Traumatic brain injury (TBI) is a major health concern worldwide. Laboratory studies utilizing animal models of TBI are essential for addressing pathological mechanisms of brain injury and development of innovative treatments. Over the past 75 years, pioneering head injury researchers have devised and tested a number of fluid percussive methods to reproduce the concussive clinical syndrome in animals. The fluid-percussion brain injury technique has evolved from early investigations that applied a generalized loading of the brain to more recent computer-controlled systems. Of the many preclinical TBI models, the fluid-percussion technique is one of the most extensively characterized and widely used models. Some of the most important advances involved the development of the Stalhammer device to produce concussion in cats and the later characterization of this device for application in rodents. The goal of this historical review is to provide readers with an appreciation for the time and effort expended by the pioneering researchers who have led to today’s state of the art fluid-percussion animal models of TBI. Traumatic brain injury (TBI) is a major health concern worldwide , 2. In tExperimental models of TBI apply various forms of mechanical energy to the brain with a goal of replicating in animals the neurological syndromes, biological responses, and pathophysiology observed in human closed head TBI. Thus, animal models provide a means of rigorously studying clinical pathological states of TBI in a scientifically controlled manner in a laboratory setting. Over the years, animal models of TBI have employed numerous techniques applied to various species in attempts to reproduce multiple aspects of human TBI. Techniques include rapid acceleration or rotation of the head , impact The earliest application of a fluid-percussion technique involved striking a fluid-filled column secured to the exposed dura of an animal. Denny-Brown and Russell attempted to produce a generalized loading to the brain rather than a focal injury by applying a localized pressure pulse to the exposed dura of cats . They caThe Richmond group at the Medical College of Virginia, led by Becker, Povlishock, and Hayes further modified the Stalhammar device and applied fluid percussion to the midline of the cat brain through an 18-mm diameter craniectomy , 30, 31.The next major advance in the fluid-percussion technique was Dixon’s development of midline fluid percussion in the rat. The impetus for developing a rat model of TBI was the goal to expand laboratory TBI model endpoints to behavioral assessment of outcome. The choice of the rat took advantage of the rich history in the field of psychology that made extensive use of behavioral analysis in this species. Dixon’s landmark study characterized the pathophysiological, histopathological, neurological, and behavioral responses to a range of injury severities in the rat . Dixon’sper diem costs of maintenance.The development of the rat fluid-percussion model also afforded several other advantages including genetic homogeneity, ethical considerations of moving away from the use of companion animals to those lower on the phylogenetic scale, ease of procurement, and availability through a network of professional vendors. Furthermore, there were practical and financial advantages of using a species that required less vivarium space, afforded ease of handling, and reduced financial burdens of procurement and lower Perhaps the main advantage of moving to a rat model of TBI was to make feasible and practical, testing of dose–response pharmacological interventions that require behavioral assessment of large numbers of subjects. The first such dose–response pharmacological intervention study administered scopolamine to examine muscarinic cholinergic receptor involvement in transient behavioral suppression (traumatic unconsciousness) and physiological responses to TBI . That stAn extremely important alteration to the central rat fluid-percussion model, instituted by McIntosh, moved the craniectomy position from the vertex to a lateral  site . The intThis innovative lateral rat fluid-percussion model provided several advantages over the central/midline model. First, rupture of the sagittal sinus underlying the central craniectomy, which could occur during surgery or upon percussive impact, was completely avoided, resulting in reduced blood loss and preservation of proper venous blood outflow from the brain. Second, the lateral model produced consistent cortical and hippocampal neuronal cell death compared to the central model. The gross pathology was always localized to the hemisphere ipsilateral to the fluid pulse, providing a relatively less injured, contralateral hemisphere useful for certain control comparisons. However, numerous studies have since demonstrated that the contralateral hemisphere sustains pathological neurochemical perturbations , 59 and Finally, the lateral fluid-percussion orientation appears to produce less direct brain stem compression allowing for application of higher fluid-percussion forces with lower mortality than central fluid percussion . CompareN-methyl-d-aspartate type glutamate receptor, demonstrated class I evidence  for improving disability rating scale scores in the chronic phase of TBI , as originally performed by McIntosh et al. . Over thWhile there are many benefits associated with the use of rodent models of TBI, there are also arguments for the use of higher order animals to better characterize the relevant pathobiology of human TBI. The use of multiple species is encouraged to address fully the complex pathobiological processes ongoing in human TBI . The lisA German group led by Bauer characterized a severe lateral fluid-percussion model over the right parietal cortex in juvenile pigs (6 weeks old mixed German domestic breed), which reliably produced secondary elevations in ICP accompanied by patterns of diffuse brain damage . More reThe robust proliferation of genetically altered mice has provided opportunity to more precisely focus mechanistic studies on pathological processes. Following the lead of the Penn group who adapted the controlled cortical impact for mice , CarboneSeveral different fluid-percussion devices have been developed since the inception of the Richmond device in the 1980s. The Dragonfly fluid-percussion device, used primarily by Shima’s group in Japan, applies the same basic principle of a closed hydraulic system as the Richmond device, but differs with smaller dimensions and stainless steel construction in place of Plexiglas™ , 83. FadThere are a number of important parameters to consider when utilizing the fluid-percussion technique regardless of the subject species. Craniectomy location  produces unique pathological responses , 50. RelFrom the early 1940s through the 1970s, pioneering head injury researchers developed and tested a number of fluid percussive methods to reproduce the concussive clinical syndrome in laboratory animals. By today’s standards, the work of these early pioneers in the field of Neurotrauma appear somewhat crude. However, it was their ingenuity and determination that laid the foundation for the level of sophistication found in today’s animal models of TBI using well-characterized and controlled devices and procedures such as fluid percussion and controlled cortical impact. The early fluid-percussion studies in cats provided a benchmark standard in the 1980s, producing groundbreaking studies of the mammalian response to brain trauma. The early cat fluid-percussion studies contributed greatly to our understanding of the brains response to head injury ranging from cerebral vascular alterations to traumatic disruption of consciousness. The development and characterization of rat fluid-percussion injury in the mid to late 1980s provided an important leap forward making possible behavioral outcome evaluations and a practical means of performing large scale studies of experimental therapeutics. Fluid percussion was later adapted for use in larger (pigs) and smaller animals (mice), providing means of studying TBI in gyrencephalic and genetically altered animals. The historical perspective of this brief review will hopefully provide appreciation for the effort, determination, and ingenuity expended by pioneering researchers who have led to today’s state of the art fluid-percussion animal models of TBI.BL wrote and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Haemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Despite their disputed taxonomic status in the literature, these subspecies have not yet been compared with molecular methods. The aim of the present study was to investigate the phylogenetic relationships of H. erinacei subspecies, in the context of the first finding of this tick species in Romania.H. e. taurica (from Romania and Turkey), four adults of H. e. erinacei  and 17 adults of H. e. turanica (from China). From these samples fragments of the cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes were amplified via PCR and sequenced. Results showed that cox1 and 16S rRNA gene sequence divergences between H. e. taurica from Romania and H. e. erinacei from Italy were below 2%. However, the sequence divergences between H. e. taurica from Romania and H. e. turanica from China were high (up to 7.3% difference for the 16S rRNA gene), exceeding the reported level of sequence divergence between closely related tick species. At the same time, two adults of H. e. taurica from Turkey had higher 16S rRNA gene similarity to H. e. turanica from China (up to 97.5%) than to H. e. taurica from Romania (96.3%), but phylogenetically clustered more closely to H. e. taurica than to H. e. turanica.After morphological identification, DNA was extracted from five adults of H. erinacei in Romania, and the first  phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses did not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank, because the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range.This is the first finding of  Haemaphysalis Koch, 1844 is the second largest genus (following Ixodes) of hard ticks (Acari: Ixodidae), including 167 species [Haemaphysalis erinacei Pavesi, 1884 occurs in Mediterranean forests, woodlands and scrub [H. erinacei are terrestrial mammals, such as hedgehogs and carnivores for adult ticks [Rickettsia massiliae [R. raoultii [R. heilongjiangensis [lt ticks . Bats, braoultii  and R. hangensis ).Fig. 1DHaemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Subspecies are conspecific taxa, representatives of which show differences in morphology and geographical range from each other, but can naturally interbreed. Accordingly, until now H. erinacei subspecies were described on the basis of different morphology and geographical range, but this resulted in a controversy in their taxonomy. Camicas et al. [H. erinacei, namely H. e. erinacei Pavesi, 1884 (described from Tunisia), H. e. ornata Feldman-Muhsam, 1956 (described from Israel), H. e. taurica Pospelova-Shtrom, 1940 (described from Crimea) and H. e. turanica Pospelova-Shtrom, 1940 (described from Tajikistan). According to Hoogstraal [H. erinacei includes three subspecies: H. e. erinacei in North Africa, H. e. taurica in the Middle East (including western states of the former Soviet Union) and H. e. turanica in Central Asia. Haemaphysalis e. erinacei also occurs in southern Europe, in particular in Spain, Italy and the western Balkans [H. e. taurica is present in the eastern Balkans [H. e. taurica and H. e. turanica are widely distributed in certain regions of Central Asia  were investigated, in the context of the first finding of this tick species in Romania.Despite this taxonomic controversy, no studies have attempted molecular phylogenetic comparison of c oxidase subunit 1 (cox1) and 16S rDNA genes are well-established barcoding genes for molecular identification and phylogenetic analyses of ticks [H. e. taurica, H. e. erinacei and H. e. turanica in the present study.Cytochrome of ticks –16. TherH. erinacei were included in this study (Table H. e. erinacei) and Filippova [H. e. turanica, H. e. taurica). Pictures were produced with a VHX-5000  digital microscope.Altogether 26 adults of dy Table . The subilippova  (H. e. tc oxidase subunit 1 (cox1) gene using the primers HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') and LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) [cox1 gene , and PCR conditions were different .DNA was extracted with QIAamp DNA Mini Kit  as described , includiTG G-3′) . ReactioTG