We report the earliest evidence for extensive fish exploitation from the hypersaline Bardawil lagoon on Egypt’s northern Sinai coast, as indicated by distinctively high δ18OPO4 values, which became abundant in the southern Levant, both along the coast and further inland, at least from the Late Bronze Age . A period of global, postglacial sea-level stabilization triggered the formation of the Bardawil lagoon, which was intensively exploited and supported a widespread fish trade. This represents the earliest roots of marine proto-aquaculture in Late Holocene coastal domains of the Mediterranean. We demonstrate the potential of large-scale δ18OPO4 analysis of fish teeth to reveal cultural phenomena in antiquity, providing unprecedented insights into past trade patterns.Past fish provenance, exploitation and trade patterns were studied by analyzing phosphate oxygen isotope compositions (δ In the southern Levant, past exploitation and trade of fish has been explored primarily based on the occurrences of fish bones in coastal, riverine and lake-side archaeological sites and through inference from the modern distribution patterns, habitat preferences and ecological niches of these fish species. In the Levant, this has mostly been done for fish that a priori were identified as ‘exotic’. For example, the identification of key Nilotic species such as Lates niloticus (Nile perch) and Bagrus sp. (Bagrid catfish) in archeological sites of the southern Levant testified that long-range trade systems between Egypt and Canaan have emerged more than 5000 years ago (during the Early Bronze Age)8.Fishing was an essential economic component of many ancient societies, as evidenced by the presence of fish remains, fishing gears, and fish-associated artifacts in numerous archaeological sites world-wideSparus aurata, Linnaeus, 1758) frequently appears in archaeological sites of the southern Levant, since prehistoric times (Late Pleistocene)6. This species is characterised by thick-enamelled, molar-like teeth  and open sea environments13. Thus, while the appearance of S. aurata in inland sites clearly indicates long range trade systems7, remains of this species in Levantine coastal sites have so far been interpreted as reflecting local fishing activity8.The gilthead seabream , these analyses showed no clustering with locality or species, and for both isotopes they demonstrated a general overlap between freshwater and marine fish, probably due to bone diagenesis20. Hence other isotopic proxies are required to assess fish provenance.State of the art research methodologies provide multiple empirical ways to explore trade and maritime connections of desirable fish source marketing to distant places. For example, past provenance and long-range trade of fish from the North Atlantic have been studied using the C and N stable isotopes of bone collagen (Atlantic cod)21 pioneering study on the distribution of the phosphate oxygen isotopes (δ18OPO4) in fish bioapatite demonstrated that δ18OPO4 values are controlled by the water temperature and oxygen isotope composition of the ambient water (δ18OWater)23. Enameloid of fish teeth is highly resistant to diagenetic alterations24, and thus in many cases preserve information regarding the original salinity and temperature26 of their past aquatic habitat 30. In closed or semi-closed water bodies with a high degree of evaporation, the δ18OWater are enriched in 18O relative to the seawater that feeds them31  of enameloid phosphate of the first molariform teeth , Egypt episodically controlled the southern Levant, whence it could extract the Mediterranean products required for its subsistence and spiritual life. Interconnections between the two regions, however, were not only dictated by cultural and political factors but by climatic fluctuations in both35. Traffic between Egypt and the Levant was conducted through marine or terrestrial routes through northern Sinai, which was both affected by it in antiquity and provides archaeological proxies for its intensity today. The main question we asked is whether the distribution of these fish to the Levant was a historically-unique and context-dependant phenomenon or rather of more sustainable nature.During this time span, southern Levantine societies evolved from hunting-gathering, to sedentary ways of lives, to complex societies, to territorial states, and were intermittently subsumed under the aegis of vast or lesser empires – from New Kingdom Egyptians in the Late Bronze Age  to the Muslims of the 718OWater23. Thus, fish record both the environmental temperature and δ18OWater in their bone and tooth δ18OPO4 values30. While the δ18OWater of the oceans generally shows small variations37, closed or semi-closed water bodies, such as coastal lagoons, show significantly elevated and variable δ18OWater values31, which are recorded in the hard tissues (i.e. teeth) of migratory fish that exploit these habitats30.Bioapatite of fish teeth forms in oxygen isotopic equilibrium with the body fluid at ambient water temperature and δ37. Variations in East Mediterranean δ18OWater are small38, varying between 1.4‰ (February–March) and 1.8‰ (July–August). Therefore, the calculated δ18OPO4 values (range: 21.2–24.2‰) for bioapatite forming in isotope equilibrium with seawater22 (see Methods) in the southeast Mediterranean reflect mostly the seasonal changes in water temperature  in teeth of modern S. aurata caught in the southeast Mediterranean littoral zone  coast display significantly higher δ18OPO4 values, between 23.5‰ and 25.4‰23  to 30 °C in summer (July–August)39. The Bardawil is connected to the sea via two small natural inlets (Boughaz Zaranik). Water exchange in the lagoon is controlled by Mediterranean Sea tides with a mean height of 50 cm. As a result, it has an elevated salinity level and δ18OWater values around 3.7‰  and hypersaline water (39.0–74.5‰) provides optimal growth conditions for several species of fish, including S. aurata11. Today, juveniles of S. aurata enter the lagoon seeking shelter and food. At about two years of age13, most fish reach sexual maturity and leave the nursery, migrating back into the open sea where they live in a variety of habitats such as sea grass beds and sandy or rocky bottoms40.The Bardawil lagoon Fig.  is a lar‰31 Fig. . The uni41. The δ18OPO4 values in teeth of modern S. aurata cover nearly the entire seasonal range of predicted δ18OPO4values for teeth formed in isotopic equilibrium with the southeast Mediterranean and the Bardawil lagoon water in the according temperature and salinity range, respectively  and a hypersaline environment reflected by higher δ18OPO4 values   to the Byzantine period is further supported by the calculated water temperatures, ranging between 16 and 28 °C (using the mean Bardawil δ18OWater of 3.7‰), which agree well with the annual water-temperature range of the present-day Bardawil lagoon39. We note that the few hypersaline fish dated to the PPN-EBA period (early to mid-Holocene), were likely caught off the southern Levantine shore, and were not exported from the Bardawil, as proposed for the LBA and onward (Late Holocene). This suggests that short-lived, hypersaline lagoons may have formed along the Levant coast when the rapidly rising sea level flooded low-lying coastal areas during the Early Holocene44.Notably, records from sediment cores of the Levant coast, dated to the Holocene, show no evidence for any hypersaline lagoon, similar to the Bardawil, neither in the dimension, nor in the unique environmental setting of this habitat45, reaching the current level (±1m) at around 3,620 ± 160 years BP46   Fig. . Sea-lev18OPO4 values indicate that S. aurata was captured mainly from southeast Mediterranean waters and to a lesser extent from hypersaline lagoons : PPNA; ~9,700 years BCE; 11,700 years BP) δons Fig. . The lat18OPO4 values indicate that S. aurata was primarily captured ‘locally’, namely along the southeast Mediterranean coast  analysed from pre-LBA contexts (PPN to EBA) had δ18OPO4 values indicative of hypersaline habitats, 84.2% of the teeth (n = 57) from the LBA to the Iron Age (IA) were characterised by high δ18OPO4 values typical for fish from hypersaline habitats. This pattern also prevails in the Byzantine period (n = 10). The δ18OPO4 values of sparid teeth from the LBA onwards are similar to those of extant S. aurata from the Bardawil lagoon , δst Table , howeveroon Figs . In addi18OPO4 values of tooth-jaw pairs of ancient S. aurata specimens from PPN to Byzantine are positively correlated , yet δ18OPO4 values of jawbones are consistently lower than those of tooth enameloid. An average offset (Δ18Otooth-jaw) of 1.8 ± 0.95‰ may reflect systematic differences of bioapatite biomineralisation in bones and enameloid. These bone-tooth δ18OPO4 differences suggest that the bone δ18O signatures likely represents a mean of the entire lifespan of S. aurata. This further supports that S. aurata spent a significant part of their adult life in the open sea, but continued to exploit hypersaline coastal lagoons as part of their trophic migration, as is the case with modern S. aurata13.The δ18OPO4 values than the teeth from the same individual/jaw, reflecting a marine origin and indicating that these fish lived predominantly in southeast Mediterranean seawater. A few jawbones have values lower than those expected for bioapatite forming in isotope equilibrium with modern southeast Mediterranean seawater. Some alteration of bone, which is known to be more prone to diagenesis than enameloid24, in low δ18O meteoric water during burial may have shifted δ18OPO4 towards lower values.Specimens from the Byzantine period have oxygen isotope signatures typical of hypersaline water both in their teeth and jawbone Fig. . This suS. aurata  and body mass-BM (kg)), can be calculated from the maximum length of the molariform tooth crown48. Body size of S. aurata reflects ontogenetic age and can thus be used as a proxy of fish exploitation  for specimens from the hypersaline lagoonal waters of the Bardawil  show significant decrease in the range and average size. The range of S. aurata decreased from a mean BM of 1.6 ± 0.8 kg (n = 33) and TL of 45.0 ± 8.3 cm during PPN–Early Bronze Age (EBA) to a mean BM of 1.1 ± 0.4 kg (n = 57) and TL of 40.7 ± 5.1 during LBA–IA, and to a mean of 0.78 ± 0.4 kg (n = 10) and mean TL of 36.4 ± 3.8 in the Byzantine period  .The body size of ion Fig. . Smalleril Table  and 5. Tiod Figs  and 5, aS. aurata captured from the Mediterranean littoral zone28. Overall, the reduction in S. aurata mean and maximum body size demonstrates similarity with present day fishery data from the Bardawil lagoon (age group 1–3 years), where S. aurata are intensively exploited due to their abundance in this unique hypersaline nursery13. The decrease in S. aurata body size, to a range similar to present-day fish