Thus far, multiple hypotheses regarding its physiological/pathophysiological roles have been proposed, especially related to Parkinson’s disease or alcohol addiction. The aim of this review was to outline studies related to salsolinol, with special focus on in vivo and in vitro experimental models. To begin with, the chemical structure of salsolinol together with its biochemical implications and the role in neurotransmission are discussed. Numerous experimental studies are summarized in tables and the most relevant ones are stressed. Finally, the ability of salsolinol to cross the blood–brain barrier and its possible double-faced neurobiological potential are reviewed.Salsolinol  is a tetrahydroisoquinoline derivative whose presence in humans was first detected in the urine of Parkinsonian patients on  The main aim of this review was to outline the most relevant studies related to salsolinol and focus on in vivo and in vitro experimental models, its possible double-faced (neurotoxic vs. neuroprotective) neurobiological potential and imply its enigmatic role in the periphery.+ ions   was proven in vitro in human brain homogenates  non-enzymatically by autoxidation -salsolinol and DMDHIQ+ ions depends on the activity of their synthesizing enzymes  to produce H2O2 and dihydroxyphenylacetic acid (DOPAC) or via autooxidation to produce O2− and reactive quinones. DA- or l-DOPA-induced neurotoxicity mediated by the generation of free radicals has been reported in damaged neurons both in vitro and in vivo , which has no clearly elucidated biological activity , the product of l-DOPA.On the other hand, the presence of acetaldehyde is usually associated with alcohol ingestion; however, in fact, rapid metabolism of acetaldehyde by the liver maintains its blood levels extremely low. Even if the blood acetaldehyde levels were significant, either because of genetic variation in alcohol-metabolizing enzymes or certain enzyme inhibitors, acetaldehyde is hardly able to cross the blood–brain barrier . l-DOPA is converted into dopamine by the aromatic l-amino acid decarboxylase , with pyridoxal phosphate as the cofactor. Dopamine is found in many types of food, but it cannot cross the blood–brain barrier; therefore, it must be synthesized locally in the brain. Dopamine can be further used as a precursor in the synthesis of the norepinephrine and epinephrine or can be broken down into inactive metabolites by monoamine oxidase, catechol-O-methyl transferase, COMT  and aldehyde dehydrogenase. (R)-Salsolinol synthase possibly catalyzes the reaction of dopamine with acetaldehyde or pyruvic acid to produce (R)-salsolinol or (R)-salsolinol 1-carboxylic acid. Both (R)- and (S)-salsolinol can be also formed non-enzymatically by the Pictet–Spengler reaction of dopamine with acetaldehyde. N-Methyltransferase catalyzes the N-methylation of (R)-salsolinol, but not that of (S)-salsolinol, into N-methyl-(R)-salsolinol. The oxidation of N-methyl-(R)-salsolinol can be either non-enzymatic (autooxidation) or enzymatic by an oxidase (sensitive to semicarbazide) and leads to the formation of 1,2-dimethyl-6,7-dihydroxyisoquinolinium ions (DMDHIQ+). Both (R)- and (S)-salsolinol can be metabolized by COMT to form 6-methoxy-(R/S)-salsolinol and 7-methoxy-(R/S)-salsolinol. It remains unknown under what circumstances dopamine might lead to salsolinol production and whether acetaldehyde comes from local production in the brain or from the periphery. It is also uncertain whether exogenous salsolinol (delivered from food) can cross the blood–brain barrier.The direct precursor of dopamine, l-DOPA with the combination of MAO (pargyline) and COMT (tolcapone) inhibitors.The endogenous synthesis of salsolinol, although relatively straightforward, requires appropriate conditions to be maintained. Thus, it seems reasonable to hypothesize that increased salsolinol levels might be rather a consequence of dysregulated enzymatic pathways due to neurodegeneration, genetic mutations, or exogenous inhibitors. Indeed, the peripheral and central catecholamine system acts as the first responder to environmental stimuli. Kocur  reportedN-methylated salsolinol -salsolinol in the human striatum. The level of a dopamine metabolite, homovanillic acid (HVA), or the ratio of HVA/dopamine, an indicator of dopamine turnover, did not correlate with the levels of catechol isoquinolines in the human brain -salsolinol should be the only enantiomer present in human brain tissue. Some of the analytical difficulties of salsolinol quantification have been recently solved because of the development of more sensitive methods for its analysis -salsolinol in cerebrospinal fluid from untreated patients with PD was significantly higher than that in controls, especially at the early stage of the disease  compared that in controls (18.9 ± 15.0 pmol/min/mg of protein) -salsolinol, and N-methyl-(S)-salsolinol were found in the caudate nuclei of PD patients compared with those in the normal human brain -salsolinol was found to occur selectively in the substantia nigra, hypothalamus, and hippocampus, possibly suggesting that the distribution should not solely depend on DA but also on the activity of the synthesizing enzymes. Indeed, the N-methyltransferase activity was found to be higher in the rat nigro-striatum than in other brain regions -salsolinol, N-methyl-(R)-salsolinol, and DMDHIQ+ ions were accumulated especially in the striatum and, to a lesser extent, in the substantia nigra. Dopamine and noradrenaline levels were reduced in the substantia nigra and in the striatum, whereas serotonin and its metabolites were not affected. In vitro experiments showed that the binding of DMDHIQ+ ions to melanin in the substantia nigra was enhanced by Fe(II) whereas Fe(III) enhanced the release of the ions from melanin, and released DMDHIQ+ ions could further cause inhibition of the mitochondrial enzymes and depletion of ATP from dopaminergic neurons , and there was a tendency toward an excess of (R)-salsolinol in brain areas with a lower dopamine concentration -salsolinol was present in high concentration in the neuro-intermediate lobe as well as in median eminence extracts of males, and intact and ovariectomized female rats. It was hypothesized that salsolinol could be synthesized in situ and could play a role in the regulation of pituitary prolactin (PRL) secretion, without an effect on the secretion of other pituitary hormones   , and R-salsolinol uncovered close structural similarities, suggesting a planar ring system, an electrophilic center and a nucleophilic moiety with H-donor properties at a constant distance (of 6.560.3 A °) as key properties of an OCT2-specifc substrate  activities in situ. Indeed, the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and, most significantly, 5-hydroxyindolacetic acid (5-HIAA) were reduced -salsolinol, (R)-salsolinol, and N-methylnorsalsolinol  by salsolinol was first reported by Yamanaka . SalsoliO-methyltransferase  and as a competitive inhibitor -salsolinol yielded almost equivalent amounts of the two possible 6- and 7-methyl ethers, whereas 7-O-methyl derivative was 88% of the product when the (R)-enantiomer was methylated (Hötzl and Thomas Hötzl and Thomas  reportedl-tyrosine. (R)-Salsolinol deleted the allostery of TH to the biopterin and reduced the activity more markedly than its (S)-enantiomer. The authors suggested that, under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain , EC 1.14.16.2, TH)—prepared from rat brain. The inhibition constant, Ki, for salsolinol was 14 μM, and the inhibition was competitive to a cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin , EC 1.14,16,2, TPH (l-tryptophan hydroxylase)). The inhibition was non-competitive in terms of either the biopterine cofactor or substrate l-tryptophan. 1,2[N]-Dimethyl-6,7-dihydroxyisoquinolinium (DMDHIQ+) ion was an extremely potent inhibitor -l-erythro-5,6,7,8-tetrahydrobiopterin, and these two elements were inhibited by salsolinol competitively and non-competitively. Thus, salsolinol enantiomers might be naturally occurring inhibitors of indoleamine metabolism -and (S)-enantiomers of salsolinol were also found to inhibit the activity of tryptophan hydroxylase prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition was found to be non-competitive with the substrate N-methyl-(R)-salsolinol led to concentration-dependent decreases in the activity of acetylcholinesterase . ACh concentrations in the striatum treated with (R)-salsolinol or N-methyl-(R)-salsolinol were increased. N-methyl-(R)-salsolinol caused a significant decrease in dopamine concentrations, and (R)-salsolinol reduced the concentrations of dopamine metabolites in the striatum . (R)-salsolinol and its derivative Salsolinol competitively inhibited the activity of debrisoquine 4-monooxygenase in rat liver microsomes, demonstrating that salsolinol has a molecular shape corresponding to the active site of CYP2D1  was incubated with salsolinol, it caused protein aggregation and enzyme inactivation. Reactive oxygen species scavengers and copper chelators inhibited salsolinol-mediated hCP modification and inactivation  influx. SH-SY5Y cells treated with salsolinol showed a reduction in transient receptor potential channel 1 (TRPC1) protein levels. Overexpression of the TRPC1 gene and increased TRPC1 protein levels protected SH-SY5Y cells against salsolinol-mediated cytotoxicity. TRPC1 overexpression also inhibited cytochrome c release and decreased the levels of anti-apoptotic bax protein required for apoptosis -salsolinol induced apoptosis in dopamine neurons, as shown in the rat model, and the mechanism of cell death was studied in SH-SY5Y cells -salsolinol was the most potent to induce DNA damage, whereas N-methyl-(S)-salsolinol and salsolinol were less cytotoxic. Apoptosis was initiated by mitochondrial permeability transition as shown by the collapse in the membrane potential, followed mainly by the release of