensated by reducing the level of SUMO modifiers. The placental deficiencies caused by the loss of SENP2 can be alleviated by the inactivation of gene encoding SUMO2 or SUMO3. SENP2 genetically interacts with SUMO2 and SUMO3 pivotal for the development of three major trophoblast layers. The alleviation of placental defects in the SENP2 knockouts further leads to the proper formation of the heart structures, including atrioventricular cushion and myocardium. Protein modification by SUMO2 and SUMO3 is essential for SENP2‐mediated placentation and organogenesis. Therefore, we used a genetic approach to test if SUMO2 and SUMO3 also modulate placental development mediated by SENP2. To perform genetic tests, mice carrying the deletion of SENP2 were crossed with those carrying SUMO2 and SUMO3 mutation to obtain the double mutants. At E10.5, three major trophoblast layers — the labyrinth, spongiotrophoblast, and TGC (trophoblast giant cell) in SUMO2+/− and SUMO3−/− placentas are comparable to the wild type (Figure 2.2The alleviation of placental defects seemed to allow the embryo to survive after E12.5. We were able to recover SENP2−/−; SUMO2+/− embryos at E12.5 Figure A‐C. TherTo test if SUMO3 also modulates SENP2‐dependent embryonic and extraembryonic development, SENP2 nulls were crossed into the SUMO3 homozygous background. We successfully obtained the double knockout of E12.5 and E13.5 embryos Figure A‐C, M‐O,3Genetic analyses described in this study clearly demonstrate that SUMO2 and SUMO3 modulate SENP2‐dependent extraembryonic and embryonic development. Placental defects caused by hyper‐SUMOylation in the SENP2 knockouts can be alleviated by the reduction of SUMO modifiers. We succeed to prove the concept that hyper SUMOylation caused by the loss of SUMO proteases can be compensated by decreasing the level of SUMO modifiers. Although SUMO1 regulates the Mdm2/p53 pathway essential for cell cycle progression,SENP2 are not primary but secondary defects due to placental insufficiency. This is further supported by the current study showing that rescue of placental insufficiencies promotes the proper formation of the AV cushion and myocardium.It remains possible that the alleviation of extraembryonic and embryonic defects is independent of each other in the double mutants. However, our data show a tight link of heart deformities to placental deficiencies, suggesting these are dependent events. Furthermore, our demonstration of SENP2 dispensable for embryonic development strongly argues against a specific requirement of SENP2 in cardiac tissue during heart development.While removing one allele of SUMO3 has no effect on the SENP2‐null defects, placental and embryonic defects are alleviated by SUMO2 heterozygosity. Therefore, SUMO2 may play a more important role than SUMO3 in SENP2‐mediated placentation. The genetic analyses also suggest nonredundant functions of SUMO2 and SUMO3 in SENP2‐dependent regulation. Although SUMO2 and SUMO3 are ~95% identical, we do not find synergistic effects when both of them are reduced in the SENP2 nulls. It is possible that SUMO2 and SUMO3 modify distinct substrates critical for placentation. In addition to the SUMO1‐mediated modulation, SUMO2/3 may be critical for the regulation of S phase during cell cycle progression.44.1The SENP2, SUMO2 and SUMO3 knockout mouse strains and their genotyping methods were reported previously.4.2Samples were fixed, paraffin‐embedded, sectioned and stained with hematoxylin/eosin for histological evaluation.The authors declare no potential conflict of interest.