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Populus euphratica and Populus pruinosa are important tree species in northwest China. In this study, the codominant EST-SSR markers related to drought resistance genes was used to reveal the genetic diversity of P. euphratica and P. pruinosa populations. The results showed that those populations exhibited rich genetic diversity levels. Compared with other P. euphratica and P. pruinosa populations respectively, the genetic diversity of populations in southern Xinjiang was all the highest. The average fixed index of different populations indicated that the genetic relationship among P. euphratica populations was relatively far, while the relationship among the P. pruinosa populations was closer. The molecular variance analysis showed that the variation within populations was bigger than that among populations in the P. euphratica and P. pruinosa populations respectively. The cluster analysis showed that P. euphratica and P. pruinosa populations could be mainly classified into two clades represented by southern and northern Xinjiang respectively. The population structure and principal co-ordinates analysis indicated that the 47 natural P. euphratica populations have been divided into 4 clades, represented by the two populations in southern or southern Xinjiang respectively. The 17 natural P. pruinosa populations had been divided into 4 clades, consisted by the three County in southern Xinjiang, and Nilka County in northern Xinjiang. At the same time, The EST-SSR fingerprintings of 47 natural Populus euphratica and 17 natural Populus pruinosa have been constructed successfully. This study laid the foundation for the later priority protection of the genetic diversity populations of excellent drought resistance.

Extractive style query-oriented multi-document summarization generates the summary by extracting a proper set of sentences from multiple documents based on the pre-given query. This paper proposes a novel multi-document summarization framework via deep learning model. This uniform framework consists of three parts: concepts extraction, summary generation, and reconstruction validation, which work together to achieve the largest coverage of the documents content. A new query-oriented extraction technique is proposed to concentrate distributed information to hidden units layer by layer. Then, the whole deep architecture is fine-tuned by minimizing the information loss of reconstruction validation. According to the concentrated information, dynamic programming is used to seek most informative set of sentences as the summary. Experiments on three benchmark datasets demonstrate the effectiveness of the proposed framework and algorithms.

Concurrent mutation of a RAS oncogene and the tumor suppressor p53 is common in tumorigenesis, and inflammation can promote RAS-driven tumorigenesis without the need to mutate p53. Here, we show, using a well-established mutant RAS and an inflammation-driven mouse skin tumor model, that loss of the p53 inhibitor iASPP facilitates tumorigenesis. Specifically, iASPP regulates expression of a subset of p63 and AP1 targets, including genes involved in skin differentiation and inflammation, suggesting that loss of iASPP in keratinocytes supports a tumor-promoting inflammatory microenvironment. Mechanistically, JNK-mediated phosphorylation regulates iASPP function and inhibits iASPP binding with AP1 components, such as JUND, via PXXP/SH3 domain-mediated interaction. Our results uncover a JNK-iASPP-AP1 regulatory axis that is crucial for tissue homeostasis. We show that iASPP is a tumor suppressor and an AP1 coregulator.

We study resonant two-photon ionization of helium atoms via the $1s3p$, $1s4p$ and $1s5p^1$P$_1$ states using the 15$^\mathrm{th}$ harmonic of a titanium-sapphire laser for the excitation and a weak fraction of the laser field for the ionization. The phase of the photoelectron wavepackets is measured by an attosecond interferometric technique, using the 17$^\mathrm{th}$ harmonic. We perform experiments with angular resolution using a velocity map imaging spectrometer and with high energy resolution using a magnetic bottle electron spectrometer. Our results are compared to calculations using the two-photon random phase approximation with exchange to account for electron correlation effects. We give an interpretation for the multiple $\pi$-rad phase jumps observed, both at and away from resonance, as well as their dependence on the emission angle.