cytochrome c, activation of caspase 3 and final fragmentation of nucleosomal DNA  cells overexpressing α-synuclein compared with normal PC12 cells and worsened α-synuclein-induced mitochondrial damage ?O-methyl-salsolinol levels. He also suggested that salsolinol present in the central nervous system of rats during ethanol intoxication should not be of peripheral origin, and salsolinol present in the cerebrospinal fluid of alcoholics during acute detoxification should be formed centrally -salsolinol (200 μM) reduced in vivo free radical formation and reduced dopamine catabolism. (R)-Salsolinol and the isoquinolinium ion reduced in vitro hydroxyl radical production from dopamine autoxidation. However, on the other hand, N-methyl-(R)-salsolinol (40 μM) increased the hydroxyl radical level in the striatum, and the free radical production by its autoxidation was confirmed in vitro. N-Methyl-(R)-salsolinol affected neither in vivo dopamine catabolism nor in vitro production of hydroxyl radicals from dopamine  was diminished, while its highest dose (500 μM) potentiated the glutamic acid effect on caspase-3 activity. Similar effects were observed for lactate dehydrogenase (LDH) release. In mouse striatum cultures, both the investigated doses of salsolinol (50 and 500 μM) revealed the neuroprotective activity. Authors concluded that salsolinol under physiological conditions could not be neurotoxic (Możdżeń et al. Maruyama et al. , for theThus, it seems reasonable to wonder whether alcohol consumption should lead to an increased risk of the development of Parkinson’s disease According to Lee et al. , no signTetrahydroisoquinoline derivatives, which are abundant in nature (Rommelspacher and Susilo Tetrahydroisoquinoline derivatives, which can mostly be either synthesized endogenously and/or delivered exogenously, can predominantly alter central dopaminergic pathways. Thus, their physiological/pathophysiological role should not be ignored. Multiple factors, such as the dose, stereoisomerism, local bioavailability, local biogenic amine metabolism or even genetic heritage, may determine their biological, possibly opposing, activity.The neuromodulatory role of salsolinol is indeed still poorly understood. In general, the available experimental data include many single-center findings for which replication by other researchers has not been reported and might be partly considered plausible. The most scrupulous attention should be paid to the role of the salsolinol’s stereoisomers. Whether salsolinol present in the brain comes solely from local production or, by contrast, there is a significant contribution from the non-cerebral sites or from dietary sources should be established at the earliest convenience.Salsolinol co-localizes with dopamine-rich regions in the brain. The same should apply for the peripheral tissues, therefore boosting further advances in this field. Because the enteric nervous system and gut microbiota are the major sources of serotonin and dopamine in the human body (Martinucci et al.


The filling of thin-walled steel tubes with quartz sand can help to prevent the premature buckling of the steel tube at a low cost. During an impact, the internal stress of the quartz sand-filled steel tube column is subjected to not only axial force but also lateral confining force, resulting in complicated internal stress. A suitable sensor for monitoring the internal stress of such a structure under an impact is important for structural health monitoring. In this paper, piezoceramic Smart Aggregates (SAs) are embedded into a quartz Sand-Filled Steel Tube Column (SFSTC) to monitor the internal structural stress during impacts. The piezoceramic smart aggregates are first calibrated by an impact hammer. Tests are conducted to study the feasibility of monitoring the internal stress of a structure. The results reflect that the calibration value of the piezoceramic smart aggregate sensitivity test is in good agreement with the theoretical value, and the output voltage value of the piezoceramic smart aggregate has a good linear relationship with external forces. Impact tests are conducted on the sand-filled steel tube with embedded piezoceramic smart aggregates. By analyzing the output signal of the piezoceramic smart aggregates, the internal stress state of the structure can be obtained. Experimental results demonstrated that, under the action of impact loads, the piezoceramic smart aggregates monitor the compressive stress at different locations in the steel tube, which verifies the feasibility of using piezoceramic smart aggregate to monitor the internal stress of a structure. Piezoceramic material has characteristics of fast response, high sensitivity, good linearity, low cost, and sensing and actuating capacity. However, the material’s brittleness limits its application in concrete structures. For this reason, a piezoceramic smart aggregate was introduced, produced by embedding a waterproofed PZT (Lead Zirconate Titanate) patch with connecting wires into a concrete block . At presThe internal stress state of the concrete structure plays an important role in the safety of concrete structures. At present, in a structural test, the structural stress is measured by attaching the strain gauge on the surface of the concrete structure. This monitoring method can only obtain the strain condition of the structural surface; however, it cannot effectively reflect the stress condition inside a structure ,18. In rIn recent years, there have been some studies on sand columns ,30,31. HPiezoceramic is the most critical component of the piezoceramic smart aggregate. Selecting the appropriate size and type of piezoceramics is an important prerequisite for the production of high-quality piezoceramic smart aggregates. At present, the most widely used piezoelectric material is Lead Zirconate Titanate (PZT). According to different physical and chemical properties, PZT can be divided into different types, including hard materials, such as PZT-2, PZT-4 , PZT-8, and soft materials PZT-5 . PZT-5H is highly sensitive to dynamic measurement and has good stability. It is widely used to build ultrasonic probes, electroacoustic transducers, sensors, accelerometers, manometers, buzzers, among others. In this paper, the PZT-5H type material is selected for this research due to its high sensitivity. The related parameters of PZT-5H are shown in A piezoceramic smart aggregate is formed by sandwiching a PZT patch between two marble blocks. The piezoceramic smart aggregate has a cylindrical shape with a diameter of 25 mm and a height of 20 mm. A total of three piezoceramic smart aggregates were fabricated and are shown in The sensitivity of the piezoceramic smart aggregate is defined as the ratio of the output voltage to the applied stress, that is:For a compressive piezoceramic smart aggregate, as a result of piezoelectric effect, the piezoceramic will produce charge under the action of external force, and the relationship between the charge and the external force isAccording to From the sensitivity definition of the smart aggregate, the following equation can be derived,−12 C·N−1, 2, and the capacitance of charge amplifier is 100 nF. Theoretical values of piezoceramic smart aggregate calculated by the (4) formula are shown in Equation (4) indicates that the sensitivity of a piezoceramic smart aggregate is only related to the performance and size of the piezoelectric material and the switching capacitance of the charge amplifier. That is, each piezoceramic smart aggregate corresponds to a fixed sensitivity. The The calibration test equipment includes an impact hammer, a data acquisition system, and a charge amplifier, as shown in According to the waveforms of the output voltages of both the piezoceramic smart aggregate and the impact hammer, their waveforms in the time domain are comparable. Taking SA-3 as an example, as shown in The outer diameter of the steel tube is 140 mm, its wall thickness is 3 mm, and its height is 300 mm. An upper cover plate with a diameter of 130 mm and a thickness of 10 mm was prepared, and the diameter of the cover plate was slightly smaller than the inner diameter of the steel tube, so that the quartz sand inside the tube can be constrained by the steel tube at the time of loading. A bottom plate with a diameter of 170 mm and a thickness of 10 mm was prepared. The test setup is shown in Piezoceramic smart aggregates SA-1, SA-2, and SA-3, were attached to a steel rod with epoxy, as shown in The test results show that the trend of the time history curve of the impact force of drop hammer in the three cases are comparable. The time history curve of the hammer impact force of case 2 is shown in The output voltage of SA-1 in case 2 is shown in Under case 2, the time history curves of the output voltage of SA-2 and SA-3 corresponding to stress are shown in In this paper, embedded piezoceramic smart aggregates (SAs) were used to monitor the internal structural stress during impacts of a quartz sand-filled steel tube column. The filling of the thin-wall steel tube with quartz sand can prevent the premature buckling of the steel tube. Several conclusions can be drawn from this research: (1) Piezoceramic smart aggregate output voltage has good linear relationship with the external force in the sensitivity calibration test, and the sensitivity test value of piezoceramic smart aggregate is close to the theoretical value. This demonstrates that the sensitivity calibration test is reliable.(2) Under the impact tests, the stress values of the piezoceramic smart aggregates embedded in the quartz sand-filled steel tube column increase with the increase in drop height. Under the same drop height, the confining stresses at different cross sections of the quartz sand-filled steel tube column are approximately the same. The feasibility of a piezoceramic smart aggregate to monitor the internal stress of a structure during an impact is verified.