G-3′) . Concernhttps://blast.ncbi.nlm.nih.gov). Representative sequences were submitted to the GenBank database , and are referenced according to accession numbers.PCR products were visualized in 1.5% agarose gel. Purification and Sanger dideoxy sequencing for samples from Romania, Italy and Turkey was done by Biomi Inc. , and for samples from China by Sangon Biotech Co. . The newly-generated sequences were manually edited, aligned and compared to reference GenBank sequences by nucleotide BLASTN program , and 99.4% identical with H. e. erinacei from Italy, but had only 94.6–95.1% similarity with isolates of H. e. turanica from China (Table H. e. taurica from Romania showed 99.3% similarity with one specimen of H. e. taurica collected in Turkey (Tokat province), and 98–98.3% similarity with H. e. erinacei from Italy. On the other hand, the 16S rRNA fragment of H. e. taurica from Romania had only 92.7–95.3% similarity with isolates of H. e. turanica from China.The cox1 and 16S rDNA gene sequence divergences between H. e. taurica from Romania and Turkey (Tokat province) and H. e. erinacei from Italy were low (below 2%). This may be consistent with allopatric separation of these two subspecies  and I. ricinus (GU074592) is 98.2% (383/390 bp), amounting to 1.8% difference [H. e. taurica and H. e. turanica.Taken together, the istances . Howeverfference , the seqH. e. taurica  than to H. e. taurica from Romania (96.3%) , which were morphologically identified as adults of ica Fig.  had high%) Table . HoweverH. e. taurica, H. e. erinacei and H. e. turanica are well reflected by the topology of cox1 phylogenetic tree, with H. e. taurica and H. e. erinacei clustering close to each other, but separately from H. e. turanica , Turkey (KR870978) and China (KF547980) with 100% 16S rRNA gene identity.Several factors may have contributed to this divergence and its maintenance. Southern peninsulas of Europe acted as major refugia during ice age(s), from which genetically distinct clades of animal species emerged , as alsopian Sea  may haveerinacei , whereasconcinna ; or H. pH. e. taurica and H. e. turanica are common, these two tick subspecies exhibit biotope isolation in overlapping parts of their geographical range [In addition, based on an extensive collection material, while the hosts of al range , which mH. erinacei in Romania, and the first  phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses do not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank. In particular, the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting  that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range.This is the first finding of


Fumihiro Arizumi appeared incorrectly as Arizumi Fumihiro. Dr. Shinichi Inoue appeared incorrectly as Inoue Shinichi. Dr. Toshiya Tachibana appeared incorrectly as Tachibana Toshiya. Dr. Keishi Maruo appeared incorrectly as Maruo Keishi. Dr. Shinichi Yoshiya appeared incorrectly as Yoshiya Shinichi.In the article, “A sewing needle in contact with the cervical dura mater and vertebral artery: A case report”,


The early evaluation of its therapeutic effect on tumours is vital for the optimization of treatment regimes. The most commonly used 32P-CP colloid tends to leak with blind therapeutic area after intratumour injection. We prepared 32P-chromic phosphate-polylactide-co-glycolide (32P-CP-PLGA) seeds with biodegradable PLGA as a framework and investigated their characteristics in vitro and in vivo. We also evaluated the therapeutic effect of 32P-CP-PLGA brachytherapy for glioma with the integrin αvβ3-targeted radiotracer 68Ga-3PRGD2. 32P-CP-PLGA seeds  and 32P-CP colloid  were implanted or injected into human glioma xenografts in nude mice. Scanning electron microscopy (SEM) of the seeds, micro-SPECT imaging, and biodistribution studies were performed at different time points. The tumour volume was measured using a caliper, and 68Ga-3PRGD2 micro-PET-CT imaging was performed to evaluate the therapeutic effect after 32P intratumour administration. The delayed release of 32P-CP was observed with biodegradation of vehicle PLGA. Intratumoural effective half-life of 32P-CP in the SG (13.3 ± 0.3) d was longer than that in the CG (10.4 ± 0.3) d (P < 0.05), with liver appearance in the CG on SPECT. A radioactivity gradient developed inside the tumour in the SG, as confirmed by micro-SPECT and SEM. Tumour uptake of 68Ga-3PRGD2 displayed a significant increase on day 0.5 in the SG and decreased earlier (on day 2) than the volume reduction (on day 8). Thus, 32P-CP-PLGA seeds, controlling the release of entrapped 32P-CP particles, are promising for glioma brachytherapy, and 68Ga-3PRGD2 imaging shows potential for early response evaluation of 32P-CP-PLGA seeds brachytherapy. Glioblastoma multiforme (GBM) accounts for approximately 60% to 70% of malignant gliomas. The prognosis of patients with GBM remains dismal, with a median survival time of less than one year. This poor prognosis primarily reflects the high proliferative, infiltrative, and invasive properties of GBM . Systemi32P is ideal because of its relatively long half-life of 14.28 day(s), maximum energy of 1.711 MeV, mean range of 3-4 mm in soft tissues, and high relative biological effect. Recent studies have focused on better vehicles or carriers, and many of these studies have used 32P bandages or films for the brachytherapy of superficial cancers and intracranial and spinal tumours  (32P-CP in the SG (13.3 ± 0.3 d) was longer than that in the CG . Pathological examination of the liver, spleen, lung, and bone demonstrated no abnormality in the SG.The biodistribution of  < 0.05] . Intratu32P-CP particles in tumour and adjacent normal tissues in the CG or SG after eradication and radioactivity counting of the remaining 32P-CP-PLGA seeds. Most of released 32P-CP particles in the SG were stagnant in the tumour core with a decreasing gradient towards the periphery in the SG  on day 16 was higher than day 8 (t1) (n the SG . This fin the SG . The gray 8 (t1) .Micro-SPECT/CT Imaging. 32P bremsstrahlung facilitates SPECT-CT imaging to monitor the intratumour administration of 32P-CP colloid  and 65.8% ± 3.1% (CG) on day 64 based on micro-SPECT/CT images. colloid  and 32P-GA seeds . As show68Ga-3PRGD2 was observed on days 2 and 4 with internal absence on day 8. Scar formation was observed on day 16 in the SG. In the P-CP-PLGA seed group, the distribution of 68Ga-3PRGD2 was uniform for 8 day(s), and internal heterogeneity was observed on day 8, reflecting natural necrosis. The tumour uptake (%ID/cm3) of 68Ga-3PRGD2 in the SG (8.50 ± 1.32) was higher than that in the P-CP-PLGA group  on day 0.5, while the tumour uptake in the SG (5.13 ± 1.06) was lower than that in the P-CP-PLGA group  on day 2. The tumour time-growth curve displayed no significant changes during 10 days in the SG or CG after 32P administration, and 40% (2/5) of the mice demonstrated tumour growth near the scar on day 20.As shown in αvβ3 and CD31 expression levels in tumour tissues from the xenografted gliomas in the SG and P-CP-PLGA group on days 0.5, 2, and 4; as shown in αvβ3 immunofluorescence levels in the SG revealed a 1.37-fold (±0.15) mean increase (±SD) on day 0.5 and 0.74-fold (±0.22) and 0.15-fold (±0.05) decreases on days 2 and 4, respectively. IHC examination was not performed on days 8 and 16 due to increased areas of necrosis and scar formation.Immunofluorescence staining was performed to examine the 32P-CP-PLGA seed is a biodegradable dosage form of 32P, utilizing PLGA as the controlled release carrier for drug delivery. In addition to its easy preparation in large amounts, the main advantage of 32P-CP-PLGA seeds is their delivery of high-dose radiation to the tumour and long intratumoural radioactive effective half-life with minimal distribution and damage to systemic tissues. A radioactivity gradient formed inside the tumour with potential effect to enhance the intratumour fire effect, reduce damage to adjacent tissues, and avoid the implantation of low radioactivity seeds in the peripheral part of the tumours.32P-CP particles in the SG and CG immediately after administration, except for the occurrence of leakage of colloidal 32P-CP in the CG. However, the dynamic radioactivity distribution indicated more retention and longer effective half-life of 32P-CP in the tumour and less or even undetectable distribution in other tissues in the SG compared with that in the CG. The principal mechanism likely involved the arrest of 32P-CP particles inside the seeds using the framework of PLGA, and their release and slow drainage was determined by the biodegradation of PLGA. A radioactivity concentration gradient, formed by the 32P-CP particles released from the 32P-CP-PLGA seeds, was observed inside the tumour. The tumour uptake (%ID/g) was extremely high, primarily because more than 95% (%ID) of the implanted 32P remained in the small tumour tissues (approximately 0.2 cm3) during the first few days. The increased uptake primarily reflected tumour shrinkage on day 16 and scar formation. The released 32P-CP particles increase the killing range and enhance the therapeutic effect without noticeable damage to adjacent tissues. The gradient gap was predominant in the first few days and narrowed with increasing release of 32P-CP particles and tumour shrinkage. However, the irregular and uneven intratumour distribution of 32P-CP colloids in the CG led to a therapeutic blind area, tumour relapse, and leakage from the injection site, resulting in damage to the adjacent tissues.No significant difference was observed in tumour uptake of 32P-CP particles drained from the tumour were primarily distributed throughout reticuloendothelial systems, such as the liver in the CG, and were less than reported [32P-CP colloids. The uptake of 32P-CP particles in normal tissues peaked at 16 days and likely involved the following three mechanisms. First, 32P-CP particles at the surface or inlaying the outer 1/3 of the seeds tended to be released, while those inlaid near the core stagnated inside, although there was gradual formation of tunnels and cavities. Second, the decrease or lack of regional circulation around the seeds resulting from tumour tissue necrosis and scar formation may inhibit the drainage of released 32P-CP particles. Third, the experimental period used in the present study may not be long enough. Pathological changes of important organs were absent in the SG.The reported . This ef32P bremsstrahlung SPECT imaging facilitates the positioning, effective for half-life evaluation under physical conditions [nditions . Any immnditions .18F-FDG PET-CT imaging was widely used to monitor therapeutic effect of tumour treatment. However, two potential disadvantages limit its use in gliomas. One disadvantage is the high distribution of glioma in normal brain tissues, particularly in the cerebral cortex, basal ganglia, and thalamus, where glioma is prone to occur. The other disadvantage is the increased accumulation of tumour tissue when inflammatory cells, such as macrophages, are present, particularly at the early stages of treatment [18F-FPPRGD2 is superior to 18F-FDG because the uptake of 18F-FPPRGD2 is not influenced by tumour-associated macrophages after chemotherapy, and the remarkable distribution of 68Ga-3PRGD2 in the U87MG xenografts is observed with low background in the normal brain tissues compared with 18F-FDG [68Ga is easily accessible from a 68Ge-68Ga generator and showed similar labelling characteristics with therapeutic isotopes, such as 177Lu, with DOTA and NOTA as linkers. In the present study, the initial increased uptake of 68Ga-3PRGD2 in glioma was observed on day 0.5 after 32P-CP-PLGA seed implantation. The possible mechanism involves the rapid upregulation of integrin expression induced by a low-to-intermediate dose of radiation, as previously reported [68Ga-3PRGD2 on day 0.5 in the SG is possibly due to radiation, while the decrease on day 2 is due to cell damage resulting from accumulated 32P radiation and therapeutic effects [68Ga-3PRGD2 imaging has potential to serve as an important tool for the therapeutic evaluation of 32P brachytherapy of glioma as early as day 2. Thus, the precise relationship between the β− radiation dose and integrin expression upregulation needs further study.reatment . 18F-FPP 18F-FDG , 19. 68Greported . Thus, t effects . This ef32P-CP-PLGA seeds, controlling the release of entrapped 32P-CP particles, are promising for glioma brachytherapy. 68Ga-3PRGD2 imaging shows potential for early response evaluation of 32P-CP-PLGA seeds brachytherapy.