from the lagoon40 is in line with observations from traditional extensive aquaculture50.This trend of reduction in fish body size accords chronologically well with cultural changes and spreads over a millennial time-scale, starting in the LBA, continuing to the Iron Age and to the Byzantine period. Interestingly, we witness the appearance of larger fish only at Tel Dor, where we identified in the past S. aurata in the Bardawil lagoon. This unique exploitation pattern continues until the present13, and agrees well with the definition of traditional extensive aquaculture50. It is similar, for example, to the Italian ‘vallicoltura’ or the Egyptian ‘hosha’ – both representing traditional fish exploitation systems which utilise natural fish traps by taking advantage of the trophic migration of juveniles from the sea into coastal lagoons. These systems therefore capitalise on naturally occurring foods from this highly productive lagoon13. Sparus aurata were traditionally exploited extensively in coastal lagoons, until intensive rearing systems for this fish were developed during the 1980s51. Therefore, the cradle of marine aquaculture may be rooted in the Bardawil hypersaline lagoon. For more than 2,000 years it functioned as the major source for Sparus aurata for the Levant. This started not later than the LBA  as extensive harvesting and still continues today as intensive aquafarming13.From the LBA onwards we find evidence for extensive and likely year-round exploitation of S. aurata were exploited by local coastal fishing communities5. The first evidence for fishing in hypersaline lagoons appears during the PPNB ca. 9,500 years BP, but these remains are scarce and are insufficient to indicate systematic exploitation of hypersaline lagoons during this period. However, the occurrence of fish remains in Neolithic inland sites of the Judean Mountains  indicates that the transportation of dry fish from the Mediterranean coast was already established in the Early Holocene52.In the southern Levant, for the last 50,000 years, S. aurata with hypersaline δ18OPO4 signatures were imported from the Bardawil lagoon, almost entirely replacing S. aurata caught locally in coastal waters. These results contradict the conventional null hypothesis assuming that in coastal sites of the Levant S. aurata remains will represent local fishing, exhibiting that regardless of the site location  most of the S. aurata were “exotic”  with δ18OPO4 signatures of the Bardawil lagoon.From the LBA period onwards, non-local (“exotic”) Lates niloticus) and the Nile catfish7 54.In archaeological sites across the southern Levant, this fundamental change in fish provenance coincides with a sharp increase in the abundance of exotic Nilotic fish, such as Nile perch , the single 14th century BCE hypersaline specimen found at Tel Rehov may hint at an earlier beginning of this trade during the LBA. Moreover, although we lack data from the MBA , it is possible that this phenomenon started even earlier. Throughout the MBA, contact between Canaan and Egypt was close, although at the end of this period administrative control was inversed: Canaanites controlled parts of Egypt at this time. Egypto-Canaanite commerce flourished in this period too, though most of it appears to have been conducted via the sea56. Dozens of MBA settlements have been surveyed in northern Sinai, especially south of the Bardawil lagoon.It is easy to envisage how the Bardawil fish industry and export emerged and functioned in this context. Although our earliest substantial evidence of 59.Importantly, our findings demonstrate that despite climatic changes and frequent socio-political, economic and demographic upheavals in both regions, once industry and marketing were in motion, they lasted at least until Byzantine times , providing a paradigmatic example of a Mediterranean exchange network driven by the diversity and interdependence between ecological micro-regionsth-century CE Caesarea Maritima—a major Southeast Mediterranean harbour city  in the presence of bromothymol blue as a pH indicator, Ag3PO4 was rapidly precipitated by adding 0.8 ml of 2 M AgNO3. Following settling of the Ag3PO4 crystals, the samples were centrifuged and the supernatant solution was removed using a pipette. The Ag3PO4 was then rinsed five times with MilliQ water and dried overnight in an oven at 50 °C.The enamel cap (~0.2–0.4 mm thickness) of each tooth was separated from the dentine using a diamond-head micro-dental drill, washed three times with distilled water, and dried overnight at 50 °C. Each sample was crushed and ground to powder using an agate mortar and pestle. Organic matter was removed from the samples soaking the samples in 2% NaOCl overnight. The phosphate fraction of the samples was separated using a method modified after Dettmann 3PO4 aliquots of 0.5 mg were placed into silver capsules and analysed in triplicate by means of high temperature reduction using a Finnigan TC-EA coupled via a Conflo III to a Micromass 100 GC-IRMS at the University of Mainz, or to a Finnigan Delta Plus XL GC-IRMS at the Universities of Tübingen and Lausanne, following Vennemann et al.63. Measured 18O/16O isotope ratios are reported in the δ-notation:18OPO4 values were measured with an external precision of ±0.3‰ (1 SD).Ag18OPO4 values were normalised to an Ag3PO4 standard produced by Elemental Microanalysis with a certified value of 21.7‰ . The analytical precision for this standard was better than ±0.3‰ (1σ). For untreated NIST SRM 120c Florida phosphate rock standard reference material, we obtained a δ18OPO4 value of 21.9 ± 0.3‰ (n = 9). This value compares well with the values around 21.7‰ initially measured by Lécuyer et al.64 and currently reported by most other laboratories as compiled in Chenery et al.65.The raw δ18OPO4 in the littoral of the southeast Mediterranean and in hypersaline lagoons, evolving from typical southeast Mediterranean water. The calculation is based on the temperature-dependent relation for isotope fractionation during biomineralisation of apatite by Lécuyer et al.22: T °C = 117.4–4.5(δ18OPO4 − δ18OSeaWater), where δ18OPO4 and δ18OSeaWater correspond to the isotope compositions of bio-apatite and seawater relative to VSMOW, respectively. This relation is valid for the temperature range of 8 °C < T < 32 °С relevant for the water temperatures encountered in the Mediterranean realm38.We calculated the equilibrium range of δ48:Body mass of ancient fish can be estimated from species-specific regressions with bone or molariform tooth size. In this study, we used the tooth length measurements recommended for the first molariform tooth Fig. , and regS. aurata first molariform tooth maximum length (FMTL) to fish total length (TL).Linear regression calculated from [2] TL = 32.21*FMTL + 154.07 −05*TL2.977 (r2 = 0.98).[3] FBM = 1.7086*eSupplementary Information


Patients with obsessive-compulsive disorder (OCD) have been found to show exaggerated error responses and prediction error learning signals in a variety of EEG and fMRI tasks, with data converging on the anterior cingulate cortex as a key locus of dysfunction. Considerable evidence has linked prediction error processing to dopaminergic function.In this study, we investigate potential dopaminergic dysfunction during reward processing in the context of OCD.n = 18) and controls (n = 18) whilst they learned probabilistic associations between abstract stimuli and monetary rewards in the fMRI scanner involving administration (on separate visits) of a dopamine receptor agonist, pramipexole 0.5 mg; a dopamine receptor antagonist, amisulpride 400 mg; and placebo. We fitted a Q-learning computational model to fMRI prediction error responses; group differences were examined in anterior cingulate and nucleus accumbens regions of interest.We studied OCD patients . In the imaging results, there was a significant interaction of group by drug . OCD patients showed abnormally strong cingulate signaling of prediction errors during omission of an expected reward, with unexpected reduction by both pramipexole and amisulpride . Exaggerated cingulate prediction error signaling to omitted reward in placebo was related to trait subjective difficulty in self-regulating behavior in OCD.There were no significant group, drug, or interaction effects in the number of correct choices; computational modeling suggested a marginally significant difference in learning rates between groups  contains supplementary material, which is available to authorized users. Obsessive-compulsive disorder (OCD) has been associated with deficits in learning and decision making  receives dense dopaminergic projections and is a target for deep brain stimulation in OCD  medication . All participants were also administered a total of 30 mg of domperidone orally for each treatment session to prevent emetic effects of dopamine receptor agonism. The administration was split up over three time points. Ten milligrams of domperidone was to be taken 12 h and 2 h before arrival, and further 10 mg of domperidone was administered together with the study medication.Subjective drug effects were assessed at two time points, 1 and 1.5 h after drug administration using the Bond-Lader Visual Analogue Scale  to investigate the group differences  and effect of drug  in stimuli choices and reaction times. We were interested in the proportion with which the subjects gave a “correct” response and whether this performance was dependent on trial types and drugs. Stimulus selection was considered correct when choosing the stimulus with the high probability of leading to winning of 50 pence in the reward trial , the picture with the high probability of receiving a neutral feedback picture in neutral trials, and stimulus with the high probability of leading to no punishment. In the neutral trials, the assignment of “correctness” is arbitrary but assigning one stimulus in each category to be the correct stimulus allows examination of to what extent response patterns differed across trial types.Computational learning model parameters (see below) were analyzed in a repeated measures ANOVA with group as a between-subjects’ variable and drug as a within-subjects’ variable.We estimated the positive and negative reward prediction error value for each trial, based on a basic Q learning algorithm , along with their temporal derivatives (used instead of slice timing correction), were defined in our model as follows: (1) reward expected value; (2) positive reward prediction error; (3) negative reward prediction error; (4) punishment expected value; (5) positive punishment PE; (6) negative punishment PE; (7) feedback presentation; (8) cue presentation. FSL requires all events to be set with non-zero duration: we set all to be 2 s. EVs 1–6 were based on the trial by trial computational model estimates of reward cue values and prediction errors. The timings of EVs1 and 4 correspond to cue presentation. EVs 2, 3, 5 and 6 correspond to the times of delivery of reward or omission of expected reward. EV2 represents reward prediction error when a reward is received , and EV3 represents the prediction error when an expected reward is omitted (negative prediction error). A positive EV3 parameter estimate, therefore, reflects lower BOLD values during omission of predicted rewards. Positive and negative reward prediction error activity were considered separately, as prior research in OCD has focused on brain correlates of errors using tasks where an error is usually accompanied by negative feedback or represents some kind of mistake, which is more analogous to negative prediction error than positive prediction error. In the present analysis, we focused on EV2 and EV3, the positive and negative reward prediction errors. We computed separate + 1 contrasts on EV2, and on EV3 at the single subject level . We defined two regions of interest . We examined total OCD symptoms (in patients only) as measured by the Y-BOCS scale , but no effect of drug (p = 0.39) or group X drug interaction (p = 0.86).  