Foetal midbrain progenitors have been shown to survive, give rise to different classes of dopamine neurons and integrate into the host brain alleviating Parkinsonian symptoms following transplantation in patients and animal models of the disease. Dopamine neuron subpopulations in the midbrain, namely A9 and A10, can be identified anatomically based on cell morphology and ascending axonal projections. G protein-gated inwardly rectifying potassium channel Girk2 and the calcium binding protein Calbindin are the two best available histochemical markers currently used to label (with some overlap) A9- and A10-like dopamine neuron subtypes, respectively, in tyrosine hydroxylase expressing neurons both in the midbrain and grafts. Both classes of dopamine neurons survive in grafts in the striatum and extend axonal projections to their normal dorsal and ventral striatal targets depending on phenotype. Nevertheless, grafts transplanted into the dorsal striatum, which is an A9 input nucleus, are enriched for dopamine neurons that express Girk2. It remains to be elucidated whether different transplantation sites favour the differential survival and/or development of concordant dopamine neuron subtypes within the grafts. Here we used rat foetal midbrain progenitors at two developmental stages corresponding to a peak in either A9 or A10 neurogenesis and examined their commitment to respective dopaminergic phenotypes by grafting cells into different forebrain regions that contain targets of either nigral A9 dopamine innervation , ventral tegmental area A10 dopamine innervation , or only sparse dopamine but rich noradrenaline innervation (hippocampus). We demonstrate that young , but not older (E14), mesencephalic tissue and the transplant environment influence survival and functional integration of specific subtypes of dopamine neurons into the host brain. We also show that irrespective of donor age A9-like, Girk2-expressing neurons are more responsive to environmental cues in adopting a dopaminergic phenotype during differentiation post-grafting. These novel findings suggest that dopamine progenitors use targets of A9/A10 innervation in the transplantation site to complete maturation and the efficacy of foetal cell replacement therapy in patients may be improved by deriving midbrain tissue at earlier developmental stages than in current practice. •Younger VM donor grafts are bigger and with enhanced A9-like dopamine neuron yield.•A9:A10-like dopamine neuron ratios in VM grafts vary between transplantation sites.•Dorsal striatum promotes A9-like dopamine neuron differentiation in VM grafts.•Nucleus accumbens slightly enriches grafts with A10-like dopamine neurons.•Transplantation site influences distribution of A9- and A10-like neurons in grafts. There is no therapy available to cure PD and current pharmacological treatments, such as levodopa, only address the symptoms by restoring dopamine transmission in the striatum, alleviating some motor deficits but often having little effect, or even impairing, cognitive functions . In contThe subtypes of dopamine neurons within embryonic VM grafts have been the focus of many studies. Based on anatomical morphology from histochemical descriptions, out of nine dopamine cell groups in the brain three are present in the developing mammalian midbrain – A9 neurons of the SNpc, A10 neurons of the ventral tegmental area (VTA), and A8 neurons of the retrorubral field . AnatomiSubsequently, A9 and A10 neurons in the midbrain can be identified based on cell morphology and expression profiles. Tyrosine hydroxylase-immunoreactive (TH-ir) A9 neurons located in the ventral tier of the SNpc as well as the ventrolateral region of the VTA are large and angular in shape and most co-express G protein-gated inwardly rectifying potassium channel 2 (Girk2), while TH-ir A10 neurons located in the VTA and dorsal tier of the SNpc are smaller and round in shape and mostly co-express the calcium binding protein Calbindin . HoweverIncreasing lines of evidence indicate that the restoration of function by dopamine grafts is mainly due to the presence of A9-like neurons . AlthougIn particular, we questioned whether targets of A9 innervation in the dSTR might have contributed to these effects. To this extent, we investigated the commitment of dopamine neuron progenitors to the A9- and A10-like fates by grafting younger, embryonic day 12 (E12), and conventional age (E14) rat VM tissue into cerebral regions containing targets of either A9, A10 or noradrenaline innervation. We examined whether different transplantation sites favour the differential survival and/or development of concordant dopamine neuron subtypes within the grafts. We believe this to be the first systematic comparison of A9-/A10-like cell populations in rat VM grafts transplanted at different developmental stages into various environments in the context of PD.22.1Sprague Dawley rats  were housed under standard conditions on a 14 h:10 h light/dark cycle with ad libitum access to food and water. All experiments were conducted under UK Home Office personal and project licences in accordance with the requirements of the UK Animals (Scientific Procedures) Act 1986 and with the approval of the local Cardiff University Ethics Review Committee. Every effort was made to minimise the number of animals used and their suffering.2.2 = 0.03, n.s.]. All transplants were made on the dopamine-depleted side of the brain. To reduce the number of animals used in the experiment each animal received 2 grafts implanted into widely separated sites in the denervated hemisphere, with the first transplant into either dSTR or N.Acc and the second transplant into either prefrontal cortex (PFC) or hippocampus (HPC). Efficacy of the grafts was confirmed with amphetamine-induced rotation tests at 4 and 6 weeks post-graft. Following the final rotation test animals were perfused and brain tissue taken for histological analysis.To test our hypothesis, female rats were unilaterally lesioned with the selective catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway and the lesions confirmed with amphetamine-induced rotations at 4 weeks post-lesion. Prior to transplantation, rats were separated into 8 balanced groups based on rotation performance . All rats, which had received grafts into the dSTR, showed a significant behavioural recovery . Therefore, behavioural data presented in Rotational asymmetry in response to the indirect dopamine agonist, amphetamine, was assessed at 4 weeks post-lesion and at 4 and 6 weeks post-graft. Post-lesion, all animals exhibited a rotational bias toward the lesioned side, a classic behavioural deficit induced by the 6-OHDA lesion to the nigrostriatal pathway see . Grafts Rotational behaviour between the E12 and E14 groups was very similar in the dSTR group. In both donor age groups, grafts in the dSTR induced an equally high significant behavioural recovery  or the transplantation site .Examination of NeuN-stained sections revealed surviving grafts in all transplantation sites with large numbers of neurons homogeneously distributed within the grafts as depicted in 3.3At 6 weeks post-transplantation, VM graft survival and integration into 6-OHDA lesioned rat brain was examined by staining for TH . Extensi = 36.18, p < 0.001]. E12 grafts produced a significantly higher yield of TH-ir cells than E14 transplants in the dSTR [p < 0.001], N.Acc [p < 0.05] and HPC [p < 0.01] but not the PFC. Interestingly, in the E12 donor age group grafts in the dSTR were the most populous in TH-ir cell numbers in comparison to grafts in any other location, whereas for the E14 donor age group the largest grafts were in the N.Acc. Also, grafts in the HPC contained unexpectedly high numbers of TH-ir cells, especially in the E12 donor age group. However, only E12 grafts in the dSTR produced a significantly higher yield of dopamine neurons than in the PFC .Younger donor tissue resulted in significantly more TH-ir cells within the grafts in most locations . In line with significant differences in the number of TH-ir neurons, grafts derived from E12 rat embryos yielded a higher percentage of TH-ir/NeuN-ir neurons than E14 grafts in the dSTR [p < 0.01], N.Acc [p < 0.01] and the HPC [p < 0.05] but not the PFC.Given that the total number of surviving neurons was not significantly different between the groups, we should expect a higher proportion of TH-ir/NeuN-ir cells in the E12 donor age group than E14 group to account for the significantly higher dopamine neuron yield in E12 grafts. Indeed, E12 grafts were more enriched for dopamine neurons than E14 grafts in most transplantation sites , despite the smaller numbers of cells implanted, whereas no significant differences were found between individual groups in graft volume. Despite the fact that brain tissue in each transplantation site is different in cell composition and density it did not affect the growth of grafts . Analysis of dopamine neuron densities revealed that E12 grafts were significantly denser than E14 grafts . TH-ir cell density was significantly higher in E12 grafts than E14 grafts in the dSTR [p < 0.01] and N.Acc [p < 0.05]. The density of TH-ir neurons in the grafts varied little between transplantation sites .Generally, E12 grafts were slightly but significantly bigger in volume than E14 grafts in all transplantation sites  influenced by both the host environment  and the VM age . In comparison to E14 grafts, E12 grafts produced a higher yield of both types of dopamine neurons in the dSTR [p < 0.001] and N.Acc [p < 0.05] and a higher yield of A10-like neurons