The obtained results observed that the ferromagnetic order is the most stable ground state and the magnetic moment will collapse at about 50 GPa. As a result, the lattice a axis becomes stiffer above about 420 GPa, ultimately presenting the same axial compressibility trends with those of nonmagnetic compounds Mo2TiAlC2 and hypothetical Cr2TiAlC2. The elastic constants and phonon dispersion curves demonstrate the structural stability during the disappearance of magnetic moment and occurrence of axial alternative compressibility. The density of states and energy band calculations confirmed the existence of magnetic moment of Cr2TiAlC2 at 0 GPa and disappearance at high pressures above 50 GPa. Evolutions of magnetic moment collapse with pressure are confirmed by a variety of properties. The obtained grüneisen parameter and thermal expansion coefficients show the maximum value among the known MAX phases, to date and to the author’s knowledge.The electronic structure and thermodynamical properties of Cr MAX phases combine the advantages of metal and ceramics and thus present many excellent properties in the field of mechanics and thermodynamics. Many such crystals could be formed by substituting the Ti atoms to those unavailable structures at ambient conditions, such as Cr2TiAlC22.5Nb2.5)AlC41.5Ti2.5)AlC3x1−Nbx)2AlC (x = 0~1.0)1−x,Tix)2AlCx = 0~1.0), Mo2TiAlC22Ti2AlC3The Mn n = 1, , 3, etc 2TiAlC2. Similarly, the novel Cr2TiAlC2 might also exist special properties. The possible origin3AlC2 is explored, the introduced Ti atom presents smaller relative intensity at Fermi level in comparison with that of Mo atom, whereas the calculated energy levels of Al/C species immigrate to higher-energy side which should give negative contribution to the crystal stability. Despite the Ti-doping stabilized compounds have been studied by the density of states, the detailed bonding processes are still unclear, in particular at higher pressure.Formation energy2TiAlC22TiAlC2. We therefore perform a systematical investigation to the newly synthesized Cr2TiAlC2 to determine its magnetically-related properties. First principles are accurate tool in the electronic structure calculations1011121314151617181920Stimulated by the novel role of Ti doping stabilized nonmagnetic Mok-point is defined using 10 × 10 × 2 Monkhorst-Pack mesh2423p63d54s1, Ti 3s23p63d24s2, Al 3s23p1, and C 2s22p2, respectively. The energetic iteration criterion is 5.0 × 10−7 eV/atom. All the simulations were finished by CASTEPThe geometric relaxation is completed by the Vanderbilt-type2AlC, Cr2GaC, and Cr2GeC2TiAlC2 are same2TiAlC2. Therefore, several collinear magnetic configurations of Cr2TiAlC2 are considered to search for its ground state, including non-magnetic(NM), ferromagnetic (FM), various antiferromagnetic (AFM), and two in-plane AFM, in which no correlation effect of the Cr d electrons is applied. These magnetic orders are same2GaC, we here ignore the schematic illustrations for simplicity. Relative to the minimum energy of the NM unit cell, the comparison for all the considered magnetic states of Cr2TiAlC2 is presented in Previous investigations for Cr2TiAlC2 is P63/mmc with twelve atoms per unit cell constituting of two formula units, the atomic coordinate is:  of Cr,  of Ti,  of Al,  of C. The obtained structural constants are: a = b = 2.9392 Å, c = 17.8341 Å, u = 0.130617, v = 0.074795, consistent with the experimental data The space group of FM Cris: 2/3, /3, u of c axis under pressure is faster than that of a axis, such as NM Mo2TiAlC2a axis is faster than that of c axis within 0~420 GPa in FM Cr2TiAlC2. Interestingly, an alternative case occurs with pressure continuous increasing, as is shown in 2TiAlC2 and find that the two axial compressibilities are almost identical below about 100 GPa, henceforth the stiffer a axis occurs. The detailed structural evolutions for FM/NM Cr2TiAlC2, and NM Mo2TiAlC2 are shown in 2TiAlC2 among the three compounds might resist the c-axis contraction. The NM Cr2TiAlC2 present almost identical c-axis contractions with that of NM Mo2TiAlC2, the slightly larger a-axis compressibility of NM Cr2TiAlC2 might originates from its smaller atomic radials. The magnetic moment definitely decelerates the c-axis but accelerates the a-axis contractions in FM Cr2TiAlC2 in comparison with those of NM Cr2TiAlC2. In addition, the volumetric compressibility of NM Mo2TiAlC2 is evidently smaller than that of NM Cr2TiAlC2 due probably to the larger Mo radius, the largest volumetric compressibility of FM Cr2TiAlC2 due mainly to the fact of its easily compressed a axis.Usually, the contraction of i.e.Our calculated elastic constants are listed in 2TiAlC2 is mechanical stable up to 500 GPa. To confirm this conclusion, we calculate the phonon spectra at 0, 420, and 500 GPa by the finite displacement method using a cutoff radium of 5 Å with a supercell volume of 16 times larger than the unit cell under the same precision settings with that of geometrical relaxation, as is shown in 2TiAlC2 is dynamic stable within 0~500 GPa. The calculated elastic constants of FM/NM Cr2TiAlC2 and NM Mo2TiAlC22TiAlC2 tends to zero with pressure increasing to about 40 GPa, suggesting that the magnetic moment are totally collapsed.It is found that FM Cr2GeC2AlC from our simulations. The difficult collapse evolution possibly means the existence of more complex intermediate transition magnetic configurations. This magnetic transition (from FM to NM) of Cr2TiAlC2 should be the internal driven force of axial alternative compressibility at about 420 GPa.Previous calculations for Cr2GeC and Cr2AlC exist net magnetic moments and the magnitudes nearly vanish at about 100 K, whereas the crude estimation of the magnetic moment of per Cr atom is 0.05μB in Cr2AlC and 0.02μB in Cr2GeC, far smaller than available calculations such as FM Cr2AlCμB at U = 1.95 and 2.5 μB at U = 2.95), AFM Cr2AlCμB), AFM Cr2GeCμB). However other measurement2AlC obtained a value of 0.64 μB. Our computed sum of the absolute magnetic moment of spin-up and spin-down directions is 5.3749 μB in AFM Cr2AlC in one unit cell , consisting well with previous calculations2AlCμB), Cr2AlCμB at U = 0 eV), Cr2GaCuB). A value of about 0.8 μB is observed in present FM Cr2TiAlC2. The difference between the present FM Cr2TiAlC2 and AFM Cr2AlC is about 0.13 μB, whereas they are nearly identical in a recent calculationμB in FM Cr2TiAlC2 and 1.0 μB in AFM Cr2AlC, respectively. The slab of Ti-C between the two nearest Cr2AlC stack blocks in unit cell of Cr2TiAlC2 possibly affect the atomic moment as the Ti atom could strongly stabilize the unavailable Cr3AlC2Previous measurements302TiAlC2 and NM Mo2TiAlC2 are shown in a-axis stiffening above 420 GPa is undoubtedly seen. The abnormal increase of bond population in Al-Cr bond above 420 GPa means that the antibonding interaction becomes stronger again after it approaches the first local minimum.The calculated bond length compressibility and bond population of FM Cre at 0 GPa and 1.01 e at 500 GPa in FM Cr2TiAlC2, which are always larger than those of C-Mo in NM Mo2TiAlC2, with individual values of 1.21 e at 0 GPa and 0.79 e at 500 GPa, revealing stronger covalent bonding of C-Cr and its degree of covalence decreases with pressure. The value of strong C-Cr bond is apparently larger than that of diamond (1.08 e)The bond populations of C-Cr are 1.35 e at 0 GPa and −0.15 e at 500 GPa in FM Cr2TiAlC2, which are always smaller than those of counterparts of C-Ti in NM Mo2TiAlC2, with individual values of 0.76 e at 0 GPa and 0.32 e at 500 GPa. Both the C-Ti bonds, belonged respectively to the FM Cr2TiAlC2 and NM Mo2TiAlC2, present smaller bond populations than their respective C-Cr and C-Mo bonds, showing the smaller covalence and larger ionicity within a large pressure range.The respective bond populations of C-Ti are 0.65 e at 0 GPa and −2.6 e at 500 GPa in FM Cr2TiAlC2, whereas the values of Al-Mo are 0.4 e at 0 GPa and −2.23 e at 500 GPa in NM Mo2TiAlC2, the critical pressures of Al-Cr and Al-Mo are about 50 and 70 GPa from positive to negative values, respectively, which means that the electronic transition from bonding to antibonding states. Conclusively, a harder ‘M-X’ and softer ‘M-A’ bond in M3AX2 phase appears. After substitution of one Mo by one Ti from the four corners of the in-plane sites, the reduction of bond population are 0.7 e from C-Cr to C-Ti in FM Cr2TiAlC2 and 0.45 e in NM Mo2TiAlC2, reduced from C-Mo to C-Ti. These large reductions might benefit the stabilization of unavailable Cr3AlC22TiAlC2, with lowest contraction within the four bonds.The bond population of Al-Cr are 0.41 2TiAlC2, we systematically studied the bond rotation angle variations under pressure and the obtained results are shown in ab plane is also more than the cases of opposite rotations, i.e., three  versus one (Al-Cr). The three rotations contribute larger to c axis than to a axis. Owing to the rapid immigration of Cr atom along c axis, all of the four bonds rotate far larger angles in FM Cr2TiAlC2 than those of NM Mo2TiAlC2, particularly for the low pressure range below 50 GPa. These behaviors might be one reasonable explanation to the stiffer c axis of FM Cr2TiAlC2 at low pressure.To deep understand the axial compressibility of FM Cr2TiAlC2, we calculate the band structure and density of states (DOS) at 0 and 50 GPa, respectively, comprising of spin-up  and spin-down (beta) components, as is seen in For understanding the chemical bonding of FM Crd dominant orbitals present substantial discrepancies between alpha and beta spin orbitals at 0 GPa, whereas such discrepancies are rapidly reduced under pressure. Their contrary shift tendencies illustrated the energy evolution shown in To explore the shift trend of the whole beta orbitals under pressure, we further test all of the orbitals crossing the Fermi level, as is shown in t2g and eg states are half-filled, whereas the low-spin states provide six more unoccupied orbitals and high-spin states fill in five more valence orbitals and therefore they provide the net magnetic to the system. Analysis to all the orbitals observed that the low-lying