in the majority of reward trials (mean percentage of correct choices for controls 83.58% ± 26.3) and patients 72.45% (± 34.9)); there was a tendency for OCD patients to make more errors across all treatment conditions , but no effect of group (p = 0.62) or an interaction (p = 0.70). Bonferroni corrected post hoc tests revealed that participants independently of group reacted to the reward trials significantly slower under pramipexole (845.29 ms ± 29.22) compared with placebo , or amisulpride  , but no effect of drug (p = 0.94) or an interaction (p = 0.31). The marginally significant group effect suggests that controls, independent of drug, have a higher learning rate  than OCD patients . For β, there were no differences across groups (p = 0.82), drug (p = 0.1) or an interaction (p = 0.78). There was no effect of drug (p = 0.38), group (p = 0.22), or interaction (p = 0.54) on model fit.For the learning rate, Results from pooled data, collapsing over group and drug conditions, are presented in p = 0.88) or drug (p = 0.44). However, there was a significant interaction of group by drug . Within each group, we then conducted a Bonferroni corrected post hoc test analyzing the effect of the drug with a repeated measures ANOVA. In controls, the drug intervention did not influence negative reward prediction error processing, as shown by a non-significant drug effect (p = 0.16). In the OCD patients, however, there was a significant drug effect . On further analysis within the patient group, amisulpride  as well as pramipexole  led to significant differences compared with placebo but not between themselves : placebo > amisulpride, p = 0.030; placebo > pramipexole, p = 0.017; amisulpride > pramipexole, p = 0.70.The mean COPE values for negative prediction error in the anterior cingulate cortex were extracted to enable a region of interest analysis in accordance with our hypothesis. Data from three OCD patients were excluded because, for at least one of the sessions, the mean COPE value from the ROI was over three standard deviations from the mean of the entire sample. Following repeated measure ANOVA, there were no main effects of group , showing that the COPE values in the controls  were lower than in OCD . For the other two interventions, no significant differences were found between groups  > OCD , p = 0.46; pramipexole: controls  > OCD , p = 0.17).Furthermore, we applied a univariate ANOVA to each drug intervention separately Fig. . For plaThere were no significant group, drug, or interaction effects for positive prediction error in the anterior cingulate cortex in a repeated measure ANOVA.There were no significant group or drug or interaction effects on negative prediction error COPE values in the nucleus accumbens.F = 11.82, dfn = 1, dfe = 30, p = 0.002, partial ƞ2 = 0.28, 1-β error probability = 0.96), but no significant effect for drug (p = 0.45) or interaction of group by drug (p = 0.54).The mean COPE values for positive prediction error in the nucleus accumbens were extracted to enable a region of interest analysis in accordance with our hypothesis. Data from three controls and one OCD patient were excluded as, on at least one of the different treatment sessions, the mean COPE values from the ROI were over three standard deviations from the mean of the entire sample. Following repeated measure ANOVA, there was a significant main effect of group , showing that the COPE values in the controls  were significantly less than in the OCD group . For the amisulpride intervention, no significant differences were found between groups  > OCD ; p = 0.63). For the pramipexole intervention, we found a marginal effect  with controls  showing less activity compared with OCD patients .We then applied a univariate ANOVA to each drug intervention separately Fig. . For plap = 0.99; OCD patients: p = 0.60).We also conducted a Bonferroni corrected post hoc test analyzing the effect of the drug within each group using repeated measure ANOVA. Neither in controls nor in OCD patients did the drug intervention influence reward positive prediction error processing , as in our analysis above without the medication factor, but no SSRI medication dose effect (p = 0.8) or interaction (p = 0.3). In the nucleus accumbens, we did not find any significant effect of high SSRI dose or interaction on positive prediction error COPE values .In order to control for a potential interaction of the patients’ individual routine medication and the study drug treatment on reward prediction error signaling, we created a dichotomized medication variable  and used this variable as a between-subject factor in a repeated measure ANOVA on the three treatment types . We found a significant treatment effect on anterior cingulate cortex negative prediction error COPE values (p = 0.4). However, there was a significant negative correlation between the cingulate negative prediction error signal and trait self-regulation as measured by the SRQ , such that patients who reported more difficulty with self-regulation also demonstrated exaggerated negative error signals in the anterior cingulate .There was no correlation between placebo cingulate negative prediction error COPE values and OCD symptoms as measured by Y-BOCS score .There were no correlations between placebo COPE values in the nucleus accumbens and rating scales  was significantly greater than in controls. In controls, we did not detect a clear cingulate prediction error signal during omitted rewards. It is possible that our paradigm was not sufficiently sensitive to detect a negative prediction error signal in the control group, although it was clearly sufficiently sensitive to detect the pronounced signal in OCD. The lack of clear prediction errors in controls may have resulted from our procedure containing only 40 reward pair stimuli trials; for comparison, other prior prediction error fMRI studies have included larger numbers of potentially rewarding trials, such as 80  showed nucleus accumbens dysfunction in OCD ESM 2(PNG 14 kb)ESM 3(PNG 14 kb)ESM 4(PNG 20 kb)ESM 5(PNG 14 kb)ESM 6(DOCX 522 kb)


Our institution found that activated AKT can up-regulate the expression of microRNA-126 (miR-126), which can promote the proangiogenic ability of EPCs. The integrin ανβ3 receptors and AKT also express in EPCs and are closely related to proangiogenic function. Therefore, we hypothesized that ANGPTL3 could improve function of EPCs by binding to integrin ανβ3 receptors and up-regulating miR-126 expression via activating AKT, thus promoting the formation of new blood vessels, attenuating myocardial ischemia and improving heart function.Angiopoietin Like protein 3 (ANGPTL3) is at present considered as a central molecular target for therapy designed to reduce atherogenic lipids and atherosclerosis. However, concerns about the safety of inactivation of ANGPTL3 in patients with coronary artery disease (CAD) especially myocardial infarction (MI) have been raised. ANGPTL3 is reported to possess proangiogenic property. Angiogenesis is critical to the recovery of MI. Endothelial progenitor cells (EPCs) have multiple differentiation potential and play an important role in the angiogenesis post-MI. Promoting the function of EPCs could facilitate the angiogenesis and recovery of MI. Previous studies have shown that ANGPTL3 can promote angiogenesis in corneal of rats and promote angiogenesis of endothelial cells by binding to integrin α Coronary artery disease (CAD) is a leading cause of mortality worldwide, myocardial infarction (MI) is one of the most serious type of this disease. Remarkably good results have been achieved in the treatment of CAD by lipid-lowering strategy. Recent research reported individuals with Angiopoietin Like protein 3 (ANGPTL3) loss-of-function mutations demonstrate a 17% reduction in circulating triglycerides, a 12% reduction in low-density lipoprotein cholesterol, and a 34% reduction in odds of CAD, in contrast, high level of ANGPTL3 is positively associated with MI . TherefoHowever, concerns about the safety of inactivation of ANGPTL3 in patients with CAD especially MI have been presented , 8. Angiνβ3 receptor and induces haptotactic endothelial cell adhesion and migration [νβ3 is a kind of integrin receptor and expressed in cardiovascular endothelial cells. Integrin ανβ3 also was reported to express in EPCs and contribute to its migration [νβ3 in EPCs and facilitate EPCs to adhere and migrate.Angiogenesis in response to ischemia after MI is critical important and believed to be initiated by endothelial progenitor cells (EPCs). The angiogenesis ability of EPCs mainly depends on its proliferation, adhesion, migration, and in vitro angiogenesis capacity. Previous study reported ANGPTL3 binds to integrin αigration . The angigration . TherefoBesides, ANGPTL3 could stimulate phosphorylation of AKT in human microvascular vein endothelial cells which contributes to its proangiogenic ability . Our insνβ3 and increasing expression of miR-126 with an aim of providing more evidence for exploring the proangiogenic function of ANGPTL3 and insight into novel therapeutic target.Taken together, we hypothesize ANGPTL3 improves proangiogenic ability of EPCs via binding to integrin ανβ3 receptors, phosphorylated AKT, and miR-126. ② We will treat bone marrow-derived EPCs obtained from MI mice with or without ANGPTL3 recombinant protein, then detect the migratory ability, proliferative ability and in vitro angiogenesis of EPCs and also measure the expression of integrin ανβ3 receptors, phosphorylated AKT and miR-126.We will design some experiments to test this hypothesis. ① C57BL/6 mice will be randomly treated with placebo or Angptl3 recombinant protein or monoclonal antibody, evinacumab, against Angptl3. After 24 h’ treatment, they will be conducted with MI surgery by ligating the left anterior descending coronary artery. After that, mice will continue the previous treatment for several days. At the end of the experiment, we will evaluate whether the recombinant protein can improve the cardiac function, infarct area and capillary density and illuminate the safety of evinacumab treatment in MI mice. Capillary density was determined by immunohistochemical staining with CD31 antibody as previously described . We will