in the HPC [p < 0.05]. This effect of the donor age group was not unexpected since E12 grafts yielded a significantly higher number of TH-ir cells than E14 grafts in all transplantation sites apart from the PFC. The number of Girk2-ir/TH-ir cells was significantly higher than Calbindin-ir/TH-ir cells in grafts in the dSTR [p < 0.001] and in the N.Acc [p < 0.001] in both donor age groups and lower in the HPC in E12 grafts [p < 0.05]. Most importantly, E12 VM grafts produced the highest yield of A9-like neurons in the dSTR than anywhere else . In the E12 donor age group , only the number of A10-like neurons in grafts in the dSTR was significantly higher than in grafts in the PFC [p < 0.05].There was a highly significant difference between A9- and A10-like dopamine neuron populations in the grafts  influenced only by the host environment and not the donor age group . The gain in the Girk2-ir/TH-ir and Calbindin-ir/TH-ir cell yields in the E12 donor age group did not outweigh the general increase in the number of TH-ir neurons between the two donor age groups. Significant differences between the percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons  = 4.68, p < 0.01] no significant differences between individual groups were found.In order to account for the difference in TH-ir cell numbers between two donor age groups, the yield of TH-ir neurons that were double-labelled with Girk2 and with Calbindin was calculated as a percentage of overall number of dopamine neurons. The percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir cells in the dSTR and N.Acc added up to 105%, confirming an overlap in Girk2 and Calbindin stained cells. Due to limited availability of tissue and technical challenges, TH, Girk2 and Calbindin triple-staining was not undertaken. However, we were able to estimate an overlap of 11–17% in Girk2 and Calbindin staining in grafted rat VM by double IHC in pilot experiments . Interes neurons K,L corre neurons I,J. A hi neurons K. Thus,  neurons L. Howeve3.5 = 47.29, p < 0.001]. The A9-like dopamine cells were affected more than the A10-like neurons by the location within the graft . Transplantation site also had an effect on the distribution of dopamine neuron populations within the grafts  while VM age did not . In both donor age groups, A9-like and A10-like dopamine neurons were found mostly in the periphery of grafts in N.Acc but in grafts in the dSTR this was true for A9-like neurons only. There was a significantly higher percentage of A10-like neurons in the periphery than in the centre in grafts in N.Acc in E14 donor age group  and N.Acc [p < 0.01] in the E12 donor age group A. In E144Improvement of functional efficacy of the cell replacement therapy for PD is still a major challenge that needs to be overcome. It is of paramount importance to understand the underlying mechanisms that lead to superior survival and integration of transplanted dopamine neurons. Here we describe the importance of two critical factors, the presence of A9/A10 dopamine innervation targets in the transplantation site and the age/developmental stage of the donor tissue, in improving the yield of functionally important A9-like dopamine neuron component in rat VM grafts in a 6-OHDA lesion model of PD.Several previous studies have highlighted the impact of donor age on the cell therapy outcomes, reporting that grafting of younger mouse and rat VM cells produces transplants with an enhanced dopamine neuron yield and a greater proportion of A9-like dopamine neurons, as a result of increased progenitor proliferation and survival and the presence of meningeal cells in tissue preparation at the time of grafting . Also, gThe degree of innervation, patterning of regenerating axons and sprouting of appropriate transplanted neurons into the target have all been shown to be influenced by the host environment in studies in the dSTR , HPC Bj and sympWithin the present study each animal received two VM grafts, the first transplant into either dSTR or N.Acc and the second into either PFC or HPC. Grafts in either the PFC or HPC did not interfere with drug-induced rotational response provided by grafts in either dSTR or N.Acc. The behavioural analysis of drug-induced rotation test revealed a significant recovery of rotational bias in rats grafted with VM tissue into the dSTR in both donor age groups A. This iThe histological findings, including the total numbers of surviving transplanted NeuN-ir neurons E and TH-As in the adult midbrain, VM grafts in this study contained two recognisable classes of dopamine neurons: large elongated Girk2-ir A9-like dopamine neurons of SNpc and smaller round Calbindin-ir A10-like dopamine neurons of the VTA . We acknConsistent with previous reports, A9-like neurons were predominantly located in the periphery of grafts while A10-like neurons were more homogeneously distributed within the graft . The effThe absence of a greater effect of the transplantation site on the A10-like neuron population in the grafts could be attributed, at least in part, to the fact that a unilateral 6-OHDA lesion of the medial forebrain bundle causes only a partial loss of dopamine neurons in the VTA and subsequently achieves only a partial denervation of the N.Acc and PFC. Indeed, previous studies have suggested that although dopamine grafts survive equally well both in the intact and lesioned striatal environment , integraA further major determinant of a successful cell therapy is to achieve adequate innervation of target tissue by the graft and restoration of dopamine supply. Irrespective of the dopamine neuron yield in a transplant, failure to repair functional connections in the dSTR does not improve motor function . MeningeTaken together, our current findings not only expand our understanding of grafted dopamine neuron differentiation and maturation in the host brain, but also have significant implications for further improving dopamine cell therapy in PD in the future. Replacement of lost neurons can be targeted to individual needs of each patient taking into account specific patterns of neurodegeneration, thereby allowing more precise restoration of the circuitry and neurotransmitter release. In the present study, we demonstrated the ability of younger VM tissue to use environmental cues in the transplantation site to guide terminal A9- and A10-like phenotypic differentiation of dopamine neuron progenitors in the graft. Thus, by transplanting younger human VM tissue into different sites in the caudate nucleus, putamen and SNpc, it may be possible to enrich grafts for a specific dopamine neuron population to achieve a more extensive restoration of circuitry and improve the quality of life for patients.MF, EMT, and SBD designed the experiment. MF and EMT ran the experiment and analysed the data. MF, EMT, and SBD wrote the manuscript.Drs. Fjodorova and Torres, and Professor Dunnett reported no biomedical financial interests or potential conflicts of interest.


In this study, scanning electron microscopy (SEM), Raman spectroscopy and high-resolution atomic force microscopy (AFM) were used to reveal the early-stage change of nanomorphology and nanomechanical properties of poly(lactic acid) (PLA) fibers in a time-resolved manner during the mineralization process. Electrospun PLA nanofibers were soaked in simulated body fluid (SBF) for different periods of time  at 10 °C, much lower than the conventional 37 °C, to simulate the slow biomineralization process. Time-resolved Raman spectroscopy analysis can confirm that apatites were deposited on PLA nanofibers after 21 days of mineralization. However, there is no significant signal change among several Raman spectra before 21 days. SEM images can reveal the mineral deposit on PLA nanofibers during the process of mineralization. In this work, for the first time, time-resolved AFM was used to monitor early-stage nanomorphology and nanomechanical changes of PLA nanofibers. The Surface Roughness and Young’s Modulus of the PLA nanofiber quantitatively increased with the time of mineralization. The electrospun PLA nanofibers with delicate porous structure could mimic the extracellular matrix (ECM) and serve as a model to study the early-stage mineralization. Tested by the mode of PLA nanofibers, we demonstrated that AFM technique could be developed as a potential diagnostic tool to monitor the early onset of pathologic mineralization of soft tissues. Poly(lactic acid) (PLA)-based biomaterials as biodegradable and biocompatible materials are widely used in bone tissue engineering ,2. Over Biomineralization is the complex process by which living organisms form minerals and biomineralization processes often lead to the hardening or stiffening of the existing tissues such as mineralized skeletons . InspireAlthough mineralization of hydroxyapatite in electrospun nanofibrous Poly(lactic acid) scaffolds has been well studied , the det3), potassium chloride (KCl), Trihydrate potassium hydrogen phosphate (K2HPO4·3H2O), magnesium chloride hexahydrate (MgCl2·6H2O), calcium chloride (CaCl2), sodium sulfate (Na2SO4), Dichloromethane (DCM) and hydrochloric acid (HCl) were purchased from Sinopharm Chemical Reagent Co., Ltd. , Tris(hydroxymethyl)aminomethane (HOCH2)3CNH2 were purchased from Sigma-Aldrich , poly(lactic acid) (PLA) were purchased from NatureWorks . All the chemicals were of analytical reagent grade. All of the solutions were freshly prepared using deionized double-distilled water.Sodium chloride (NaCl), sodium bicarbonate  was dissolved in DCM (5 mL) to get a viscous solution (0.15 g/mL) for electrospinning for 12 h at room