energy levels are contributed mainly by Cr 3d (t2g) states in the vicinity of Fermi level, whereas the high-lying energy levels are composed mainly by Cr 3d (eg). Energy levels crossing Fermi level present highly hybridized characters. Energy levels sited in the conduction band are contributed mainly by Cr 3d (eg) and Ti 3d (eg) states. These distributions consist with the bond population features shown in Both the 2TiAlC2, far smaller than the current 7.15 states/eV of FM Cr2TiAlC2, denoting the significant influence of magnetic moment. In 2TiAlC2, particularly for its spin-up components, present similar metallic and energy-level features with that of NM Mo2TiAlC2 at 0 GPaThe values (per unit cell) of the spin-up DOS at Fermi level are 4.74, 4.27, 3.51, 1.08 states/eV for 0, 20, 50, and 500 GPa, respectively. The correspondent values of spin-down counterparts are −2.41, −2.73, −3.39, and −1.08 states/eV. The total DOS valuee) mainly from Ti (0.81 e) and Al (0.32 e) atoms, respectively. Moreover, Cr atom loses its minor charges (0.03 e) at ambient conditions in FM Cr2TiAlC2. However the number of charges in C atoms keeps almost unchanged even under higher pressure, which is important to sustain the extensive stability of C-Cr and C-Ti bond populations under pressure. The Al atoms lose its charge firstly and gain charge from others subsequently under pressure, which is just contrary with that of Cr atom, whereas Ti atoms monotonically lose its charges with pressure. These charge transfer direction forms significantly larger bond populations along Al-Cr orientation and relatively small values along C-related bonds including C-Cr and C-Ti, which probably means the strong charge saturation of C atoms.Electron density difference (EDD) shown in d states present strong anisotropy feature, whereas such feature rapidly decreased under pressure, indicating the strong hybridization of Cr d states. Moreover, the partial-filling feature of Cr d states is undoubtedly shown, such as the prominent t2g (dxy) at low pressure and eg  presents similar variations under pressure with its counterpart in the two compounds FM Cr2TiAlC2 and NM Mo2TiAlC2.Electron localization function (ELF) generally reflects the general and total orbital bonding features and the charge transfer trends as well as the atomic charge distributions, as is shown in 2TiAlC22TiAlC2 behaves similar variations with those of FM Cr2TiAlC2. The large electron localization could be seen in the region between adjacent atoms in NM Mo2TiAlC2, indicative of nearly completely filled and slightly stronger covalent bonding, consisting well with its relatively larger mechanical quantities. Both FM Cr2TiAlC2 and NM Mo2TiAlC2 show similar polarized bonding features with directional orientations around the C atoms at high pressure, which means the anisotropic bonding characters and different chemical bonding styles. The small ELF value between C and Cr and the nearly spherically symmetry distribution of Cr sites in FM Cr2TiAlC2 demonstrate the mixture bonding character. A directional bonding between Cr and Al is polarized towards the Cr sites judged from an arc shape. There is a maximum value between C and Ti, indicative of covalent bonding. The nearly square distribution around C means its partially ionic constitutions. In addition, a predominantly antibonding orbital along Cr-Ti is built.Under pressure, the giant reduction of ELF value in NM MoThe calculated Fermi surfaces (FS) of orbitals crossing Fermi level are shown in The whole FS profiles of orbitals 51 and 52 are shifted to first BZ at 20 GPa. Meanwhile, the higher energy of the orbital exists, the nearer distance of its FS to the center of the first BZ will be, which is just contrary to the case of beta spin at 0 GPa. Orbitals 49 and 50, with beta spin belonging to higher-energy orbitals in comparison with the other orbitals crossing Fermi level, site farer from the center of the first BZ relative to the other ones at 0 GPa, whereas they transform to the lower-energy orbitals with same orbital sites at 20 GPa, meaning the huge shift of the beta orbital towards lower-energy side under 20 GPa. Such orbital energy shift is about 0.6 eV, far larger than the case of alpha orbital which shows a global shift of about 0.2 eV towards higher-energy side. The just opposite shift tendencies will cause more overlap between them and ultimately induce them are totally overlapped at 500 GPa. Despite the magnetic moment has already been disappeared at 50 GPa, the orbital energies and the FS profiles are not totally merged, still existing an energy discrepancy of about 0.1 eV between the different spin orbitals.c33 means that the c-axis direction is relatively soft, which is partially inconsistent with the axial compressibility. An evident dip of c33 corresponds to the c-axis softening above 420 GPa in FM Cr2TiAlC2, as is shown in A = c33/c11, A = 1, denoting isotropic crystal, any ratio higher or lower than 1 means an elastic anisotropy. The obtained A are 1.1671 at 0 GPa and 0.9978 at 500 GPa, respectively. The judgment of the anisotropy in shear is obtained by A1 = 2c44/(c11 − c12). When c44/cs = 1, a crystal is isotropic. The FM Cr2TiAlC2 presents anisotropic nature at 0 GPa, whereas the degree of anisotropy decreases with pressure, with results of 0.4259 at 0 GPa and 1.2231 at 500 GPa, respectively. Another shear anisotropy ratio is A2 = (c11 + c33 − 2c13)/4c44, a crystal is isotropic when A2 = 1. The variation range of A2 is 0.9803~0.3822 within 0~500 GPa in FM Cr2TiAlC2. The detailed variation trends are shown in A behaves oscillation phenomenon within 0.9~1 in the pressure range 50~500 GPa, which is different with the other two shear anisotropic factors. In comparison with the cases of NM Mo2TiAlC22TiAlC2 behave significant variations at low pressures below about 40 GPa.The small B) and shear (G) moduli are G ≈ 1.1B and G ≈ 0.6B, respectively. For FM Cr2TiAlC2 the simulated values of G/B are 0.8643 at 0 GPa, 0.4576 at 100 GPa, and 0.2977 at 500 GPa, respectively, indicating that the mixed bonding is suitable for FM Cr2TiAlC2 at 0 GPa and the degree of ionicity increases with pressure. All of these values are larger than their counterparts in NM Mo2TiAlC2, corresponding to 0.715 at 0 GPa and 0.403 at 100 GPa, respectively2TiAlC22TiAlC2 at 50 GPa mean that the pressure could effectively tune the chemical bonds of magnetic materials.For covalent and ionic compounds, the relationships between bulk  at 0 GPa, and the degree of brittleness increases with pressure, such as the values are 0.4576 at 100 GPa and 0.2977 at 500 GPa, respectively. However those values are 0.715 at 0 GPa and 0.4031 at 100 GPa in NM Mo2TiAlC22TiAlC2 is more brittleness owing to the appearance of the magnetic moment. Moreover, the current B/C44 is 1.2068, which is also slightly smaller than that of 1.442TiAlC2 but just within the range (1.2–1.7) of Mn+1AXn phases.Pugh 2TiAlC2, respectively, far smaller2TiAlC2, meaning that the FM Cr2TiAlC2 is covalent and ionic materials at 0 GPa, and a mixed bonding with partial metallic and certain ionic participation combination could be assumed at higher pressure. The Poisson’s ratios of NM/FM Cr2TiAlC2 present similar variations with the only exception of lower counterparts within 10~40 GPa as there is an obvious reduction in FM one, which could be attributed to the influence of magnetic moment.The Poisson’s ratios are 0.1634 (0 GPa), 0.3015 (100 GPa), and 0.3646 (500 GPa) in FM Cr2TiAlC2 by the quasi-harmonic Debye model, the calculation detailsγ characterizes the anharmonicity of lattice, the calculated γ are shown in γ of FM Cr2TiAlC2 is far larger than that of NM2TiAlC2, particularly at low pressure, such difference is obviously decreased with pressure. In 2TiAlC2 and NM2TiAlC2 at 0 GPa within 0~1500 K, the significantly larger value of Cr-containing compound represents the stronger phonon-phonon interaction originating probably from the effect of magnetism at low temperature, whereas the value of γ approaches its maximum limit at about 600~700 K and then decreases gradually with temperature in FM Cr2TiAlC2, such inverse variation indicates probably the opposite strength response of the phonon-phonon interaction with the volume change. Previous calculations for Ti2SCγ in FM Cr2TiAlC2 are similar with those2TiAlC2 and thus we here neglect the detailed discussions. Generally, the values of γ are within 1.5~22GeC presents largest γ with a value of 2.38, which is still smaller than the present FM Cr2TiAlC2 which is 2.63 at 0 GPa and 0 K, meaning that the present compound behaves the largest γ among all known MAX phases till now, to the author’s knowledge.Several thermodynamical properties are studied for FM Crα with temperature and pressure of FM Cr2TiAlC2 is generally the same with that2TiAlC2, as are shown in α approaches its upper limit at about 1200 K in FM Cr2TiAlC2, with a value of 3.5343 × 10−5 × K−1, which is far larger than those of counterparts2TiAlC2, with a maximum value of 2.064 × 10−5 × K−1 at 1500 K. Such larger α can also be found in other Cr-containing MAX compounds, such as Cr2GaC and Cr2AlC−5 × K−1 and 2.4312 × 10−5 × K−1 at 1500 K. Moreover, previous calculations for Ti2SC−5 × K−1 K at 900 K, and it decreases to 1.8007 × 10−5 × K−1 at 1500 K. The detailed summary of α and other coefficients could be found in a recent reviewα probably is the largest value among all the MAX phases.The variation trend of the thermal expansion coefficient BT) and adiabatic (BS) bulk moduli present similar variation trends with those2TiAlC2 with the only exception of 0 GPa, as is shown in BT and BS exist small difference owing to the small α and γ, BS = BT(1 + αγT), where T means temperature. Variations of the discrepancy in (Mo2TiAlC2-Cr2TiAlC2) for BT and BS present just opposite variation trends between 0 GPa and higher pressures because the novel variation trends of FM Cr2TiAlC2 , which approaches the minimum value 130.63 GPa at about 1400 K in BT and 144.08 GPa at 800 K in BS. Such nonlinear variation is closely related to the variations of α and γ. Previous calculations for Ti2SCBT and BS.The isothermal .