Contact resistance and current crowding are important to nanoscale electrical contacts. In this paper, we present a self-consistent model to characterize partially overlapped parallel contacts with varying specific contact resistivity along the contact length. For parallel tunneling contacts formed between contacting members separated by a thin insulating gap, we examine the local voltage-dependent variation of potential barrier height and tunneling current along the contact length, by solving the lumped circuit transmission line model (TLM) equations coupled with the tunneling current self consistently. The current and voltage distribution along the parallel tunneling contacts and their overall contact resistance are analyzed in detail, for various input voltage, electrical contact dimension, and material properties . It is found the existing one-dimensional (1D) tunneling junction models become less reliable when the tunneling layer thickness becomes smaller or the applied voltage becomes larger. In these regimes, the proposed self-consistent model may provide a more accurate evaluation of the parallel tunneling contacts. For the special case of constant ohmic specific contact resistivity along the contact length, our theory has been spot-checked with finite element method (FEM) based numerical simulations. This work provides insights on the design, and potential engineering, of nanoscale electrical contacts with controlled current distribution and contact resistance via engineered spatially varying contact layer properties and geometry. The electrical contact properties have been extensively studied in metal-semiconductor9, metal-insulator-semiconductor and metal-insulator-metal13 junctions. The growing popularity of novel electronic circuits based on graphene, carbon nanotubes (CNTs) and other new materials has made contact engineering crucial. CNT based devices, in particular, experience significant challenges because of the inter-tube connections. On macroscopic level, the exceptional intrinsic electrical properties15 of CNTs become elusive16. Contact resistances between CNTs profoundly affect the electron transport and reduce the electrical conductivity of carbon nanofiber (CNF)17, and greatly limit the performance of CNT thin film based Field Effect Transistors (FETs)21. One can naturally expect these issues also arising from other novel two-dimensional materials  as well as new nano-composites. While the work presented here is generalizable to these other material systems, here we choose carbon materials as examples.Nanoscale electrical contacts have attracted substantial attention due to the advancements in nanotechnology, material sciences and growing demands for miniaturization of electronic devices and high packing density. Contact resistance and their electro-thermal effects have become one of the most critical concerns of very large scale integration (VLSI) circuit designers, because of the excessive amount of Joule heating being deposited at the contact region25 are commonly found for CNT-CNT30, CNT-Metal33 and CNT-graphene35 contacts, where the contacting members are separated by very thin insulating layers. Tunneling effects in contact junctions significantly lower the electrical conductivity of the CNT/polymer composite thin films23. It is also found that tunneling resistance plays a dominant role in the electrical conductivity of CNT-based polymeric or ceramic composites27.Tunneling type of electrical contacts38 in 1960s. Simmon's formula have since been used for evaluating tunneling current in tunneling junctions39. Though there have been attempts to extend Simmons’ models to the field emission and space-charge-limited regimes41, it is always assumed that the tunneling junctions are one-dimensional (1D), i.e. there is no variations on the voltages drops along the length of the tunneling junction and the insulating film thickness is uniform. Thus, these existing models of tunneling junctions give no hint on the variation of tunneling current along the contact length and the importance of current crowding near the contact area when the two contacting members are partially overlapping  transmission line model for partially overlapped parallel contacts with spatially varying specific contact resistivity. Spatial dependence of specific contact resistivity of the contact interface may be introduced by many factors, such as nonuniform distribution of the resistive contaminants, oxides, or foreign objects at the contact interface, formation of contact interfaces with spatially varying thickness, or the presence of tunneling contacts between contact members. In the latter case, because of the nonlinear current-voltage characteristics of the tunneling junctionsess Fig. . For the43. Here we further extend the TLM model with the effects of spatially dependent contact resistivity. In the results and Discussion section, we consider three cases of parallel contacts: (1) constant specific contact resistivity, (2) linearly varying specific contact resistivity, and (3) tunneling contact resistivity depending on local junction voltages along the contact length. The first case of uniform specific contact resistivity along the contact length has been verified with COMSOL45 2D simulations. For the last case, for simplicity, we use the Simmons’ model38 to determine the local current-voltage characteristics across the tunneling junction. Though full scale quantum mechanical calculations may have to be used to accurately evaluate the nanoscale circuits, our model based on Simmons formula reveals the fundamental scalings and parametric dependence of current and voltage profiles, as well as electric contact resistance of tunneling contacts. Summary and suggestions for future research are given in the last section.The formulation of our 2D contact resistance TLM model is given in the next section. We would like to point out that, albeit an application of the standard transmission line theory based on the Kirchhoff's laws, the TLM has been used extensively with great success to characterize mesoscale and nanoscale electrical contacts47, and Klein tunneling junctions48.Note that, although this paper is focused on the normal Schrödinger tunneling type electrical contacts, the proposed TLM with spatially varying contact resistivity can be used for many other types of electric contacts, such as nanoscale Schottky contacts based on 2D materials heterostructureD, and the contact length is L. A thin resistive interface layer is sandwiched between them. Both contacts in Fig. Rsh1 and Rsh2 for conductor 1 and 2, respectively, such that there is no variation along the width w in the transverse dimension. The spatial dependent specific interfacial resistivity  is ρc(x), which is either predefined, or calculated from the local tunneling current in case of insulating tunneling layer38. We use the DC equivalent lump circuit transmission line model (TLM)44, as shown in Fig. Consider a parallel contact formed between two nanowires or nanotubes or between two conducting thin films or layers, as shown in Fig. In the contact region PQNM in Fig. I1(x) and I2(x) represent the current flowing at x through the lower contact member, MN and the upper contact member, PQ respectively, and V1(x) and V2(x) the local voltage at x along MN and PQ, respectively, and w is the effective transverse dimension of the contacts. When Δx → 0, Equation (where Jc(x) = Vg(x)/ρc(x) and Vg(x) = V1(x) − V2(x) are the local current density and the local voltage drop across the contact interface at x, respectively.where I1(x) + I2(x) = Itot = constant, where Itot is the total current in the circuit, to be determined from the boundary conditions. The boundary conditions for Eq. 2c), I1 = Itot. From Eqs ,,x = L isIo = wV0/Rsh1L, ρc0 = V0wL/Io, and Rc0 = Rsh1L/w. In normalized forms, Eq.   6) are solve Eqs  and (6)    6) can b and Eqs  and (6) ρc, the TLM equations, Eqs ,,ρc, the 49, as the current density is distributed to follow the least resistive path . It is important to note that as the specific contact resistivity 44 and field theory50.Figure }x¯ Fig. , whereas}x¯ Fig. , with thRsh2/Rsh1, with fixed specific contact resistivity Figure entclass1pt{minima42.The normalized contact resistance, from Eq.  is plott= 0, Eq.  becomes,45, for various combinations of −9 for each point.To verify the results obtained from our analytical solution, we performed numerical simulations using the COMSOL multiphysics softwareA increases, the overall contact interface becomes more resistive, therefore, the voltage drop A < 0, and at A > 0), while the maximum interface current A < 0, and at A > 0). The effects of A on the voltage We assume the specific resistivity varies linearly along the contact length Fig.  as \docuving Eqs  and 6)  \documenses Fig. , whereasral Fig. . The maxA increases, A.The normalized contact resistance, from Eq.  for line52.Here, we assume the parallel contacts are formed through a tunneling interface layer between the two contact members. For simplicity, we have made the following assumptions: (1) the thickness of interfacial insulating film in the contact area is uniform, and (2) the insulating film is sufficiently thin  so that charge trappings are ignored37,φ1 = W1 − χ and φ2 = W2 − χ. W1 and W2 are the work functions of contacting member 1 and 2 respectively, χ is the electron affinity of the insulating layer, which is 0 for vacuum. For For dissimilar contact members, the  current density at any location along the contact from contact member 1 to contact member 2 is calculated using Simmons’ formulaOn the other hand, the definitions of 36, in Eq.  and work function  of contacting members 1 and 2, permittivity (εr), thickness (D), and electron affinity (χ) of the interfacial insulator layer, and the contact length L. Using Eq. ,,V0, sheeρL = 20 kΩ/μm54, and diameter (or the width w) of 3 nm, an equivalent sheet resistance for both CNT contact members are estimated as 49. The work function of CNTs is W1 = W2 = 4.5 eV55. The interfacial layer is assumed to be vacuum . The voltage drop Vg(x) across and the tunneling current density Jc(x) through the contact interface are shown in Fig. L, vacuum gap distance D, and applied voltage V0. The profiles of both Vg(x) and Jc(x) are symmetric about the center of the contact, as expected