temperature.Electrospinning solution (about 5 mL) was drawn into 15 mL plastic syringes connected by a plastic tube with a metal capillary tube. The syringe was maintained at a flow rate of 1 mL/h. Electrospinning was carried out at room temperature of 20 °C and at environmental humidity of 30%. In electrospinning, the nozzles on its axis at a linear speed of 100 mm/min, and the lateral distance of 150 mm to keep moving. Fibers were collected in aluminum foil surface and drying process was conducted at the condition of 40 °C for 24 h.The SBF was prepared according to literature ,30 to miFor morphological observation, the mineralized PLA fiber pieces were observed using SEM  at an accelerating voltage of 15 kV. −1 were used in this work. The spectra were recorded in the range of 400–3200 cm−1.Chemical structures of the products were detected by Raman . An excitation wavelength of 780 nm and a spectral resolution of 1 cm−1. Tip radius was calibrated against a polystyrene standard provided by Bruker. The measured value of the tip radius was 10 nm. Both samples imaging was carried out in Scan AsystPeak Force QNM  mode. Force-distance curves were collected by nanoindentation of the sample in a point-by-point fashion. The maximum force (peak force) was controlled at each pixel to obtain force-distance curves, which are then used as feedback signal. Image processing and nanomechanical properties analysis was performed with the NanoScope Analysis software . 3D topography images were generated from NanoScope Analysis software. Surface Roughness measurements were carried out using NanoScope Analysis software too.An Icon AFM  was used to image the mineralized samples. For imaging mineralized samples, we used a 500 nN force, a scanning rate of 1 Hz and RTEAPA (MPP-21110-10) cantilevers with spring constant of 3 NmAfter being immersed in SBF for 0–21 days, the morphology of the PLA nanofibers was observed by SEM. Minerals were found to be gradually deposited onto PLA nanofibers as time going on. As shown in −1 and 2800 cm−1 were attributed to C–H stretching modes of CH and CH3 groups. The CH3 stretching modes appeared at 3014 cm−1 and 2960 cm−1. The CH stretching mode was observed at 2877 cm−1, which is consistent with the previous literature [3 symmetric and asymmetric deformation modes were observed at 1382 cm−1 and 1453 cm−1. The typical stretching of C=O group was centered at 1769 cm−1. Besides, bands at 1085 cm−1, 873 cm−1, 739 cm−1, 398 cm−1 and 308 cm−1 can be assigned to the COC, C–COO stretching modes, C=O, CCO, and COC deformation vibration of PLA [43− groups at 960 cm−1 appeared after PLA fiber sample was immersed in SBF for 21 days, indicating that the apatite minerals were deposited on the surface of PLA nanofibers [The Raman spectra of PLA nanofibers soaked in SBF for different periods of time were shown in terature . The CH3n of PLA ,34,35. Tnofibers . −1 gradually increased with the mineralization time, indicating the amount of deposited apatite on the surface of PLA fiber was increasing. Time resolved Raman spectroscopy analysis could confirm that apatites were deposited on the PLA nanofibers after 21 days of mineralization. However, there is no apparent signal change among several Raman spectra before 21 days as shown by As shown in Surface morphology of PLA nanofibers was observed by high-resolution atomic force microscopy. As shown in ® . To obtain the Young’s Modulus, the retrace curve was fitted by the software using the Derjaguin-Muller-Toporov (DMT) Model [F − adhF was the force on the cantilever (F) relative to the adhesion force (adhF), R was the tip end radius, and d − d0 was the deformation of the sample.Analysis of the force-curves was performed by the Peak Force Tapping technology and the topography and Young’s Modulus were measured by ScanAsystT) Model : (1)F−FaAs shown in Roughness data  were obtSome pathologic calcification including artery calcification, cardiac valve calcification, and myositis ossificans are associated with the undesired biomineralization ,44. The An early-stage mineralization study from electrospun nanofibrous PLA scaffolds was carried out in the SBF. In the study, high-resolution SEM, Raman spectroscopy and AFM were used to reveal nanostructural changes to mineralized PLA nanofibers. The results indicated that the deposited minerals were apatite with plate-like or flaky-like structures. The Surface Roughness, Young’s Modulus and Surface Area of the PLA nanofibers significantly increased with the time of mineralization, which could be used to monitor early-stage nanomorphology and nanomechanical changes of PLA nanofibers. Using electrospun PLA nanofibers as a model to study early-stage mineralization, we demonstrated that AFM techniques could be developed as a potential diagnostic tool to monitor the early onset of the pathologic mineralization of tissues.


The present paper aims to study the preparation of polyaniline nanofibers through simple interfacial polymerization. Ammonium persulfate, hydrochloric acid and chloroform were used as oxidant, dopant and organic solvent respectively. Field Emission Scanning Electron Microscopy (FESEM), X-ray diffraction and Fourier Transform Infrared Spectroscopy (FTIR) techniques were used to analyze the product. FESEM results show that polyaniline has nano-fiber morphology. XRD results show the crystalline properties of polyaniline nanofiber, and FTIR results confirmed the formation of polyaniline in different monomer/oxidant molar ratios. This study provides a better understanding on the synthesis of uniform polyaniline nanofibers through interfacial polymerization. Conductive polymers belong to an interesting group of polymers, which have unique electrical, electrochemical and optical properties ,2. They Different methods used to fabricate polyaniline nanofibers include electrospinning , ultrasoAniline monomer, ammonium persulfate, chloroform and hydrochloric acid were purchased from a chemical company and they were used as received without further purification process. In conventional synthesis method, 4mmol aniline was dissolved in 20 mL hydrochloric acid (1 M) and stirred with magnetic stirrer for one hour. 4mmol ammonium persulfate was also dissolved in 20 mL hydrochloric acid (1 M) and magnetically stirred for one hour. Then the ammonium persulfate solution was added into the aniline solution and was left at room temperature for 6 hours. Finally, the black green precipitate was collected and washed several times by acetone and distilled water. The pure polyaniline was then dried at ambient temperature for 24 hours. In the interfacial polymerization, two types of solutions were prepared: (a) 20 mL hydrochloric acid (1 M) which contains 4 mmole ammonium persulfate; and (b) 20 mL chloroform which contains 4 mmole aniline. Each solution was stirred with magnetic stirrer for one hour. Then the oxidant based solution was carefully transferred to aniline based solution. After a short induction time, the reaction took place and the polyaniline was initially formed at the interface of the immiscible solutions. The green polyaniline nanofibers increased in amount over time and filled the water based solution. After 24 hours, the black green polyaniline nanofibers were filtered and dried at room temperature for 24 hours. The morphology of polyaniline nanofibers was characterized using the field emission scanning electron microscopy . The structures of polyaniline (PANI) were analyzed using Fourier Transform Infrared Spectroscopy (VERTEX70 infrared spectrometer (using pellets with KBr)). The crystalline properties of nanofibers were measured by using X-ray Diffraction (Ultima III X-ray diffractometer with Cu Kα radiation).etc.) according to their solubility in the organic and aqueous phases. After a short induction period green PANI appears at the interface, moving into the water phase and finally filling the whole water layer  nucleation; (2) initial growth; (3) secondary growth. In conventional methods, polyaniline nanofibers are formed initially due to the linear chain of polyaniline . The forFrom the FESEM results in −1. The main peaks at 1570 and 1490 cm−1 can be associated with C=N and C=C stretching vibrations of quinone and benzene rings, respectively. The peaks at 1300 and 1240 cm−1 are attributed to the C-N stretching mode. The peak at 1150 cm−1 is due to quinonoid unit of doped PANI. The peak at 802 cm−1 is attributed to the out-of-plane bending of C-H. These related peaks confirm the successfully formation of PANI at different aniline/oxidant molar ratios. As shown in the spectra of all samples the peaks are approximately presented at 802, 1240, 1300, 1490, 1570 cmet al. [XRD analysis indicates the crystallinity of the product. High crystalline products could display metallic behavior and they are more useful than amorphous products. Generally, the ratio of half-width to height (HW/H) of an X-ray diffraction peak reflects the order of the polymer crystallinity. Rahy et al.  shows thPANI nanofibers were successfully prepared chemically by an interfacial polymerization. It has been found that different aniline/APS molar ratios affected the uniformity and morphology of polyaniline. The more uniform and thinner polyaniline nanofibers have been achieved at aniline/APS molar ratios = 4.0. XRD results show that polyaniline nanofibers with more uniformity at aniline/APS molar ratios = 4.0 has higher crystallinity rather than irregular shaped polyaniline. FTIR results also show that the polyaniline products have similar molecular structure in different aniline/APS molar ratios.