Varicella zoster virus (VZV) reactivation spans the spectrum from uncomplicated segmental herpes zoster to life-threatening disseminated CNS infection. Moreover, in the absence of a small animal model for this human pathogen, studies of pathogenesis at the organismal level depend on analysis of human biosamples. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuroinfection and neuroinflammation. We therefore applied a targeted metabolomics screen of CSF to three clinically distinct forms of VZV reactivation and infectious and non-infectious disease controls in order to identify biomarkers for CNS involvement in VZV reactivation.n = 14), facial nerve zoster (n = 16), VZV meningitis/encephalitis (n = 15), enteroviral meningitis (n = 10), idiopathic Bell’s palsy (n = 11), and normal pressure hydrocephalus (n = 15).Metabolite profiles were determined by targeted liquid chromatography-mass spectrometry in CSF from patients with segmental zoster  that were particularly associated with VZV meningoencephalitis. SM(OH) C14:1 accurately distinguished facial nerve zoster from Bell’s palsy. Random forest construction revealed even more accurate classifiers (signatures comprising 2–4 metabolites) for most comparisons. Some of the most accurate biomarkers correlated only weakly with CSF leukocyte count, indicating that they do not merely reflect recruitment of inflammatory cells but, rather, specific pathophysiological mechanisms. Across all samples, only the sum of hexoses and the amino acids arginine, serine, and tryptophan correlated negatively with leukocyte count. Increased expression of the metabolites associated with VZV meningoencephalitis could be linked to processes relating to neuroinflammation/immune activation, neuronal signaling, and cell stress, turnover, and death , suggesting that these metabolites might sense processes relating to end-organ damage.The results provide proof-of-concept for the value of CSF metabolites as (1) disease-associated signatures suggesting pathophysiological mechanisms, (2) degree and nature of neuroinflammation, and (3) biomarkers for diagnosis and risk stratification of VZV reactivation and, likely, neuroinfections due to other pathogens.Not applicable .The online version of this article (10.1186/s12974-017-1041-0) contains supplementary material, which is available to authorized users. Varicella zoster virus  is the causative agent of chicken pox. After this primary infection or immunization with attenuated VZV, latent viral reservoirs persist in neuronal ganglia along the neuraxis, and upon reactivation, the virus spreads along the nervous system. The lifetime risk of reactivation is 20–30% but increases with age or immunosuppression, presumably due to waning T cell immunity , 2. EvenIf VZV reactivation is suspected based on clinical examination, current diagnostics include serology, PCR, and culture. The value of VZV culture is limited due to the low sensitivity of around 45% , but CSFAs demonstrated in recent studies on neurodegenerative disorders , metabolomic and/or lipidomic profiling of CSF has considerable potential for identifying biomarker candidates and suggesting disease-associated metabolic networks, also in the context of neuroinflammation –19. The The lack of a small animal model for VZV infection and reactivation has been impeding studies of its pathogenesis, meaning that such studies at the organismal level depend on analyses of biosamples from well-characterized patients or samples obtained at autopsy. The use of the latter is limited due to well-known artifacts from post-mortem changes. Considering (1) the successful applications of CSF metabolite profiling for biomarker discovery in the aforementioned studies and (2) the need for diagnostic and prognostic biomarkers for VZV reactivation and a better understanding of its pathogenesis including the nature of the associated neuroinflammation, we aimed to identify novel small-molecule CSF signatures and biomarkers for this multifaceted disorder.n = 14; defined by the presence of the typical cervical, thoracic, or lumbar rash); (2) facial nerve zoster ; and (3) zoster meningitis and/or encephalitis . Three control groups were included: (1) enteroviral meningitis  as infectious disease control; (2) idiopathic facial paresis ; and (3) normal pressure hydrocephalus  and was conducted according to the Helsinki Declaration. Informed consent was waived due to the use of anonymized patient data. Inclusion criteria for evidence of VZV reactivation were either detection of VZV in CSF by PCR, intrathecal synthesis of VZV IgG, or the typical skin rash. Patients were categorized according to three different VZV reactivation patterns: (1) segmental zoster , IgG-inim triad ), and PCim triad . A cliniv/v/v), and extracted using 5 mM ammonium acetate in methanol. Remaining extracts were diluted and measured according to the manufacturer’s instructions. The following lipid nomenclature is used: sphingomyelin (SM) and hydroxysphingomyelin (SM(OH)): Cx:y: x = total number of carbon atoms and y = total number of double bonds in the amide bond. Phosphatidylcholines (PC): aa: both side chains are fatty acids linked to glycerol backbone by ester bonds, ae: one of the side chains is a fatty alcohol linked to glycerol backbone by an ether bond, and Cx:y: x = total number of carbon atoms and y = total number of double bonds in both fatty acid chains. Peak integration and calculation of metabolite concentrations were performed with the Analyst®  and MetIDQ™ software  [We applied the AbsoluteIDQ®-p180 kit  for CSF analysis  on a triAustria) . MetabolP value of < 0.05 unless stated otherwise. Corrections for multiple-hypothesis testing were not performed in those cases where data were shown to visualize the distribution of differential expression , according to the SPSS Missing Values Manual , using aion Fig.  or correion Fig. c–f. Nonmn = 80 patients) and a validation subset (n = 1 patient) and computes the best classifier on the training subset. The selected classifier is then tested on the validation subset. In an iterative approach, every sample is used as the validation subset and all classifiers are ranked according to the frequency of selection, with the most frequently selected classifiers constituting the best validated biomarkers. In detail, values <LOD were imputed with the k-nearest neighbors algorithm and the Heterogeneous Euclidean Overlap Metric (HEOM) [The leave-one-out (jackknife) method was used for internal cross-validation, which was necessary because an external validation based on additional samples was not feasible due to the low incidences of some of the etiologies studied. Leave-one-out cross-validation is recommended as a validation strategy if no independent test dataset is available and sample sizes are limited , 35. Bric (HEOM) . FeatureTable n = 54) and “75–99%” (n = 34) were merged and used for all subsequent analyses. The analytes detected in 75–99% and <75% of all samples did not comprise metabolites that were completely absent in any particular patient groups but present in the others, but were rather missing due to low abundance, or lack of detection due to technical reasons, across all groups. Nevertheless, the concentrations measured and the analytical coverage for the analyzed metabolites agreed with the information provided by the manufacturer [All metabolites detected above LOD were initially considered and were grouped into four categories according to completeness of detection Fig. . The grofacturer  and the facturer . The subAs a first proof of concept, we addressed the question whether the metabolite patterns would reflect the expected clinical relationships among the disease entities. Using MDS, the non-infected controls could be clearly separated from the virally infected groups along the first dimension, whereas the three forms of VZV reactivation could be separated along the second dimension in order of increasing potential severity and CNS involvement Fig. . InteresP < 0.05 in both cases), indicating that there was a tendency toward higher CSF metabolite concentrations in VZV infection. Regarding the individual VZV reactivation forms, there was a steady increase in the fraction of significantly (P < 0.05) elevated (with respect to controls) analytes from Z. segmental  to Z. facial  and to Z. meningoencephalitis . In contrast, the fraction of downregulated analytes even decreased . Of note, the downregulated analytes mostly comprised amino acids, with concentrations of serine being decreased in all three VZV reactivation forms. The discriminatory potential of the analytes was then assessed by the magnitude of the area under the ROC curve (AUC), the asymptotic P value of the ROC curve, and the ROC curve lower bound confidence interval , and consistent with these differences between the two viral etiologies, concentrations of 24 markers differed (P < 0.05) between Z. meningoencephalitis and ent. men  is shown in Additional file A combination of differential concentration analysis and binary ROC curve analysis was then used 1) to assess the degree of metabolite reprogramming in VZV reactivation and 2) to obtain a first overview of differences in concentration of the 88 metabolites within the clinically most relevant paired comparisons. We first tested whether CSF metabolite concentrations predominantly decreased or increased in the different disease states. Comparing the three forms of VZV reactivation combined against controls, concentrations of 27 metabolites 31%) were increased and only 3 metabolites (3.4%) were decreased  in all other disease etiologies, even in ent. men, which was included as disease control . When the cross-validation was applied to identify markers for the two milder forms of VZV reactivation in clinically relevant comparisons, there was more heterogeneity among the best validated markers for the various paired comparisons. Nonetheless, markers of moderate to excellent discriminatory ability could be validated for all comparisons (see below).Jackknife internal cross-validation see the “” sectionTable P = 0.18, χ2 = 7.181). Overall, there were weak-to-moderate correlations between analyte concentrations and leukocyte count, as indicated by comparatively low Spearman coefficients  and the sum of hexoses were also among the analytes with decreased concentrations in VZV reactivation identified in Fig. CSF leukocyte count (“cell count”) is a currently used indicator for the degree of overall CNS inflammation, independent of underlying disease etiology, but is also used to aid in making important clinical decisions such as institution or termination of anti-infective medications. In order to test whether the four metabolites specific for Z. meningoencephalitis merely reflect the degree of CNS inflammation, as does CSF leukocyte count, or whether they are indicative of distinct pathophysiological mechanisms, we performed a Spearman’s correlation analysis between metabolite concentrations and CSF leukocyte count. Correlation with leukocyte count was not preferentially associated with any of the analyte classes  to diagnose Z. meningoencephalitis among all VZV-positive samples Table . Both fiUsing targeted mass spectrometric analysis, we identified metabolic signatures and small-molecule CSF biomarker candidates for three forms of VZV reactivation of differing extent of neuroinflammation and CNS involvement and evaluated these for the differentiation from non-inflamed controls and from ent. men as a viral CNS infection of different etiology.Elevated CSF concentrations of four analytes  were most closely associated with Z. meningoencephalitis, suggesting that they may represent potential biomarkers for this diagnosis and also reflect aspects of its pathogenesis. Their concentrations did not correlate directly with CSF leukocyte count, indicating that they do not