for similar contact members  across the contact interface decreases, so does the tunneling current density Jc(x), as shown in Fig. L, since the total contact resistance of the tunneling junction decreases as the contact length increases  increases, but the current density Jc(x) decreases, which is because the tunneling junction becomes more resistive36. Figure Vg(x) and current density Jc(x) increase when the applied voltage Vo increases. More importantly, both Vg(x) and Jc(x) exhibit a stronger spatial dependence as V0 increases. This strong voltage dependence of electrical properties of the tunneling junction is in sharp contrast with those of ohmic contacts, where the shape of the profiles of Vg(x) and Jc(x), and the total contact resistance are independent of the applied voltage, and the current density scales linearly with the voltage drop, as discussed in Cases 1 and 2 above.We consider CNT-vacuum-CNT parallel contact as an example. Both contact members are made of the same single-walled CNTs. Using the typical value of linear resistivity of single-walled CNT cf. Fig.  below. AL , by (a), setting Vg = V0 and using Eq. (Vg → 0 (dotted lines)37. In the latter case, the tunneling current density is a linear function of Vg.A and Vg = 0. D decreases or the applied voltage Vo increases. As the tunneling junction resistance becomes nonlinear in these cases, it is necessary to use the coupled TLM equations, Eqs , Rc is almost independent of V0, as shown in Fig. V0 > 0.3 V, Rc decreases sharply with V0. This is because the junction is no longer ohmic and the tunneling resistivity ρc decreases nonlinearly with the junction voltage, as a function of position along the contact length. Ohmic approximations  for the simplicity of calculations. The resistivity of Ca, Al, Cu and Au are known to be 3.36 × 10−8 Ωm, 2.7 × 10−8 Ωm, 1.68 × 10−8 Ωm and 2.2 × 10−8 Ωm respectively57.Next, we extend our calculations for contacts of CNT with different metals – calcium (Ca), aluminum (Al), copper (Cu) and gold (Au). The work functions of Ca, Al, Cu and Au are taken as 2.9, 4.08, 4.7 and 5.1 eV respectivelyW2) and the permittivity of the thin insulating layer (εr), on the current and voltage characteristics in CNT-insulator-metal contacts. As the two contact members are different, the voltage drop Vg(x) and the tunneling current density Jc(x) are no longer symmetric along the contact length L. Figure W2. Figure D = 0.5 nm, the ohmic approximations using Eq. Vg = V0 could be a good approximation for the self-consistent TLM model  constant specific contact resistivity, (2) linearly varying specific contact resistivity, and (3) spatial dependent specific contact resistivity along the contact length due to current tunneling. The analytical solutions for the first case have been verified with FEM based numerical simulations. Our study provides a thorough understanding of the contact tunneling resistance, current and voltage distributions across nano and sub-nano scale metal-insulator-metal (MIM) junctions in parallel electrical contacts. The effects of contact geometry , and material properties  on the spatial distributions of currents and voltages across these contacts, and the overall contact resistance are studied in detail. While predominately classical in nature, the inclusion of tunneling current starts to address quantum effects in these small scale objects.D or the permittivity εr of the tunneling film is relatively large, or the applied voltage across the contact V0 is relatively small. When the 1D models become unreliable for small D or εr, or large V0, the self-consistent TLM equations coupled with the tunneling current  is only a simplified approximation of the 2D electrical contacts, where the current crowding and the fringing fields near the contact corners cannot be fully accounted for49. In order to accurately evaluate these effects as well as the impact of finite thickness in the contact members and the contact junction, and the possible parallel component of current flows in the interface layer, field solution methods need to be used58. Future studies may also include the effects of various contact geometry, insulator layer non-uniformities and AC response on the electrical properties of tunneling type contacts. An additional feature might include the role of capacitance and inductance in nano- and micro-scale structures, especially when a large contact resistance coupled with these reactive effects might introduce new time scales into time-dependent dynamic problems.In this formulation, we have ignored the effects of space charge and exchange-correlation inside the tunneling gap


However, this inclination is often lost in vivo due to multiple mechanisms mediated by tumor cells and local microenvironment. Multiple myeloma (MM) is a paradigm disease in which antitumor immunity is selectively impaired at the tumor site. By interrogating the immune reactivity of bone marrow (BM) Vγ9Vδ2 T cells to phosphoantigens, we have revealed a very early and long-lasting impairment of Vγ9Vδ2 T-cell immune functions which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even in clinical remission after autologous stem cell transplantation. Multiple cell subsets  are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the redundant expression of multiple immune checkpoints (ICPs). This review will address some aspects related to the dynamics of ICP expression in the BM of MM patients in relationship to the disease status  and how this multifaceted ICP expression impairs Vγ9Vδ2 T-cell function. We will also provide some suggestions how to rescue Vγ9Vδ2 T cells from the immune suppression operated by ICP and to recover their antimyeloma immune effector functions at the tumor site.Vγ9Vδ2 T cells are non-conventional T cells with a natural inclination to recognize and kill cancer cells. Malignant B cells, including myeloma cells, are privileged targets of Vγ9Vδ2 T cells  These date (ZA)  leading d BTN3A1 . Wilhelmefficacy –13. Undevia soluble and cellular factors  microenvironment to establish a protective niche to support their growth, immune evasion, and drug resistance. MM is invariably preceded by a precursor asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) with an estimated risk of progression to symptomatic disease ranging from less than 1% to more than 3% per year. This range depends on risk factors traditionally ascribed to intrinsic features of myeloma cells. Genomic alterations determining clonal advantage are already detectable in MGUS indicating that the probability of progression is also dependent on extrinsic factors such as the composition of the surrounding TME . TME in Immune escape in the BM of MGUS and MM patients is achieved by shifting the balance between immune effector and immune suppressor functions as in many other cancers. The immune suppressive mechanisms include the local recruitment and/or activation of immune suppressor cells like Tregs, MDSC, the protumoral polarization of tumor-associated macrophages and/or mesenchymal stem cells, and the differentiation and activation of Th17 cells . The wanFor many years, the conventional wisdom has been that the immune balance is tipped in favor of myeloma cell control in MGUS and early stages of MM, whereas the balance is tipped in favor of myeloma cell growth in advanced disease. The wisdom was based on the results obtained from experiments exploring the phenotype and function of T cells, NK cells, and NKT cells in MGUS and early MM stages compared to advanced MM stages –26. NowaImmune checkpoints/ICP-L are expressed by a variety of immune cells to control the strength and duration of immune responses and maintain T-cell homeostasis and self-tolerance . Smartlyin vitro studies showed enhanced myeloma cell killing by T and NK cells after PD-1 and/or PD-L1 blockade  and dexamethasone proved synergistic effects in phase I/II trials, nurturing hopes for therapeutic exploitation of PD-1 blockade in MM –50. Immu+ Vγ9Vδ2 T cells in breast cancer patients produced less IFNγ had lower cytotoxic activity and CD107 degranulation than PD-1− cells after challenging with PD-L1+ tumor target cells. Zumwalde et al. , is the prolonged TCR engagement by pAg in the TME. Preliminary results from our lab indicate that myeloma cells are not the only IPP producers in the TME, and that BMSC in MGUS and MM also produce and release very high amounts of IPP in the extracellular microenvironment   and the release of suppressive factors like IL-10  in Vγ9Vδ2 T cells could affect E Figure , may conE Figure  and polaE Figure . This hyWhether the mechanisms reported above are operative under baseline conditions or sharpened by pAg stimulation in the presence of PD-1 blockade is a matter of current investigation in our lab. Preliminary data suggest that the intracellular metabolic and signaling pathways evoked by PD-1 blockade could worsen the immune competence status of pAg-experienced Vγ9Vδ2 T cells.Vγ9Vδ2 T cells are programmed by default to behave as very effective professional killers of malignant B cells, including myeloma cells. We propose that Vγ9Vδ2 T cells are very precociously neutralized by myeloma cells in cooperation with neighboring cells in the TME of MGUS and MM patients. Finalistically speaking, it makes sense that myeloma cells inactivate or co-opt in their favor those immune cells mostly well suited to threaten their survival. This is done very early at the stage of MGUS and the uncontrolled production of IPP by BMSC and myeloma cells is probably a relevant initiating event.Functional interrogation of BM Vγ9Vδ2 T cells from MM patients in remission has revealed that clearance of myeloma cells does not automatically imply the recovery of a fully immune competent TME.in vitro, especially in MM at diagnosis or in relapse. These data indicate that additional immune suppressive mechanisms are involved in the anergy of Vγ9Vδ2 T cells. A working knowledge of these mechanisms may yield insight into the development of more effective interventions to fully exploit the immune potency of Vγ9Vδ2 T cells in MM and other cancers. This knowledge could be profitably implemented by next generation sequencing studies investigating the genetic and epigenetic consequences of cell-to-cell interactions of Vγ9Vδ2 T cells and other cell subsets in the TME of MGUS and MM patients.ICP/ICP-L abundantly expressed by tumor cells, immune effector cells, and immune suppressive cells are major promoters of immune suppressive cross talks in the TME at any stage and hamper the antimyeloma activity of BM Vγ9Vδ2 T cells. Single agent PD-1 blockade is insufficient to fully recover the antitumor activity of Vγ9Vδ2 T cells All authors have made substantial contributions to text and figures and have approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Each site in the (diploid) genome can be in one of five states, 0/0, 0/-, 0/1, 1/-, or 1/1, where 0 denotes reference allele, 1 denotes variant allele, and - denotes a deletion.