CD99 is a cell surface protein with unique features and only partly defined mechanisms of action. This molecule is involved in crucial biological processes, including cell adhesion, migration, death, differentiation and diapedesis, and it influences processes associated with inflammation, immune responses and cancer. CD99 is frequently overexpressed in many types of tumors, particularly pediatric tumors including Ewing sarcoma and specific subtypes of leukemia. Engagement of CD99 induces the death of malignant cells through non-conventional mechanisms. In Ewing sarcoma, triggering of CD99 by specific monoclonal antibodies activates hyperstimulation of micropinocytosis and leads to cancer cells killing through a caspase-independent, non-apoptotic pathway resembling methuosis. This process is characterized by extreme accumulation of vacuoles in the cytoplasmic space, which compromises cell viability, requires the activation of RAS-Rac1 downstream signaling and appears to be rather specific for tumor cells. In addition, anti-CD99 monoclonal antibodies exhibit antitumor activities in xenografts in the absence of immune effector cells or complement proteins. Overall, these data establish CD99 as a new opportunity to treat patients with high expression of CD99, particularly those that are resistant to canonical apoptosis-inducing agents. MIC2  and it is involved in essential functions including apoptosis  , thereby emerging as a novel therapeutic target. CD99 had promising preclinical effectiveness in xenograft models, and thus paves the way for further development of antibodies to be used in clinical settings. Of note, CD99 engagement increases natural killer (NK) cell-mediated tumor lysis by inducing heat shock protein 70 (HSP70) expression  and a short form with 161 amino acids  generated by alternative splicing of the cytoplasmic region.CD99 acts through homophilic interactions and enables homo- or heterotypic cell aggregation.CD99 can mediate diverse cellular processes such as adhesion, transendothelial migration, differentiation, and cell death, thereby affecting immune functions, inflammation and cancer metastasis.CD99 can act as an oncogene or as an oncosuppressor depending on the cellular context.MIC2, which is located in the pseudoautosomal region (PAR) of both the X (Xp22.33-Xpter) and Y (Yp11-Ypter) chromosomes in humans  encoding the Xga antigen ) and CD99L2 (CD99 antigen-like 2)  is related to exons 1, 4, and 5 of MIC2. Transcripts from the MIC2R locus have been detected in all human tissues but none of them contains a functional open reading frame, making the role of MIC2R still unknown  with 185 aminoacids (corresponding to a molecular weight of 32 kDa) and a truncated form or CD99 type II (CD99sh) with 161 aminoacids (corresponding to a molecular weight of 28 kDa)  CD99 genes are more similar to CD99 type I in humans, as is the cytoplasmic region of Xenopus CD99.Additionally, the search for CD99 homologs has been successful only in primates, indicating a high level of sequence divergence of this gene during evolution   such as DN16, 12E7, O13, F21, MSGB1, YG32 and 0662 see Fig. . NaturalCD99 is considered an important player in lymphocyte development. In studies on anencephaly, CD99-deficient fetuses typically demonstrated a marked impairment in thymic development, which suggests a role of CD99 in normal thymus ontogeny , with the highest levels observed in the most immature stage 1. The alternatively spliced CD99 type II mRNA is either absent in normal BCPs or present at extremely low levels with no effect on maturation . Furthermore, CD99 engagement did not influence apoptosis, differentiation, or antibody secretion of PCs but it reduced chemotactic migration of PCs toward CXCL12 and reduced ERK activation by CXCL12, suggesting that CD99-engaged PCs were less sensitive to the chemoattractive stimuli of CXCL12   expression reflects maturation-associated patterns of normal B lymphopoiesis, with CD34+ cells expressing the highest levels of CD99  components; 2. extravasation across the endothelium; 3. cell survival, proliferation, differentiation and response to stress; 4. regulation of stem cell fate; 5. communication between immune cells as well as cancer cells and the tumor microenvironment via complex and still poorly defined molecular interactions.Being a cell surface molecule, CD99 can be easily targeted by antibodies. Most of these antibodies have been reported to activate cell death signals and inhibit cell migration. This opens interesting therapeutic perspectives for tumors in which CD99 acts as on oncogene, such as EWS, ALL, MDS, AML and glioblastoma. CD99 engagement induces death in tumor cells via non-conventional, caspase-independent programmed cell death or through a non-apoptotic pathway resembling methuosis  and are selective for malignant stem cells. Of note, a human diabody has been recently developed  with Due to the lack of sufficient reports on the different roles for the two CD99 isoforms, a more detailed study of their expression in normal and diseased cells is necessary.The mechanisms of action of CD99 isoforms are strictly dependent on the cellular context and need further investigation.CD99 likely acts by interacting with other cell surface molecules, which are responsible for its signaling. Hovewer, very limited data are available on the heterophilic interactions of CD99.The functional relationship between human and murine CD99 is unclear.CD99 has been largely ignored by the scientific community. However, increasing evidence highlights a crucial role for this peculiar transmembrane molecule in cancer and inflammation. CD99 regulates fundamental biological processes and may have has promising clinical application in several fields of medicine. Efforts should thus be made to improve our understanding of the mechanisms of action of CD99, as it is time to give proper attention to this molecule.


The announcement of “Artificial Life” by the Craig Venter group, and the media stir that arose from the news, provoked thoughts about the current technologies in contemporary science and the cultural tension of such projections on the media. The increasingly blurred boundaries between specialist and generalist media, while promising a wider appreciation of scientific discovery, potentially allow unrealistic, ideological claims to dictate scientific research. This is particularly evident in biology, where the pervading paradigm is still dominated by a physically naïve reductionism in which the only relevant causative layer is the molecular one. The reductionist hypothesis is that everything one observes is the result of an underlying molecular mechanism almost independent of the context in which it operates. Molecular mechanisms are often necessarily studied in isolation and therefore operate in unnatural conditions. The mechanistic view of biological regulation implies that we think of genes as intelligent agents. Here we try to critically analyze the motivations behind the spread of such unrealistic simplifications.


TiC nanofibers reinforced Al matrix composites were produced by High Frequency Induction Heat Sintering (HFIHS).The titanium carbide nanofibers with an average diameter of 90 nm are first prepared by electrospinning technique and high temperature calcination process. A composite solution containing polyacrylonitrile and titanium isopropoxide is first electrospun into the nanofibers, which are subsequently stabilized and then calcined to produce the desired TiC nanofibers. The X-ray diffraction pattern and transmission electron microscopy results show that the main phase of the as-synthesized nanofibers is titanium carbide. The TiC nanofibers is then mixed with the aluminum powders and introduced into high frequency induction heat sintering (HFIHS) to produce composites of TiC nanofibers reinforced aluminum matrix. The potential application of the TiC nanofibers reinforced aluminum matrix composites was systematically investigated. 99.5% relative density and around 85 HV (833 MPa) Vickers hardness of the Al reinforced with 5 wt % TiC nanofiber has been obtained. Furthermore, the sample of Al contains 5 wt % TiC, has the highest value of compression and yield strength of about 415 and 350 MPa, respectively. The ductility of the Al/5 wt % TiC showed increasing with increasing the TiC contents. In particulates ,7,8,9. Wiculates ,11,12. Ticulates . In ordeiculates ,15,16. Ticulates .On the other hand, the novel technique of high-frequency induction heat sintering (HFIHS) has been shown to be an effective sintering method that successfully consolidates ceramics and metallic powders to near their theoretical densities. The HFIHS process involves the rapid sintering of a nanostructured hard metal in a very short time with high-temperature exposure and the application of pressure. This process is advantageous because it allows for rapid densification to near the theoretical density of the associated materials and inhibits grain growth in nanostructured materials. Furthermore, the use of conventional methods of powder consolidation often results in grain growth when sintering compacts and also the presence of porosity, thus leading to compacts with lower density than the ones obtained by pressure-assisted processes. It is therefore essential to minimize grain growth through careful control of the consolidation parameters, particularly the sintering temperature, pressure and time. Furthermore, retention of the nanostructure of the final product after exposure to elevated temperatures during processing is one of the main challenges of using nanostarter powders. In this regard, high-frequency induction heat sintering (HFIHS) has been shown to be an effective sintering method that successfully consolidates ceramics and metallic powders to near their theoretical densities in a very short time. This allow rapid densification to near full density of the associated materials and inhibits grain growth in nanostructured materials. Another important reported aspect of the HFIHS process is the role of rapid heat transfer to the product via electromagnetic