merely reflect CNS inflammation but also pathological processes in CNS parenchymal cells including neurons. These markers belong to three different molecular classes, suggesting that VZV reactivation with CNS involvement does not preferentially affect a particular metabolic pathway. The association with Z. meningoencephalitis was strongest for SM C16:1. Sphingomyelins are integral components of cell membranes, including neuronal membranes, and their levels are regulated partially through a dynamic exchange with ceramides. Dysregulated SM levels are seen in processes associated with cell stress and death  , 39 and P < 0.05, lower CI > 0.5) between ent. men and controls and, in addition, it had low discriminatory potential . Preliminary analyses of samples from patients with HSV CNS infection (n = 9) have revealed a different set of metabolite markers specific for this diagnosis . Taken together, these results do suggest the possibility that the above identified four metabolites may be specific for VZV CNS infection. Pathogen-specific CSF metabolite signatures may result from differences in host-pathogen interactions such as viral tropism, type of infection (persistent vs. lytic), host immune responses, or blood-CSF-barrier function. On the other hand, differences that non-specifically reflect severity of infection and extent of tissue damage may blur, for instance, in cases of mild infections with normally highly pathogenic viruses  compared to severe cases of infection with a virus of normally relatively low virulence . Clearly, additional cohorts also including different viral etiologies now need to be studied in order to address the above question more conclusively, and we are currently collecting samples for an external validation cohort.From the point of view of biomarker research, it would be desirable to identify markers that are specific for selected pathogens or pathogen families. In the presented study, we used ent. men as a viral CNS disease control. Of note, of the four metabolites most closely associated with Z. meningoencephalitis, lysoPC a C26:1 was a highly accurate marker to differentiate Z. meningoencephalitis also from ent. men, and only glycine was among the 22 metabolites with significantly different concentrations  are useful to differentiate bacterial from viral meningitis , but lacEven though we used a relatively broad screen encompassing several analyte classes, the 88 analytes that passed the quality assessment represent only a subpopulation of potentially important molecules in CSF. Therefore, broader and more sensitive screens might unveil additional important metabolite changes. The annotated sphingo- and glycerophospholipids measured with the targeted MS/MS approach inherent to the Biocrates AbsoluteIDQ®-p180 kit can also stem from isomers and isobars , which mThe results suggest that assessing CSF metabolite patterns may be a powerful tool for identifying small-molecule biomarkers for diagnosis and risk stratification of CNS infections, extent and nature of neuroinflammation, and to improve our understanding of the pathogenesis of VZV reactivation at the organismal level. Considering the observed differences between Z. meningoencephalitis and ent. men, it is tempting to speculate that patterns characteristic of certain pathogens or pathogen families can be identified which could aid in establishing a diagnosis in patients in whom a pathogen cannot be detected.


Ex-vivo quantification of gadolinium showed significantly higher uptake of EP-3533 compared to EP-3612 in tumours, but not in surrounding tissue . Uptake of EP-3533 visualized in T1-weighted MRI correlated well with spatial distribution of collagen determined by second harmonic generation imaging. Differences in the tumour pharmacokinetic profiles of EP-3533 and EP-3612 were utilized to distinguish specific binding to tumour collagen from non-specific uptake. A model-free pharmacokinetic measurement based on area under the curve was identified as a robust imaging biomarker of fibrosis. Collagen-targeted molecular MRI with EP-3533 represents a new tool for non-invasive visualization and quantification of fibrosis in tumour tissue.Fibrosis with excessive amounts of type I collagen is a hallmark of many solid tumours, and fibrosis is a promising target in cancer therapy, but tools for its non-invasive quantification are missing. Here we used magnetic resonance imaging with a gadolinium-based probe targeted to type I collagen (EP-3533) to image and quantify fibrosis in pancreatic ductal adenocarcinoma. An orthotopic syngeneic mouse model resulted in tumours with 2.3-fold higher collagen level compared to healthy pancreas. Animals were scanned at 4.7 T before, during and up to 60 min after i.v. injection of EP-3533, or of its non-binding isomer EP-3612.  Desmoplasia, i.e. fibrosis, is typical for many solid tumours including pancreatic ductal adenocarcinoma (PDAC)3. The desmoplastic reaction is characterized by excessive deposition and abnormal structuring of ECM components. Type I collagen, the major protein constituent of ECM, is also dysregulated in tumour tissue and undergoes extensive remodelling6. It has been recognized that abnormal collagen distribution contributes to cancer progression and resistance to therapy in several ways. Excessive collagen increases mechanical stress that promotes tumour growth and is associated with poor prognosis8. Linearized collagen fibres act as movement tracks for cancer cells, facilitating their migration and increasing metastatic potential10. Dense collagen networks also impede diffusion and delivery of drugs, especially those of the macromolecular and nano-sized character13. Thus, suppression of fibrosis and collagen emerged as a promising strategy in cancer therapy, particularly in pancreatic cancer19. However, the current tools to noninvasively characterize the tumour ECM are limited. There is an unmet need for techniques that would allow visualization and quantification of collagen in tumour tissue in a non-invasive and repeatable way6.In mammalian tissue, the extracellular matrix (ECM) provides integrity and mechanical support for cells, as well as performs important regulatory functions. Homeostasis of ECM is important for normal tissue function, while its dysregulation is tightly associated with diseases such as fibrosis and cancerin-vivo imaging20. However, it is somewhat invasive in that the tumour must be exposed to light, and limited in the ability to evaluate macroscopic tissue volumes. Magnetic resonance imaging (MRI) is an attractive modality to complement biopsy and SHG imaging, as it offers combination of three-dimensional tumour coverage and deep tissue penetration.Fibrosis has been traditionally assessed by biopsy and histopathological analysis. Although biopsy remains the gold standard, it is invasive and provides only a static snapshot of a small tissue sample. Second harmonic generation (SHG) imaging has been increasingly used for visualization of collagen in recent years. This two-photon microscopic technique is specific to fibrous collagens (type I and III), requires no exogenous stains, and can be adapted for 24, pulmonary fibrosis25 and cardiac fibrosis26. In this work we sought to determine whether EP-3533 enhanced MRI could selectively detect and quantify tumour fibrosis in an orthotopic model of PDAC that closely mimics the stroma observed in human disease27. A particular challenge of molecular MRI of cancer is the non-specific uptake of most molecular probes into tumours. A permeable vasculature coupled with poor lymphatic drainage results in non-specific uptake of the probe28. Here we validated the specificity of EP-3533 for tumour fibrosis by direct comparison with a non-binding isomer. We then showed that a model-free pharmacokinetic analysis provides a robust imaging biomarker of fibrosis that can be used to visualize and quantify fibrosis in pancreatic cancer non-invasively.EP-3533 is a peptide-based gadolinium-containing magnetic resonance (MR) probe targeted to type I collagen that has previously been shown to detect and stage fibrosis in animal models of liver fibrosisp = 0.70). At later times (13–16 days from cell implantation), the disease advanced into stiff, palpable tumours (~10 mm in diameter). These mature tumours manifested with prominent fibrosis as observed by strong positive staining for collagen  .The cancer cell line used in this animal model resulted in an aggressive tumour growth in the pancreas. To establish the best window for imaging we investigated the disease at two different time points. Early tumours (7–9 days from cell implantation) were relatively small (3–5 mm in diameter) and in some cases the boundaries could not be confidently distinguished from the surrounding pancreas (during the dissection and/or in MR images). Histological analysis showed only modest increases in positive staining for collagen in early tumours compared to healthy pancreas Fig. . This wagen Fig. . The disgen Fig. . Distinc−5) Fig. . CollageSeveral imaging sequences were used to confidently define regions of interest for image analysis Fig. . Good coin-vivo EP-3533 enhanced MR images compared to the spatial distribution of collagen within the tumour volume, we performed some ex-vivo imaging studies. An ex-vivo T1-weighted MR image of a tumour that was previously imaged in-vivo after EP-3533 injection was compared to a collagen image obtained with SHG imaging on a 30 μm thick tumour section. Autofluorescence images that carried no information about collagen were acquired simultaneously. In agreement with histology, the SHG images revealed highly heterogeneous distribution of collagen throughout the tumour mass  of a collagen-targeted MR probe (EP-3533) or a non-binding control probe (EP-3612). Some animals were scanned consecutively with both probes in randomized order , with a 24 h delay between imaging sessions to insure elimination of the first injected probe . This was followed by a gradual decrease of the signal enhancement with the non-binding control probe EP-3612. Differently, the signal enhancement continued increasing up to 35 min after injection of the collagen-targeted probe EP-3533, and then showed a modest decrease. Overall, EP-3533 provided higher signal enhancement in tumour compared to EP-3612 for all time points except 5 min post-injection. At 55 min post-injection, tumour signal enhancement was 29% higher with targeted probe compared to control. Quantitative ex-vivo analysis of gadolinium further confirmed higher uptake of EP-3533 than EP-3612 in tumour tissue , while similar Gd levels were observed in blood and muscle for both probes  but this was followed by a rapid washout from healthy pancreas  for each voxel. A mean AUC5–55 was then calculated for the whole tumour volume. Figure 5–55 values could be either positive or negative, depending on the profile of the curve. This is notably different from the usual meaning of the area under the curve (in this work AUC0–55), where the reference is taken from the baseline image and the values are thus always positive , while for EP-3612 they were almost exclusively negative (−1.12 ± 0.63 AU.min). The difference between AUC5–55 of the two probes was statistically highly significant (p = 0.0007). The AUC5–55 values obtained with EP-3533 in healthy pancreas were exclusively negative (−4.97 ± 1.71 AU.min) and statistically significantly different from tumours .Figures ive Fig. . Figure 5–55. The top and middle rows of images depict a best-matched cross-section of the same tumour specimen imaged with EP-3612 (top) and EP-3533 (middle). The bottom row shows images of the pancreas of a healthy animal imaged with EP-3533. At 5 min post-injection in tumour, both EP-3612 and EP-3533 provided heterogeneous increase in the MR signal that was comparable in its intensity and localization