λd: the rate of recurrent point mutation at a site.λl: the combined rate of deletion and loss of heterozygosity (LOH).We introduce two rate parameters: Q:Using these five states and rate parameters, we have the following instantaneous rate matrix Genotype 0 corresponds to states 0/- and 0/0.Genotype 1 corresponds to state 0/1.Genotype 2 corresponds to states 1/- and 1/1.We now abstract the genotypes as follows: Q given in Eq. (5) in the main text.Under this abstraction and the assumptions detailed in the caption of Table the following statement should be removed: “LOH events can result in the genotype transitions 1→0 and 1→2 whereas deletions can result in the genotype transitions 1→0,1→2 or 2→1. To compute the infinitesimal rates for these transitions, we introduce two parameters λd and λl that account for the effects of deletion and LOH respectively.”Given the explanation above, The statement“It is important to note that out of the three different types of events that could hint at a deviation from the infinite-sites assumption, SiFit currently models events  that affect the same genomic site more than once and the FP and FN errors in SCS data.”should be replaced by“It is important to note that out of the three different types of events that could hint at a deviation from the infinite-sites assumption, SiFit currently models events  that affect the same genomic site more than once and the FP and FN errors in SCS data.”The statement“These parameters being relative quantities , we choose a beta distribution as their prior.”should be replaced by“These parameters being relative quantities , we choosea beta distribution as their prior.”The following clarifications also apply to the main text:


Cataracts were induced in viable rat lenses cultured for 48 hours in TC-199 bicarbonate media through physical trauma, 10 mM ouabain as Na+/K+ ATPase ion transport inhibitor, or 1 mM of an experimental compound that induces water influx into the lens. Subsequent 48-hour incubation with 15 mM of lanosterol liposomes failed to either reverse these lens opacities or prevent the further progression of cataracts to the nuclear stage. Similarly, 3-day incubation of 47-year old human lenses in media containing 0.20 mM lanosterol or 60-year-old human lenses in 0.25 and 0.50 mM 25-hydroxycholesterol failed to increase the levels of soluble lens proteins or decrease the levels of insoluble lens proteins. These binding studies were followed up with in silico binding studies of lanosterol, 25-hydroxycholesterol, and ATP as a control to two wild type (2WJ7 and 2KLR) and one R120G mutant (2Y1Z) αB-crystallins using standard MOETM (Molecular Operating Environment) and Schrödinger’s Maestro software. Results confirmed that compared to ATP, both oxysterols failed to reach the acceptable threshold binding scores for good predictive binding to the αB-crystallins. In summary, all three studies failed to provide evidence that lanosterol or 25-hydroxycholesterol have either anti-cataractogenic activity or bind aggregated lens protein to dissolve cataracts.The paradigm that cataracts are irreversible and that vision from cataracts can only be restored through surgery has recently been challenged by reports that oxysterols such as lanosterol and 25-hydroxycholesterol can restore vision by binding to αB-crystallin chaperone protein to dissolve or disaggregate lenticular opacities. To confirm this premise,  At its anterior surface, the lens contains a single layer of proliferating epithelial cells. As these cells reach the equator, they elongate and differentiate into fiber cells that then make up the bulk of the lens. These elongated fiber cells become completely internalized with their ends joined by collagen at sutures that run from the lens center to anterior and posterior poles. The lens grows throughout life with new fiber cells continually laid on top of the older fiber cells so that the fiber cell depth within the lens is directly related to the age and stage of lens development4. The transparent lens is unique because this “enclosed bag of regularly ordered cells and proteins” has evolved into an internal micro-circulatory system composed of ions that are coupled to fluid movement that causes the lens to demonstrate behavior similar to that of a single cell7.The ocular lens is a transparent organ whose function is to focus light onto the retina. It is composed of epithelial cells that are enclosed in a thick capsule formed from epithelial basement membrane9. Since the lens fiber cells lack the capacity for protein turnover and repair, specific antioxidant defenses and protein chaperones are present within these fiber cells to protect lens proteins from post-translational changes and aggregation13. Among these are the small heat shock proteins (sHSP) α-crystallins with chaperone-like activity that play a central role in maintaining lens transparency by trapping the denaturing or unfolding proteins that are responsible for light scattering in the highly ordered lens fibers17.The lens is transparent because light scattering within the lens is minimized. It lacks blood vessels that can scatter and absorb light as well as light scattering cellular organelles such as nuclei, mitochondria, and endoplasmic reticula that are removed during differentiation of the epithelial into fiber cells. Light scattering is further minimized by the specialized organization and composition of the tightly packed fiber cells which contain structural crystallin proteins that also assist in maintaining the proper refractive index in the lens19. Since this aggregation and denaturation of lens proteins appears irreversible, the surgical removal of the opaque lens is currently the only treatment for restoring vision loss from cataracts13. As a result, the development of anti-cataract agents has primarily focused on supplementing the lens with biochemical intermediates or redox agents to reduce or prevent the post-translational modifications that eventually result in irreversible changes in lens protein structure and aggregation. An exception is the pharmacological prevention of diabetic cataracts where a specific enzyme that initiates sugar cataracts has been identified22.Cataracts develop from the loss of lens transparency associated with increased light scattering and changes in refractive properties. The protective lens antioxidant defenses and the molecular chaperone reserve of α-crystallin decrease with age so that the aging lens can no longer adequately protect itself from post-translational modifications of lens proteins. This leads to increased light scatter as a function of protein aggregation of post-translationally modified structural proteins which has been experimentally and clinically established in pre-cataractous lenses by dynamic light scatteringet al.23 and Makley et al.24 who report that interaction of lanosterol or 25-hydroxycholesterol with α-crystallin chaperones enhance the ability of these chaperones to restore lens clarity by increasing their ability to physically dissolve protein aggregates and/or the denatured amyloid-like fibril proteins present in cataractous lenses. These reports have subsequently been expanded to include dissolving aggregated proteins in cataracts from two additional congenital mouse models26. The striking possibility of a non-surgical cataract removal has received worldwide coverage by the news media and encouraged investigators to focus on developing anti-cataract drugs that reverse rather than prevent cataract formation. It has also led to the commercialization of lanosterol eye drops29. However, the ability of these compounds to restore lens clarity has not been independently confirmed. For example, a recent report has shown that culturing 25 mM lanosterol with the lens nuclei from 40 age-related cataractous human lenses for 6 days at room temperature failed to either dissolve the aggregated proteins or restore the clarity of the lens nuclei30. Similarly, clinically administering eye drops containing 5 mM lanosterol dissolved in olive oil two times daily for the first week and three times daily for the next seven weeks to a patient with idiopathic unilateral juvenile nuclear cataracts failed to produce any relevant clinical effect in reversing either the cataract or halting the progressive worsening of visual acuity with an increasing of myopic shift31. Triparanol, an inhibitor of the conversion of lanosterol to cholesterol, has also been shown to not only induce cataract formation but also increase tissue lanosterol levels34. The presence of 25-hydroxycholesterol in human lenses has also been linked to the presence rather than absence of cataracts35. To clarify these contrasting findings, we pursued a series of studies to evaluate whether oxysterols such as lanosterol or 25-hydroxycholesterol can reverse experimentally induced cataracts or to re-dissolve aggregated lens crystallin proteins. We also explore if either oxysterols adequately bind to αB-crystallin chaperones at the molecular level.This paradigm that vision can only be restored through cataract surgery has recently been challenged by Zhao All procedures involving live animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Nebraska Medical Center. Human lenses were obtained from donor eyes from the Saving Sight Eye Bank, Kansas City, MO and stored at −85 °C prior to use.2 at 37 °C38.Eyes from young  Sprague Dawley rats were immediately enucleated upon death from carbon dioxide asphyxiation. The intact lens from each eye was removed by careful dissection from a posterior approach and cultured as previously described in sterile TC-199 bicarbonate media containing 30 mM fructose and 20 U/mL of penicillin-streptomycin in a humidified incubator under an atmosphere of 95% air and 5% COFollowing overnight pre-incubation to ensure intact lenses were not damaged during dissection, all clear lenses were transferred into 24-well culture plates containing 2 ml of the standard culture media for 48 hours according to the following 4 groups (6 lenses/group). The first group served as the untreated control, while each lens in the second group was squeezed at the equator with forceps to induce blunt trauma opacities. In the third group, opacities were induced by addition of 10 mM of the  Na/K ATPase inhibitor, ouabain, and in the fourth group opacities were induced by culture with 1 mM of an experimental toxic glycoprotein chaperone that induces osmotic cataract. After 48 hours the culture media for each lens was replaced with fresh TC-199 bicarbonate media containing 15 mM of lanosterol liposomes and the lenses were cultured an additional 48 hours. At the end of the second 48-hour culture period, each lens was carefully washed with PBS solution and transferred