waves. The main objective of the present study was to help clarify and optimize the overall processing parameters of the HFIHS process, focusing on the sintering of conventional nanostructured powders and the suitability of HFIHS for the compaction of various ceramic and metallic materials. Efforts were made to reduce the grain size of the target materials to less than 100 nanometers by optimizing the overall parameters of the novel HFIHS process. The effects of processing parameters such as sintering temperatures, pressures, holding time and heating rates on the mechanical and microstructural properties were investigated systematically. In this paper, the TiC ceramic nanofibers are synthesized by electrospinning and subsequent thermal calcinations. The TiC nanofibers are then mixed with aluminum matrix. The mixture is then introduced into HFIHS using high heating rate (500 °C/min). Therefore, in this article, HFIHS method was employed to fabricate Al/TiC nanofiber nanocomposites. The effects of TiC on the microstructure and mechanical properties of Al were carefully investigated.4, Aldrich), and 9 gm of acetic acid were dissolved into 30 gm of N,N-dimethylformamide (DMF). The mixture was vigorously stirred at room temperature for two hour to obtain 45 grams of a homogeneous solution. Second, polyacrylonitrile (PAN)  7 wt % solution was dissolved in (DMF) with vigorous stirring for at least 2 h to get a transparent solution. The prepared Ti precursor solution was then dropwise added to the PAN solution with vigorous stirring to prepare a transparent PAN/Ti precursor composite solution. The sol-gel is then introduced into electrospinning machine. A schematic diagram for the used electrospinning device of polymer nanofibers in this study is shown in PAN/Ti composites were prepared via sol-gel by two steps. First, 4.5 gm of titanium isopropoxide (Ti (OiPr)In a typical electrospinning setup, a high-voltage source is connected to a metallic needle, which is attached to a solution reservoir (syringe) and located 15 cm from the rotating collector. The needle has a relatively small orifice of 0.2 mm that concentrates the electric charge density on a small pendant drop of solution. In the electrospinning process a high voltage (20 kV) is used to create an electrically charged jet of polymer solution with a flowrate of 0.5 mL/h. Stretching of the viscoelastic thread under the electric field combined with solvent evaporation leads to the formation of Ti/PAN nanofibers deposited on the grounded collector. Before reaching the collecting drum, the solution jet evaporates or solidifies, and then collected as an interconnected mat of small fibers. The as-spun fibers obtained from the previous step were initially dried for 12 h at 75 °C under vacuum In order to remove the polymer; binder used in making sol-gels. Samples of the dried nanofibers were placed into ceramic boats, which were then transferred to a tube furnace for calcination process. Calcination process normally takes place at temperatures below the melting point of the product materials and above the thermal decomposition temperature for decomposition and volatilization reactions) or the transition temperature (for phase transitions). The calcination process was performed by using tube furnace (CARBOLITE Type 3216CC). The tube furnace was fitted with an alumina tube with gastight fittings on each end. The samples were first stabilized thermally in the tube furnace at 270 °C in air for 3 h, and then carbonized and pyrolyzed under argon at 1000 °C for 3 h with heating rate of 10 °C/min  was done by JEOL JEM 2100F operating at 200 kV, JEOL Ltd., Tokyo, Japan. Before measurement, these samples were transferred onto a Formvar-coated copper grid. Powder X-ray diffraction patterns  were used to measure the crystallinity of samples, and Fourier transform infrared (FTIR) spectra were obtained using Bruker, TENSOR Series FT-IR Spectrometer,  to detect the chemical structural changes of the pyrolyzed samples.Pure aluminum powders  with average size of 44 μm (325 mesh) were weighed carefully in an argon-filled glove box. All powders were milled in a Fritsch mechanical alloying machine using zirconia balls and a rotation speed of 200 rpm for 30 min with a ball-to-powder weight ratio of 5:1. Presintered electrospun TiC nanofibers with 1 wt %, 2 wt %, 3 wt %, and 5 wt % were added to the slurries and mixed with the de-agglomerated Al powder for 15 min. After mixing, the slurries were dried at 60 °C in a vacuum drying oven. The mixed composites were placed in a graphite die  and then introduced into the HFIHS machine .The samples were densified by heating to a sintering temperature 580 °C and then rapidly cooled to room temperature. 50 MPa compaction pressure was applied to achieve highest density and improve the mechanical and microstructural properties. The pressure was applied in two steps. First, the pressure was maintained at a constant level during heating, and then the maximum pressure was applied during the holding time. High heating rates of about 500 °C/min were applied. Intense magnetic field is applied through the electrically conducting pressure die and, in some cases, also through the sample. Thus, the die also acts as a heating source, and the sample is heated from both the outside and inside. Temperatures was measured using a pyrometer focused on the surface of the graphite die. In this work, the uniaxial pressure is applied and an induced current (frequency of approximately 50 kHz) is then activated and maintained until densification, indicating the occurrence of sintering and the concomitant shrinkage of the sample is observed. Sample shrinkage is measured by a linear gauge that measures the vertical displacement.3 for aluminum and 4.93 g/cm3 for titanium carbide. The microstructural observations of the polished and coated fracture surfaces of the composite were investigated using a field-emission scanning electron microscope (FE-SEM) (JEOL FESEM 7600 F) coupled with energy dispersive X-ray analyzer (EDX) analysis. The surfaces were coated with platinum to avoid charging during FE-SEM observation. Phase identification was performed on samples cut from the center of hot-pressed bodies using X-ray diffractometry . Hardness testing was carried out using a Vickers diamond indenter on an automated hardness tester . Sintered specimens of Al-X wt % of TiC were tested and evaluated for their hardness. During the hardness test, a load of 2.94 N with a loading time of 10 s was applied. The specimens used in hardness testing had an average diameter of 20 mm and an average thickness of 5 mm. Three samples were tested for their hardness at three different locations. The averages of these readings were computed, reported and compared. This determination of hardness was performed at ambient temperature and followed the ASTM C 1421-99 standard. To determine the mechanical strengths of the samples under uniaxial compressive loading, the sintered Al-TiC specimens were tested in a fully automated tensile tester from Instron  with a constant crosshead speed of 0.5 mm/min. The compressive strengths of three samples were determined, and the averages for all conditions were calculated and compared for analysis.After sintering, the samples were ground and polished for subsequent indentation and microscopy studies. The densities of the sintered samples were measured by the Archimedes principle, and the theoretical density of the composites was calculated using the rule of mixtures with a theoretical density of 2.71 g/cmIn −1, attributed to the C≡N band and the decrease of those in the regions 2930–2870, 1670–1660, 1460–1450 and 1260–1220 cm−1, assigned to the aliphatic CH group vibrations of different modes in CH, CH2 and CH3 [−1 (the peaks of reaction products of Ti precursors), concomitant with the advent of the peaks in 1660 (due to cyclic C=O), 1608 (due to C=C), 1391 (due to C=N), and 1273 cm−1 (due to C–N) [etc. [2. The electrospun nanofibers were further carbonized and pyrolyzed to produce the TiC–C composite nanofibers.To produce high strength carbon fibers, oxidative stabilization of PAN prior to carbonization is the preferred route, ,19. The  and CH3 , 1094  ,22,23, tN) [etc. ,25,26, aThe XRD patterns are used to analyze the phase of the pyrolyzed fibers and the curves are shown in The reduction of the fibrous diameter is due to the fibrous shrinkage and the reduction in composition. The inset in The basic configuration of a HFIHS unit  is reported elsewhere ,28,29. TDue to mixing process, the TiC electrospun fibers were broken into small parts with aspect ratio nearly 5 to 15. Some fragmented TiC nanoparticles also exist. Notably, the relative density of the Al/TiC electrospun nanofiber almost did not vary with the increasing the TiC contents more than 3 wt %, being on the same level of approximately 99.5% as shown in SEM micrographs of the fracture surfaces of Al containing TiC electrospun nanofibers contents are shown in XRD patterns of the pure Al/TiC nanofibers after HFIHS pressing in the same conditions are shown in High Frequency Induction Heat Sintering (HFIHS) have been used to produce nanocomposites of TiC nanofibers reinforced Al matrix composites. The titanium carbide nanofibers were first synthesized by sol-gel followed by electrospinning technique and high temperature calcination process. The average diameter of the calcined nanofibers was 90 nm. The formation of the as-synthesized titanium carbide nanofibers was confirmed by X-ray diffraction pattern and transmission electron microscopy. The TiC nanofibers is then mixed with the aluminum powders and introduced into (HFIHS) to produce composites of TiC nanofibers reinforced aluminum matrix. Compaction of the mixture was at a temperature of 580 °C under the pressure of 50 MPa. A high dense Al-TiC of 99.5% relative density and around 85 HV (833 MPa) hardness has been obtained in the Al reinforced with 5 wt % TiC nanofiber. With respect to mechanical properties, the samples of Al contains 5 wt % TiC, have as high as 415 and 350 MPa, respectively value of compressive and yield strength.


PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer . Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Adding location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts.Until recently, no prediction models for Lynch syndrome (LS) had been validated for The online version of this article (doi:10.1007/s10689-017-0039-1) contains supplementary material, which is available to authorized users. Lynch syndrome (LS) is a hereditary predisposition to colorectal cancer, endometrial cancer and other extra-colonic cancers at a young age , 2. MorbMLH1, MSH2, MSH6 or PMS2, or in the 3′ end of the EPCAM gene and consequent hypermethylation of the MSH2 promoter region  and 0.83 [95% CI 0.73–0.94] for PREMM5 and AUC of 0.79 [95% CI 0.69–0.89 and 0.67–0.91] for MMRpredict. Both models had a fair discriminative power for MSH6 mutation carriers (AUC of 0.69 [95% CI 0.58–0.80] for PREMM5 and AUC of 0.66 [95% CI 0.56–0.76] for MMRpredict). MMRpredict still had fair performance for PMS2 mutation carriers (AUC of 0.72 [95% CI 0.57–0.87]), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers at all with an AUC of 0.51 [95% CI 0.35–0.66].Overall, PREMM5 predicted higher probabilities of carrying a LS mutation than MMRpredict . For mutation carriers, risk scores varied from 0.02 to 0.99 for PREMM5 and from 0.002 to 0.99 for MMRpredict. Both prediction models could fairly discriminate between index patients with and without an MMR mutation.. This higher sensitivity came at the expense of a lower specificity (46 vs. 67%). For PREMM5, using a cut-off of 5%, resulted in a sensitivity for MLH1 mutation carriers to 83% for PMS2 mutation carriers . Overall, the revised Bethesda guidelines had a sensitivity of 90% with a specificity of 24%. In order to reach the same sensitivity, PREMM5 and MMRpredict had a similar specificity (25%).Sensitivity of the revised Bethesda guidelines decreased from 96% for PMS2 mutation carriers and non-mutation carriers. Only one significant difference remained; PMS2 mutation carriers more often had proximal CRC than patients without an MMR mutation    and MSH6 (AUC 0.75 [95% CI 0.65–0.84] vs. 0.69 [95% CI 0.58–0.80]) mutation carriers . Overall, the adjusted PREMM5 model performed better than the original PREMM5 model (AUC 0.81 [95% CI 0.76–0.86] vs. 0.72 [95% CI 0.66–0.79]) and MMRpredict (AUC 0.81 vs. 0.73 [95% CI 0.66–0.79]). The adjusted prediction model can be found as supplemental material.Adding tumour location also improved the performance of PREMM5 for identifying At a 5% cut-off, sensitivity of the extended PREMM5 model was higher than the sensitivity of the original PREMM5 model (92 vs. 78%) with similar specificity (45 vs. 46%). Sensitivity and specificity of the extended PREMM5 model at a 5% cut off were both higher than those of the revised Bethesda guidelines (sensitivity 92 vs. 90% and specificity 45 vs. 24%).MLH1, 26 MSH2, 28 MSH6 and 73 PMS2 mutation carriers. Similar to the results in the initial cohort, the extended PREMM5 model had better predictions than the original PREMM5 model for PMS2 mutation carriers (AUC 0.90 [95% CI 0.86–0.94] vs. 0.82 [95% CI 0.76–0.87]) and overall (AUC 0.92 [95% CI 0.89–0.95] vs. 0.87 [95% CI 0.84–0.91]). Performance for MLH1, MSH2 and MSH6 mutation carriers was also slightly better for the extended PREMM5 model than for the original PREMM5 model .In our validation cohort, 60% of the patients were male and median age was 55 years (IQR 45–63 years). The cohort included 31 PMS2 mutation carriers. The performance of the PREMM5 model improved considerably by adding the location of CRC to the model. In our clinical cohort of 734 CRC patients as well as in a validation cohort of 376 CRC patients, this extended PREMM5 model not only identified PMS2 mutation carriers more accurately, its overall performance was also better than the original PREMM5 model and the MMRpredict model.The results of our study indicate that while the models MMRpredict and PREMM5 can adequately predict whether an individual is likely to have Lynch syndrome, they fail to identify 1,2,6 model had a slightly better overall performance than MMRpredict [1,2 also performed better than MMRpredict in several studies [Our results are in line with those of previous studies, where the PREMMRpredict , 32, 33. studies , 24, but studies , 26 or l studies  predicti studies .PMS2 mutation carriers specifically. An explanation for this could be that the location of CRC is incorporated in the MMRpredict model but not in the PREMM5 model. Proximal location of CRC is a known predictor for Lynch syndrome and in our cohort was the only significant difference between PMS2 mutation carriers and non-mutation carriers. After adding this new variable to the existing PREMM55 model, this new model performed better than MMRpredict for PMS2 mutation carriers. The extended PREMM55 model also performed better than the original model for MLH1, MSH2 and MSH6 mutation carriers and had a better overall performance.Although PREMM5 had better overall predictions, MMRpredict had a better performance for PMS2 mutation carriers. Selection of patients for analysis of MSI and IHC may have been less stringent at the Erasmus Medical Center Rotterdam than at the Leiden University Medical Center. Therefore, mutation carriers in our validation cohort, who were all from Leiden University Medical Center, may have had a family history more suspect for Lynch syndrome than family history of the patients in our original cohort. This could explain the higher AUCs in the validation cohort. However, in both cohorts we showed that the extended PREMM5 had better performance.In our validation cohort, all AUCs were much higher than in our original cohort, including those for Prediction models for Lynch syndrome are not yet regularly used in current clinical practice. However, the US Multi-Society Task Force on Colorectal Cancer recommends genetic evaluation if an individual’s risk of carrying an MMR gene mutation is ≥ 5% according to one of the prediction models MMRpro, MMRpredict or PREMM . The Ame1,2,6 [The US Multi-Society Task Force on Colorectal Cancer recommends the use of either PREMM, MMRpredict or MMRpro to assess the probability of an individual carrying an MMR mutation . Since w1,2,6 –27, 32.MLH1 and MSH2 mutation carriers than for LS patients carrying a mutation in MSH6 or PMS2. This finding is in line with a previous study that showed that carriers of mutations in MSH6 or PMS2 had lower risk scores than carriers of a mutation in MLH1 or MSH2 [PMS2 mutation carriers was the least accurate, in line with its more limited penetrance.Both PREMM5 and MMRpredict were far more accurate for  or MSH2 . In our PMS2 mutations [PMS2 mutation carriers. To our knowledge, our study is the first to validate LS prediction models for PMS2 mutation carriers specifically since the development of the PREMM5 model. At a 5% cut-off, our extended PREMM5 model was able to detect 5/6 PMS2 mutation carriers who would have been missed by the original PREMM5 model at the same cut-off. Identification of Lynch syndrome carriers is highly important, since this allows not only them, but also their family members carrying the same mutation, to undergo intensive surveillance in order to prevent the development of cancer. Our new model would also identify more Lynch syndrome patients overall than the original PREMM5 model.Around 15% of all Lynch syndrome cases are estimated to be caused by utations . In our The performance of prediction models can differ between high-risk settings and population-based cohorts. Further validation studies should indicate whether our results can be generalized to settings with patients at low to median risk of having Lynch syndrome. Since patients in our study cohort were all referred for genetic counselling, family histories were obtained in detail and in many cases also verified by medical documents. In other settings where patients are at lower risk of having Lynch syndrome, family history is not verified and might be less reliable. Therefore, prediction models should also be validated in population-based cohorts. However, in a meta-analysis, prediction models performed better in population-based cohorts than in clinic-based cohorts .1,2,6 was more accurate than MMRpro and MMRpredict, but still only reached an AUC of 0.71 [PMS2 was not performed in the Korean study, so there might have been more mutation carriers in their cohort. Another non-Western population has been studied by Khan et al., who analysed the performance of prediction models in 15 African American patients [1,2,6 both had a high AUC of 0.89.It is not known whether the current prediction models for Lynch syndrome are useful in non-Western populations. In a recent study among Korean patients, PREMM of 0.71 . There wpatients . In thesMSH6 and PMS2 mutations. Since 12 patients were identified as a PMS2 mutation carrier, we were able to evaluate the prediction models for each MMR mutation specifically, admittedly with considerable uncertainty [PMS2 mutation carriers.A main strength of our study was the large cohort, which consisted of more than 700 index patient including 83 Lynch syndrome patients. Also, our cohort included patients with ertainty . FurtherA limitation of our study was that germline mutation analysis was not done for all index patients. Patients who had microsatellite stable tumours with normal IHC were assumed to be non-mutation carriers. However, some of these patients might still have an MMR mutation. Also, the sample size per gene was still relatively small and it is unclear whether our results from a high-risk population apply to a population-based setting.PMS2 mutations. Adding the location of CRC to the PREMM5 model improves the performance of the model for PMS2 mutation carriers as well as its overall performance. These findings should be validated in large cohorts from population-based settings.In conclusion, we have shown that although MMRpredict and PREMM5 can accurately predict an individual’s risk of carrying a causative MMR mutation, neither model is able to identify patients with Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 14 KB)Supplementary material 2 (DOCX 15 KB)Supplementary material 3 (DOCX 19 KB)