within the tumour mass, as is apparent from the T1-weighted images (E) and signal enhancement maps (F). Compared to 5 min post-injection, the MR signal and signal enhancement in tumour at 55 min post-injection decreased with the non-binding EP-3612 probe, but further increased with collagen-targeted EP-3533. The difference between the two probes in tumour tissue is even more pronounced in the AUC5–55 maps that were windowed to show positive AUC5–55 values (G). With EP-3612 most of the tumour volume remains dark (negative AUC5–55). On the contrary, EP-3533 provides clear distinct regions that display strongly positive AUC5–55 values. The behaviour of EP-3533 in the pancreas of healthy mice  was consistent with low specific binding of the probe, showing only modest signal enhancement at 5 min post-injection (F) that decreased at 55 min. In consequence, the AUC5–55 map of pancreas with EP-3533 shows mostly negative values and is dark (G). Additional T1-weighted MR images of tumours imaged with EP-3533 are provided in Figure Figure 5–55 and total tissue collagen . The correlation was strong despite the fact that the tissue specimens greatly differed in perfusion characteristics and hence in the amount of probe reaching the tissue. Conversely, the full area under the curve, AUC0–55, displayed a poor (r = 0.16) correlation with tissue collagen  also correlated positively with tissue hydroxyproline, but this correlation was weaker (r = 0.46) than when using AUC5–55. We also correlated other MR measurements with tissue hydroxyproline. Figure Figure gen Fig. . Apparen31 and is also host to proliferating myofibroblasts (pancreatic stellate cells), immune, and vascular cells32. The stiff, fibrotic tumour microenvironment may hold the key to understanding the nature of PDAC chemoresistance through its role in regulating growth, immune response, dissemination, and limiting blood flow to the tissue37. Currently, no non-invasive tools for specifically monitoring the desmoplastic response exist, which hinders efficient translation of preclinical observations to approved therapies that are desperately needed by PDAC patients.PDAC is notable for the intense fibrotic reaction associated with the tumour, known as the “desmoplastic reaction”, which influences tumour survival and progression in a complex manner. Desmoplastic stroma is characterized by up to 3-fold increase in collagen  compared with the normal pancreas38. We took a simpler, model-free approach based on the comparison of EP-3533 with its size- and pharmacokinetic-matched negative control probe EP-3612. We observed that the initial pattern and extent of tumour enhancement was very similar immediately after injection. This is expected since the signal at these early time points will be dominated by distribution. At later time points, as unbound probe washes out of the tumour the observed signal will have a stronger contribution from the collagen-bound probe. Quantifying the AUC of the tumour signal from 5 min post injection  to 55 min post injection was a useful biomarker of tumour fibrosis. The AUC5–55 values for the non-binding probe were almost exclusively negative indicating washout from the tumour, while the AUC5–55 values for EP-3533 were consistently positive. Considering tumour and healthy pancreas tissue, this simple metric correlated very well with hydroxyproline content, a biochemical measure of fibrosis.Here we showed in a mouse model of PDAC that the collagen-targeted MR probe EP-3533 can specifically detect and quantify Type I collagen, a protein that tightly correlates with the extent of desmoplastic reaction and with poor prognosis. Molecular imaging in cancer is confounded by the leaky vasculature and poor lymphatic drainage within tumours which leads to a non-specific enhancement with almost all imaging probes. Separating the specific, collagen-bound signal from non-specific distribution is challenging. One approach would be to employ a kinetic model to describe the time-dependent tumour signal after probe injection5–55 values with EP-3533 may be contributed by two factors. First, EP-3533 may accumulate in collagen-rich tissue as long as its concentration in blood is in favour of binding over detachment. Consistent with this, the blood half-life of EP-3533 in mice was previously determined as 19 min22. Additionally, we speculate that signal at early time points may be subject to partial volume loss because the probe is restricted in location. As the probe diffuses into the tumour, this partial volume effect is diminished and signal increases.The source of the positive AUC39. A benefit of EP-3533 is its small size, which should result in faster and deeper penetration into tumour tissue compared to high molecular weight probes. There have been other indirect MR approach to assess tumour fibrosis such as through activity of matrix metalloproteinases40, magnetization transfer MRI41 or diffusion-weighted MRI42. Differently, EP-3533 specifically binds to type I collagen, which is directly related to fibrosis. We note that molecular MR with EP-3533 could be combined with magnetization transfer or diffusion weighted MRI, and it is possible that a combined readout could be an even more sensitive measure of tumour fibrosis.Other collagen-targeted probes have been described such as those based on collagen-adhesion protein14, but these preclinical results did not translate to human trials43. However, drugs that target the desmoplastic stroma still have high potential to benefit PDAC patients. One promising mechanism to enhance therapeutic drug delivery involves use of recombinant PEGylated hyaluronidase (PEGPH20) to degrade the hyaluronic acid (HA) within the desmoplastic stroma, thus decompressing blood vessels and improving flow45. Recombinant hyaluronidase is currently undergoing clinical trials  and preliminary data in patients with previously untreated stage IV pancreatic cancer suggested promising efficacy, especially in patients with a high HA content47. Stromal collagen burden was also shown to be an important variable in eliciting increased vascular perfusion and drug delivery in animals treated with the angiotensin II receptor inhibitor losartan48. Losartan increased perfusion through reduction in stromal collagen, reduction in hyaluronan, and decreased expression of pro-fibrotic signals such as transforming growth factor-β1 and Type I collagen expression. Importantly, these effects occurred only in collagen rich tumours, suggesting that non-invasive assessment of collagen burden could be beneficial in treatment planning during clinical trials .To date, results of “anti-stromal therapy” to enhance the delivery of chemotherapeutics to PDAC by breaking down the stromal barrier have been mixed. Targeting the sonic hedgehog pathway with a smoothened inhibitor reduced type I collagen levels, decreased the desmoplasia reaction, increased intratumoural vascular density, and improved the delivery of gemcitabine, leading to prolonged survival in miceNoninvasive monitoring of changes in tumour stroma will be valuable in assessing treatment efficacy. For combination therapies that combine an anti-stromal therapy with a conventional chemotherapy, molecular imaging of tumour fibrosis could be used to guide the timing of the chemotherapy regimen, i.e. to begin chemotherapy after the anti-stromal therapy has shown a reduction in fibrosis.Here we focused on developing a technique to assess and quantify tumour fibrosis with molecular MRI. However other MR readouts could also be incorporated to further characterize the tumour and its response to treatment. For instance, the similarity in initial tumour enhancement with EP-3533 and its non-binding control EP-3612 suggests that immediate dynamic imaging with EP-3533 could be used to assess vascular permeability, while the later AUC measurement could be used to assess fibrosis. Such techniques may be used to monitor and guide therapeutic approaches, where suppression of fibrosis is part of the treatment strategy. Further studies in this direction are underway.−1s−1 (5.4 per Gd ion) at 4.7 T). EP-3612 has an identical structure to EP-3533 except that one of the cysteine moieties is changed from L-Cys in EP-3533 to D-Cys in EP-3612. This change in chirality results in >100-fold loss in collagen affinity for EP-3612, however its relaxivity remains equivalent to EP-3533. EP-3533 and EP-3612 were synthesized according to a published procedure49.EP-3533 comprises a ten amino acid cyclic peptide conjugated to three gadolinium moieties with affinity for type I collagen (Kd = 1.8 µM) and strong MR signal enhancement  on an inbred FVB/n strain background was used to generate tumours27. Cells (106) in 25 uL of PBS were mixed with 25 uL of Matrigel (BD Biosciences) were directly implanted in the pancreas of wild type FVB mice and the tumours were allowed to grow to up to 10 mm in diameter in 7–16 days. Sham control animals were injected with medium containing no cells.All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. A mouse pancreatic cancer cell line, from Ptf1-Cre; LSL-Krasi.v.) delivery of the imaging probe while the animal was positioned in the scanner. Imaging was performed at 4.7 T using a small-bore animal scanner with a custom-built volume coil. EP-3533 or EP-3612 were administered at a dose of 10 μmol/kg as 5 mM solutions in PBS. Thus 2 µL per gram of body weight was administered using a calibrated 100 µL Hamilton syringe, and accounting for the dead volume of the tubing.Animals were anesthetized with isoflurane (1–2%) and continuously maintained under this anaesthesia up to the end of experiment (return to cage or sacrifice). Animals were placed in a specially designed cradle with body temperature maintained at 37 °C. The tail vein was cannulated for intravenous  and coronal orientation , T2-weighted rapid acquisition with refocused echo (RARE) sequence  and T1-mapping sequence (RARE inversion recovery (IR), TR/TE = 3200 ms/9.6 ms, matrix = 96 × 96, 9 inversion times from 47 to 3000 ms, RARE factor 8, single 1 mm slice). Following the injection, the axial T1-weighted sequence and T1-map were repeated at approximate times 5, 20, 35, 45 and 55 min post-injection.ex-vivo MR imaging the animal was first imaged with EP-3533 as described above. After sacrificing at 80 min post-injection, the tumour was excised and fixed with a suture in a plastic tube filled with OCT compound. Ex-vivo images were acquired with a T1-weighted 3D gradient echo sequence . The tumour was then frozen and sectioned for two-photon microscopy so as to preserve the orientation.For Axial T1-weighted MR images were used for the analysis. Images in DICOM format were opened in OsiriX software, regions of interests (ROIs) were drawn to outline tumours and dorsal muscle in all slices that contained the tumour. ROI masks were exported from OsiriX using CreateROIMask plugin. The DICOMs and masks were further analysed in freeware Octave. See Supplementary data for the data processing scripts. The analysis was performed on the entire tumour volume and included the following steps: First, the image intensity was normalized to average dorsal muscle signal . Then, maps of signal enhancement for each post-injection time-point were calculated as: signal enhancement, SE = /. Areas under the curve (AUC) were calculated from the enhancement time courses for each voxel individually before being averaged for the entire tissue volume. AUC values are expressed as SE  × time (minutes) with units AU•min. No co-registration of images was applied.Tissue perfusion was assessed based on signal enhancement at 5 min post-injection. A distinction between well- and poorly-perfused voxels was based on the median signal enhancement at 5 min of all analysed voxels . Data for both EP-3533 and EP-3612 probes were considered together. Taking the median as a threshold, a