to culture plates containing PBS solution. The appearance of each lens was immediately photo-documented by placing the lens over a light source containing a grid. Each photograph was then standardized by adjusting the pixel densities of each outer grid line to a standard value.Lanosterol (Alpha Chem) was dissolved in a 250 mL round bottom flask containing acetonitrile and the solvent was removed under vacuum in a rotary evaporator. The resulting glassine layer of lanosterol coating the inner surface of the round bottom flask was then removed by scraping with a spatula and then an appropriate amount of TC-199 bicarbonate media was added to the round bottom flask to give a 15 mM solution. The lanosterol sheets were converted to liposomes by sonication using a micro homogenizer tip (MISONIX Fisher Scientific Sonicator Ultrasonic Processor XL) until the solution appeared homogenous (~5 min). Equal aliquots (2 mL) were pipetted into the 24-well culture plates for experimental use.2HPO4, and 1.5 mM KH2PO4, pH 7.4, containing 0.03% sodium azide. Lanosterol dissolved in 12.5 µL of ethanol was then added to some of the lens pieces in PBS to give a 0.20 mM lanosterol test solution in a total volume of 500 µL. All lens pieces (with or without lanosterol) were incubated at 37 °C in the dark for 72 hours and then homogenized. The soluble and insoluble protein fractions were separated by centrifugation at 13,000 rpm for 20 min at room temperature in an Eppendorf centrifuge. The protein content of the soluble protein in the supernatant was determined using the Bio-Rad Protein Assay . Control samples were similarly prepared using only 12.5 µL ethanol.Two human lenses from a 47-year-old donor were cut through the center of the lens into four equal quadrants and weighed. One piece from each lens tissue was placed in PBS media composed of 137 mM NaCl, 2.7 mM KCl, 10 mM NaSix frozen lenses from 60-year-old donors were each cut into three equal pie shapes with each sample composed of two pie shapes from different lenses. These formed 9 samples were each immersed into 500 µL of PBS containing 0.03% sodium azide and 10% ethanol containing either 0.0, 0.25 or 0.50 mM of 25-hydroxycholesterol. The samples were then incubated at 37 °C for 72 hours in the dark. Following incubation, the samples were homogenized and the water-soluble fraction was separated from the insoluble fractions by centrifugation at 13,000 rpm for 20 min at room temperature. The water-insoluble fractions were treated with 100 µL of 0.1 M NaOH for 2 hours to solubilize the proteins. The protein content in all samples was measured using the Bio-Rad Protein Assay. Samples incubated only with 10% ethanol served as controls.Attempts to solubilize lens proteins by lanosterol and 25-hydroxycholesterol were also carried out using lens homogenates prepared from 72 year old human lenses. Tissue homogenates containing 0.25 and 1.0 mg protein in 0.5 ml PBS (containing protease inhibitor cocktail) and oxysterols at a final concentration of 0, 100 or 200 µM were incubated at 37 °C for 24 hours. At the end of the incubation, the tubes were centrifuged at 13,000 rpm for 15 min at 4 °C. The protein content in soluble and insoluble fractions was measured using Bio-Rad protein assay reagent as described previously. These experiments were conducted three times separately using homogenates prepared from a pair of lenses.39. To investigate the binding activities of these three compounds to αB-crystallins, docking studies were conducted using MOE’s internal dock and Schrödinger’s Glide dock40. The target proteins were three αB-crystallins obtained from the Protein Data Bank (PDB): a human wild type dimer 2WJ741, a human wild type αB-crystallin 2KLR42, and a R120G mutant, 2Y1Z43.The Chemical Computing Group’s Molecular Operating Environment 2016 (MOE) and Schrödinger LLC’s program, Maestro 11, were used for this study. The structures  = e(docking_score*1000/(1.98*298.15))/10−6.Once the binding pocket was identified, ligands were docked to the binding site using MOE docking with the “Triangle Matcher” placement and the flexible “Induced Fit” method. Output was a London ΔG binding score. Predicted K2WJ7 and 2KLR and the R120G mutant 2Y1Z, followed by Glide dock with scoring function of Extra Precision (XP). The docking scores of each protein-ligand docked complex were expressed in kcal/mol, with −9 kcal/mol approximated to an IC50 or Kd in the high nM/low μM range. A more negative docking score indicates stronger ligand binding to the protein.The Maestro docking studies were conducted using Glide Dock by first defining the binding pocket using the Glide Grid Generation method using centroids identified above for all three proteins, wildtypes 48. Therefore, lens culture studies were conducted to investigate the ability of lanosterol to reduce lens opacities. Cataracts were experimentally induced with blunt trauma, by ATPase ion transport inhibition with 10 mM of ouabain, or by osmotic stress induced with 1 mM of a toxic experimental compound that induces water influx into the lens. Untreated lenses were used as the control. Within 48 hours of culture, all the lenses developed opacities except the control untreated group. As illustrated in Fig. The new paradigm suggests that lanosterol and other oxysterols are able to clear cataracts, presumably by boosting the α-crystallin’s chaperone activity to dissolve the light scattering, denatured, aggregated lens proteins responsible for opacification. Since transparency and biochemical viability can be maintained for up to two weeks, lens organ culture has become a valuable technique in investigating the mechanisms of both lens homeostasis and cataractogenesisThe above described lens culture studies suggest that lanosterol either inadequately binds to lens proteins or that the chaperone activity of the assumed bound lanosterol to α-crystallin complex is not adequate to reverse or alter the progression of cataracts as previously proposed. To evaluate this possibility, two lenses from a 47-year-old human were equally divided into 4 pie shaped portions and incubated in the dark for 3 days at 37 °C in media containing 0.2 mM lanosterol dissolved in 10% ethanol. As shown in Fig. 24. For these studies, 6 frozen lenses from three 60-year-old donors were each cut into three equal pie shaped fragments, thawed and incubated in the dark for 3 days at 37 °C in media containing either 0.0, 0.25 or 0.50 mM of 25-hydroxycholesterol. For each group, two pieces from different lenses were combined for each experiment. The percentage of protein in soluble and insoluble fractions were estimated separately for all samples in each group and averaged. As summarized in Fig. Since in our experiments lanosterol failed to increase soluble protein levels, the binding studies were expanded to include 25-hydroxycholesterol whose binding power has been reported to be superior to that of lanosterolThe lack of binding demonstrated by both oxysterols could possibly be due to the failure of both oxysterols to adequately penetrate the lens fibers in the incubated lens fragments. To investigate this possibility, protein solubilization studies with 100 and 200 µM concentrations of lanosterol and 25-hydroxycholesterol were carried out with lens homogenates of 0.5 and 2.0 mg/mL protein concentrations in PBS (containing protease inhibitor cocktail) prepared from 72 year old human lenses. Changes in the soluble protein levels after 24-hour incubation at 37 °C are illustrated in Fig. in silico studies to gain insight into chaperone binding to lanosterol and 25-hydroxycholesterol at the molecular level were conducted. As specified in the Method Section, lanosterol and 25-hydroxycholesterol were docked to three small αB-crystallins heat shock chaperones, two human wild type dimers, 2WJ741 and 2KLR43, and an ARG120GLY mutant, 2Y1Z43. As an additional control, ATP, which has been reported to bind these αB-crystallins, was also examined. Molecular modeling and docking studies were conducted using MOE dock and Schrödinger Glide dock standard docking methods. As specified in the Method section, the appropriate binding modes were determined by docking the ligands to two potential pockets, an ATP pocket and the dimer interface. The top five docked poses were collected and the best docking scores along with predicted Kd values are presented. The MOE dock scores are reported as London ΔG binding scores. London ΔG binding scores generally indicate that a value of −12.00 corresponds to the low nM range and a dissociation constant, Kd, of −12.3 kcal/mol, a value of −8.00 corresponds to the low µM range and a Kd of −8.2 kcal/mol, and a value of −4.00 corresponds to the low mM range and a Kd of −4.1 kcal/mol.Since both oxysterols failed to increase the anticipated soluble protein levels in lenses or lens homogenates where age-related protein denaturation had occurred, this suggests that oxysterol binding to the crystallin chaperones may be inadequate. Therefore, additional 2WJ7 and 2KLR  were observed. This indicates that the methods employed successfully predicted the expected feasible nanomolar range binding of ATP to the ATP binding pocket of these chaperones. However, ATP binding to mutant 2Y1Z appeared to be lower as suggested by the lower docking score . In contrast, the MOE docking results suggested only weak binding of lanosterol  and 25-hydroxycholesterol  to the dimer interface containing the PHE centroid in the wild-type 2WJ7 model. Furthermore, the results predict that no binding of either oxysterols to 2KLR and 2Y1Z is anticipated because positive docking scores suggest unfavorable binding. A value of “n.d.” indicates that no docking data could be acquired.The MOE docking results, summarized in Table 2WJ7 and 2Y1Z mutant models were obtained; however, oxysterols were not able to bind to the ATP binding pockets of these αB-crystallins. Adequate ATP binding onto the dimer interface of the 2WJ7 model was also observed, however, the binding of ATP to the interface of the mutant 2Y1Z was much weaker, suggesting that ATP would preferentially bind to the ATP pocket rather than the dimer interface. This is not surprising since natural selection has preferred ATP to bind to the ATP binding pocket. Only very weak oxysterol binding to the dimer interface of the αB-crystallins was observed, suggesting that the oxysterols are only very weak binders or do not bind at all to either the ATP binding pocket or at the dimer interface.Could the oxysterol binding perhaps occur at the ATP binding site? To address this question, docking studies with ATP, 25-hydroxycholesterol and lanosterol were also conducted using the Glide dock program to the β4-β8 groove, which is the ATP interactive binding region, as well as the dimer interface containing the PHE 55 centroid  β- and γ-crystallins. Solubilizing these insoluble, light scattering aggregated lens proteins should result in an increase of soluble proteins and a decrease in insoluble proteins, i.e. “the cataract should be dissolved”.The mammalian lens contains millions of densely packed fiber cells that are continuously formed throughout life from differentiating lens epithelial cells. These fiber cells contain three major crystallin proteins families: α-crystallin, which resembles small heat shock proteins, and β- and γ-crystallins, that have structural and functional roles in maintaining transparency and the high refractive index of the lens. Increased light scattering leading to the appearance of lens opacities is directly linked to lens protein aggregation of the β- and γ-crystallins. To counteract this aggregation, Horwitz50. With the loss of its organelles during fiber cell differentiation, the plasma membrane forming the external boundary of the fiber cell cytoplasm becomes the only remaining membrane in mature fiber cells51. Its extremely high cholesterol content makes this membrane one of the most saturated and ordered (stiff) membranes in the human body. While the need for this high lipid content is unclear, disturbances of cholesterol homeostasis can lead to cataract formation.Cholesterol derivatives, in addition to interacting with crystallins, also play an integral role in the regulation of cholesterol-dependent processes in fiber cell plasma membranes and in the maintenance of fiber cell membrane homeostasis52. This compound inhibited cholesterol synthesis at the desmosterol step, several steps downstream of lanosterol and resulted in the cellular accumulation of lanosterol34. Although lens lanosterol levels were never specifically measured after triparanol administration, it can be assumed that downstream inhibition of cholesterol synthesis would similarly increase lanosterol levels in the lens. Therefore, altered cholesterol homeostasis may lead to cataract formation despite an increase in lanosterol. Similarly, cataracts have been associated with the lenticular accumulation of cholesterol oxides such as 25-hydroxycholesterol35. Cataract formation has also developed with other cholesterol lowering agents such as statins and fibrates. Animal studies and several clinical studies report that patients undergo cataract surgery at higher rates with long-term statin or fibrate administration56. Statins inhibit cholesterol biosynthesis at the initial mevalonic acid level, while fibrates modify lipids, decrease triglycerides, and alter cholesterol levels of HDL/LDL by activating alpha peroxisome proliferator-activated receptors (PPAR-α). Studies suggest that cataract formation is induced by statins because they lower the isoprenylation of small GTPases57. At present, the relationship between the regulation of cholesterol-dependent processes in lens fiber cells and their plasma membranes and cataract formation is not well established and more studies in this area are required.The first synthetic cholesterol lowering drug associated with irreversible cataract formation was triparanol (MER-29)In vitro organ culture of lenses has become a powerful experimental tool for not only investigating the relationship between lens metabolism and lens clarity, but also for elucidating the mechanism(s) of cataract formation by drugs and biochemical agents58. These culture studies require carefully excised lenses that are cultured in specially buffered and osmotically compensated culture media at 37 °C in order to maintain their viability and clarity60. Moreover, because the use of freshly excised lenses are ideally required, the majority of culture studies employ readily available rat lenses. Rat lens organ culture studies have been used to elucidate the effect of statins on cataract formation57, the role of osmotic changes on sugar cataract formation61, and the role of oxidative stress on oxidation induced cataracts63.65, while ouabain alters the intracellular Na+ and K+ levels and affects the anabolism and catabolism of protein during cataract formation66. Experimentally induced osmotic changes initiate increased endoplasmic reticulum (ER) stress in lens epithelial cells that subsequently results in the generation of reactive oxygen species (ROS) and oxidative stress69. These biochemical insults all lead to similar protein destabilization, the presence of partially unfolded aggregation-prone intermediates, and the formation of insoluble, light-scattering protein aggregates that appear as lens opacities13. Subsequent 48-hour exposure of these lenses to 15 mM of lanosterol liposomes failed to decrease the insoluble, light-scattering protein aggregates that had developed into lens opacities. More importantly, the presence of lanosterol during this 48-hour period also had no effect on influencing the further progression of protein aggregate development because all lens opacities progressed to the advanced nuclear stage  with wild-types 2WJ7, 2KLR and mutant 2Y1Z αB-crystallins chaperones  that were in good agreement with Palmisano et al.79 who determined the binding constant for ATP to the α-crystallin to be . Significantly better ATP binding scores were obtained for binding at the ATP binding pocket of 2WJ7 and 2Y1Z , and at the interface with 2WJ7 (−9.43). In contrast to ATP, the present MOE and Glide docking scores suggest that lanosterol and 25-hydroxycholesterol binding to α-crystallin is unlikely at less than micromolar concentrations. For example, the Kd for lanosterol and 25-hydroxycholesterol, respectively, are predicted to be 653 µM and 11 µM against wt 2WJ7  is normally considered to be low affinity. Both our docking results, and the fact that the Makley’s and Zhao’s experiments used high concentration (from 100 µM to 15 mM), suggest that under 10 µM or lower concentration, it is unlikely for lanosterol to have significant and specific binding to α-crystallin.Our docking results were not contradictory to previously reported data in Makley 2WJ7 contains residues ASP 109 and ARG 120,  and surrounded by positively charged residues (blue). Inspection of this dimer interface reveals that the central open groove binding pocket identified in 2WJ7 becomes narrower in 2KLR and is blocked in 2Y1Z. 2KLR has a second salt bridge between ASP 80 and ARG 107 which further stabilizes the dimer interface so that a closed-groove conformation can be maintained. Such a scenario may result in a poor binding environment for both lanosterol and 25-hydroxycholesterol. This structural difference helps to explain why the docking scores of ATP and oxysterols are smaller in 2KLR than those of 2WJ7. Similarly, the open-structure observed in wild type 2WJ7 is disrupted in the ARG120GLY mutation 2Y1Z so that the mutation of ARG120 to GLY makes it impossible to form a stabilizing salt bridge between ARG120 and ASP109. Instead, salt bridges between ASP 80 and HIS 83 are formed in which the side chains of these two residues block the central pocket so that no suitable binding with either oxysterol can occur.Additional protein-ligand interaction in our studies further disclosed the underlying cause for the poor binding of oxysterols, such as lanosterol, to the current αB-crystallins models. The human wild-type dimer 20, Fig.  which weet al.24, have not identified specific binding residues used for docking oxysterols to αB-crystallins, residue PHE 55 of the “A” chain was chosen as the centroid to define the binding pocket for the dimer interface in 2WJ7, 2KLR and 2Y1Z. In addition, the Walker-B ATP-binding motifs containing the sequence 82KHFSPEELKVKVLGD96 were used for docking on both wildtypes, 2WJ7 and 2KLR and mutant 2Y1Z to determine whether the oxysterols can also bind to the ATP binding pocket. This ATP binding pocket was previously reported by Ghosh et al.44. Both binding regions are highlighted in Fig. Since no crystal structures of the αB-crystallin/oxysterol complex are available, and the published papers, including that of Makley 79. Depending on the αB-crystallin model employed, the binding constant for ATP was estimated to be in the range of 0.1 to 80 µM  of αB-crystallin incubated with 25-hydroxycholesterol. Consistent with the present docking and binding studies, this suggests that 25-hydroxycholesterol is held within αB-oligomers without any apparent binding interaction with the protein. This interaction may be similar to the partitioning of dexamethasone with α-crystallin as reported by Augusteyn and co-workers and interpreted as a non-functional interaction80.Despite the poor binding results of 25-hydroxycholesterol with the three αB-crystallin models and the inability of 25-hydroxycholesterol to solubilize lens proteins Figs  and 5, sLens protein aggregation occurs within the millions of mature lens fiber cells where αB-crystallin chaperones are located. Therefore, the oxysterols must penetrate into the millions of fiber cells to bind to the αB-crystallin chaperones. While investigators have focused on lens protein aggregation and the role of oxysterol induction of chaperones in reversing lens protein aggregation in solutions, missing is the consideration of the unique properties of the lens fiber membranes and the required demonstration that oxysterols can actually penetrate through the highly saturated, stiff cholesterol containing membranes of these fiber cells which are tightly interconnected through ball and socket junctions. In fact, in the rat lenses cultured with lanosterol liposomes Fig. , the ambin silico molecular modeling and docking studies, provide no evidence that oxysterols have anti-cataractogenic activity or can bind aggregated lens protein to dissolve cataracts. We join two other laboratories in failing to independently confirm the premise that oxysterols are effective in reversing cataracts.In summary, while the concept of a medical treatment for cataracts that can reverse protein aggregation to restore lens clarity is exciting, independent confirmation that oxysterol treatment, such as with lanosterol or 25-hydroxycholesterol, can restore vision is required. The results of the present studies using the established technique of whole lens culture and human lens protein binding studies, complemented with


Scientific Reports 10.1038/s41598-019-42440-2, published online 08 May 2019Correction to: This Article contains a typographical error in the Results section under subheading “Interaction with Lipopolysaccharides” where,“The LAL test was used to assess the ability of the AMPs to interact and neutralize the LPS. Both melimine and Mel4 inhibited LPS’s ability to stimulate amoebocyte lysate in a dose-dependent manner as demonstrated in Table 3.”should read:“The LAL test was used to assess the ability of the AMPs to interact and neutralize the LPS. Both melimine and Mel4 inhibited LPS’s ability to stimulate amoebocyte lysate in a dose-dependent manner as demonstrated in Table 2.”