voxel was assigned as well perfused if the signal enhancement at 5 min surpassed this threshold. The well-perfused tissue volume fraction (PerfVF) was calculated for each tissue specimen as: (number of well-perfused voxels in ROI)/.50. Gadolinium was quantified in nitric acid digests by inductively coupled plasma-mass spectrometry (ICP-MS) using dysprosium as an internal standard. Hydroxyproline and gadolinium are expressed as μg or nmol per g of wet tissue weight, respectively.Formalin-fixed samples were embedded in paraffin, cut into 5 µm-thick sections and stained with Masson’s trichrome, Sirius red or hematoxylin and eosin (H&E) stain according to standard procedures. Quantitative analysis of hydroxyproline was used as a measure of total amount of collagen in tissue. Hydroxyproline was quantified by HPLC analysis using a reported methodex-vivo tumour tissue slices was visualized by second harmonic generation (SHG) imaging using a commercial multiphoton imaging system . Unstained 30 µm-thick slices on glass slides were imaged in an epifluorescence configuration using a 4x microscope objective . Excitation light (λ = 880 nm) was delivered using a tuneable, pulsed Titanium:Sapphire laser  and scanned across the field of view using galvanometer scanning mirrors. Using dichroic beamsplitters and optical bandpass filters, emitted SHG light from excited collagen and autofluorescence signal were collected through the objective, spectrally separated, and directed to separate photomultiplier tube detectors .Collagen content within 51. Coregistration with MRI was performed in Octave by rotation and resizing of micrographs using bicubic interpolation and re-slicing of 3D MR data so as to achieve the best visual agreement between the tumour boundaries.For a single image, the field of view 2.35 × 2.35 mm was captured with 9.18 µm per pixel resolution. The whole tissue slice was imaged in sections by moving the sample in the xy-plane with an automated translation stage. The entire thickness of the tissue slice was imaged by collecting a depth-resolved z-stack of images at each location . A complete image was obtained as a maximum projection image first for each z-stack, followed by stitching of multiple fields of view using ImageJ software with a stitching pluginp < 0.05 as significant.All data are shown as mean ± standard error of the mean (SEM). Error bars represent SEM. Box plots show the 0 and 100 percentile (whiskers), 25 and 75 percentile (box) and median value . Statistical significance was tested by unpaired two-tailed Student’s t-test (Excel), with The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supporting Information


A novel method for preparing eco-biodegradable lubricant based on hydroxypropyl methylcellulose (HPMC) via hydration process is demonstrated. The smooth and homogeneous HPMC coating has a uniform thickness (~35 μm). It has been demonstrated that the preparation parameters play a critical role in controlling the lubricating behavior of the coating; in addition, excess HPMC and water concentration suppress the tribology properties. Nevertheless, a remarkable friction-reduction and anti-wear performance has been obtained. Impressively, the preparation parameter of 5% HPMC + 30 mL water significantly improves lubricant performance and durability. A simple approach for the water-degradability evaluation of HPMC is proposed. Microelectromechanical system/Nanoelectromechanical system (MEMS/NEMS) is a technology combining electronic and mechanical devices by using a mature semiconductor processing technique in the scale of micron/nanometer. NEMS/MEMS products are successfully entering the market and created a thousand billion US dollars in revenue in the year of 2014. It has been treated as a power source of the next industrial revolution. However, severe wear occurring in silicon materials has been a bottleneck for wide application. Therefore, a very effective solution to improve MEMS durability is to find a novel material with potential tribology performance [In the past two decades, environment and energy issues at a global scale have increased seriously year by year. However, very recently, there has been increased activity in the development of green technology, with quite a few reports on bio/eco technology. Biofriendly synthesis techniques have been reported ,4,5,6. BManufacturing enterprises ask for lubricants without phosphorus or heavy metals as well as non-toxic substances for the reasons of environmental and toxicological considerations. For this reason, tribology techniques expand into the direction of saving energy, reducing materials usage and waste, decreasing noise and shock, increasing recycling, and developing environmentally friendly lubrication ,21,22,23Numerous important applications of cellulose derivatives, including pharmaceutical, food, adhesive and textile fields, have been widely studied for many years. Cellulose automatically qualify as an ideal polymer in the formation of film coating ,43. HighThe surface properties of HPMC play an important role when the coatings are used in domains such as protection layers. Nevertheless, very few studies concern the protection and anti-wear property of HPMC film. In this study, we focus on understanding the effects of hydration on the coating characteristics of the formulation films. Moreover, we report on the application of a biofriendly HPMC coating as an anti-wear lubricant. A novel method is demonstrated for HPMC coating manipulation to enhance the tribological performance. Furthermore, a simple approach to identify the water-degradability of HPMC is proposed and discussed.Raw material HPMC particles , ethanolThe sample preparation parameters are shown in −1 (C–O stretch vibration) and smaller peaks are found between 1200 and 1500 cm−1. These smaller peaks overlap with the carboxylic acid groups. The bands around 1600 cm−1 correspond to the bending mode of water. The other major bonds are found at 2900 cm−1 and mainly due to absorptions of the C–H stretch bond. O–H stretch bands were found at 3420 cm−1. These FTIR peaks are signatures of pure HPMC as reported previously [The Fourier transformed infrared (FTIR) spectra in eviously . Comparevs. cycles can be used to determine the lubricating behavior during the tribological test.HPMC is soluble in cold water and some organic solvents. In this study, HPMC solution was prepared by dissolving HPMC powder into ethanol and water. First stage, we check the lubrication properties by using 2–7 g HPMC. Secondly stage, we want to optimize the lubrication property by adding water to the solution to enhance the tribology performance. Three parameters were set by different water concentrations, varying from 30 to 60 mL. Tribological tests were carried out for all the HPMC preparation parameters, using a ball-on-disk tribometer . The varvs. cycles is reported. The variation in friction coefficient vs. long-term cycle is shown in vs. cycles for five preparation parameters—bare silicon, 5% HPMC, 5% HPMC + 30 mL water, 5% HPMC + 50 mL water, and 5% HPMC + 60 mL water, respectively. For bare silicon, the friction coefficient jumps very fast due to absence of lubricating coating to protect the surface and leads to a high friction coefficient. For 5% HPMC coating, thre friction coefficient increased to 0.4 gradually as the test reachers 500 cycles. With regards to 5% HPMC + 30 mL water, the friction coefficient is low from the start of the experiment and it remains stable throughout the test, even when the test completes 6000 cycles. However, higher water concentrations do not further reduce the friction coefficient more than the 30 mL water concentration sample. We suppose that the concentration of water is too high to soften the coating and lose the protection property. HPMC coating is easily worn out due to the softening behavior of adding too high a water concentration. Consequently, a high friction coefficient is observed when there is a breakdown or absence of protecting coating. The comparison of duration cycles vs. HPMC preparation parameters are shown in To further understanding the tribological performance of HPMC coating on long-term lubricating behavior, duration and stability tests are carried out. The evolution of the friction coefficient of 5% HPMC with different water concentration The SEM images in −1 shows no difference in the range of soaking time from 4 min to 20 min. When the soaking time reaches 24 min, the characteristic peak vanishes. It is expected that the HPMC coating will totally dissolve in water exposing the silicon surface beneath. As a result, there is no FTIR signal for the silicon surface. This finding is reasonable, since the penetration depth of FTIR is only around a few micrometers. To further understanding the mechanism, microtribology tests were carried out using the samples that had a soaking time longer than 24 min. It reveals a similar behavior to bare silicon  powder with a hydroxypropoxy content of 9% and viscosity of 6 mPa·s was obtained from Shin-Etsu Chemical Co., Ltd., Tokyo, Japan. Ethanol (95%) was used to improve hydration of HPMC, and help to decrease the bubbles in the coating-forming process and eventually in drying the coating formation. HPMC coating was prepared by dissolving 5 g of HPMC in a solution of ethanol (100 mL) and distilled water. The solution was stirred for 30 min at 55 °C by using a heating and stirring hot plate. After a homogenous solution was achieved by the stirring and mixing process, films were made by a pipette injection on the silicon substrate and left to dry at room condition: 25 ± 2 °C and 60% ± 5% related humidity (RH) for 24 h. Micropipette  was used for precise volume-control during the injection process. The thickness of the films was measured using cross-sectional SEM images. The thickness was measured in 10 randomly selected samples, as shown in The surface morphology was characterized using high-resolution thermal field emission scanning electron microscopy , including top view and cross-sectional image. Wear phenomenon was recorded using microfigure measuring instrument .−1.Fourier transform infrared spectrometer  was used to study the structural properties of HPMC coatings with a software-assisted interface (OMNIC). The spectra were obtained by reflection mode in scanning speed of 50 scans/s with the scanning range of 500–4000 cmi.e., friction force) was measured by a load cell connected to the rotating disk. The friction coefficient was recorded for further tribological property analysis. The friction experiments were repeated to ensure reproducibility of the results. This study designed to investigate the effect of the HPMC structure and preparation parameters on friction and wear, was done and by using a ball-on-disk tribometer . Test seThe water-degradability of HPMC coating was measured by monitoring the characteristic peak signal of HPMC in FITR. The HPMC coated (5% HPMC + 30 mL water) silicon substrates were prepared and immersed into DI-water. The test was carried out at environment temperature of 25 °C for 28 min.The tribological performance of HPMC coating on silicon has been demonstrated. Lubricating behavior is highly related to the weight percent of HPMC and water concentration. It is further shown that by adding suitable HPMC and water concentration, the friction coefficient can be significantly lowered at greater stability. The HPMC coating is a promising lubricant material with great friction-reduction and anti-wear characteristics, especially in the green tribology and MEMS applications. FTIR technology was performed to analyze the structure and water-degradability of HPMC. In this study, we report on the first application of biofriendly HPMC coating lubricating behavior. A novel approach for HPMC coating manipulation to enhance the tribological performance is demonstrated.