Aim: This work aims to understand potential inhibitory structural requirements and identify lead compounds for non-small cell lung cancer through 3D-QSAR pharmacophore-based virtual screening, molecular docking, CoMSIA and CoMFA QSAR modelling. Materials and Methods: QSAR pharmacophore models were developed by HypoGen Module and validated by test data set, Fischer’s randomization and Guner-Henry equation. The well-validated pharmacophore model was employed to perform virtual screening to identify potent hits from ZINC database. The retrieved hits were subsequently subjected to filtering using ADMET descriptors and Lipinski’s Rule of Five. CoMSIA and CoMFA were then utilized to produce QSAR models on phenylpyrimidine derivatives also. Results: Validations on 3D-QSAR pharmacophore model indicate that the enrichment factor is 6.34, GH is 0.517 and a correlation coefficient is 0.83, implying its highly predictive ability. Top three hits: ZINC29356266, ZINC06589615, and ZINC03375633 were identified as promising potent inhibitory candidates with IC50 value of about 0.54 µM and fitness value of about 59.4. Interestingly, the top three hits indicate dual inhibitory activity targeting EGFR and PD-L1 from structure-based docking. Two developed QSAR models from CoMSIA and CoMFA modelling indicate a potential predictive ability (q2=0.67, and 0.71 respectively). The designed compound C indicates a more potential (dual) inhibitory activity (pIC50=7.39) targeting EGFR (fitness=59.78) and CTLA-4 (fitness =47.90). Conclusion: Validations indicate that the developed 3D-QSAR pharmacophore model is highly predictive. Top three hits were identified as promising potent inhibitory candidates and indicated dual inhibitory activity targeting EGFR and PD-L1. The designed compound C indicates a more potential (dual) inhibitory activity targeting EGFR and CTLA‐4. These important 3D-QSAR and molecular docking bioinformatics results achieved from this work should be valuable in designing more promising potent inhibitory candidates and developing novel lead compounds against advanced NSCLC in future.

Arylsulfatase and β-glucuronidase are two important enzymes in human, which play important role on dynamic equilibrium of steroidal estrogens. This work probably for the first time reported that hydrogen peroxide (H2O2), hypochlorite and peracetic acid (PAA) could effectively inhibit the activities of arylsulfatase and/or β-glucuronidase. The 50 percent of inhibitions (IC50) of H2O2, and PAA on arylsulfatase were found to be 142.90±9.00, 91.83±10.01, and 43.46±2.92 μM, respectively. The corresponding respective IC50 of hypochlorite and PAA on β-glucuronidase were 704.90±41.40 and 23.26±0.82 μM, while H2O2 showed no inhibition on β-glucuronidase. It was further revealed that the inhibition of hypochlorite on both arylsulfatase and β-glucuronidase was irreversible. On the contrary, the inhibition by H2O2 and PAA was reversible. Moreover, it was found that the inhibitions of arylsulfatase and/or β-glucuronidase by these three chemicals were pH-dependent, among which the inhibition by H2O2 was competitive and non-competitive for PAA. In general, H2O2 and hypochlorite can be endogenously produced in human, which suggested that the two compounds are potential endocrine disruption compounds (EDCs) as they can cause endocrine disruption via inhibition of arylsulfatase and β-glucuronidase. This work further indicated that any agent that can induce production of H2O2 or hypochlorite in human is potential EDC, which explains why some EDCs with very weak or no estrogenic potency can cause endocrine disruption that confirmed in epidemiological studies.

Background: Total knee arthroplasty (TKA) has recently become an almost irreplaceable and effective means to relieve pain, reconstruct knee motor function and improve the quality of life of patients with end-stage knee osteoarthritis (KOA). However, the muscle strength after TKA is usually difficult to recover. Although electroacupuncture (EA) can enhance the muscle strength of lower extremity, there is hardly any literature on the effect of EA on the muscle strength of lower extremity in patients after TKA. To address this problem, this trial is intended to evaluate the efficacy of EA after TKA for recovering the muscle strength of lower extremity, esp. in the early post-TKA period.Methods/design: This is a double-blinded, randomized and controlled trial. It will be conducted between January, 2020 and June, 2020. A total of 94 participants with KOA will have undergone unilateral TKA and they will then be randomized into a treatment group and a control group, viz. the EA group and the sham EA group. The former will receive EA at acupuncture points of ST37, ST36, SP10 and SP9. The latter, the control group will receive sham EA at sham locations for the acupuncture points of ST37, ST36, SP10 and SP9. The participants will be given 5 sessions of treatment per day for 2 weeks. The primary outcomes include a change in the amount of muscle strength and the Hospital for Special Surgery (HSS) score at the second week from a baseline (POD 3). The secondary outcomes include a 6-minute walking test, Numerical rating scale (NRS), the Hamilton Anxiety Scale and additional use of analgesia. The additional outcomes include incidence of analgesia-related side effects and rate of participant satisfaction. Blinding of the participants will also be assessed. The participants will be asked to guess whether they have received EA after the latest intervention. Adverse events of EA will be documented and assessed throughout the trial.Discussion: EA is helpful for the recovery and enhancement of muscle strength of the lower limb after TKA.

Hypoxia plays a critical role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated the by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybrid screen, is an asparaginyl hydroxylase that negatively regulates transcriptional activation by HIF. This study aimed to define mechanisms that govern transitions of FIH between nucleus and the cytoplasm. We report that FIH accumulates in the nucleus within a short time window upon hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by “coupling” with HIF1α for importin β1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway.

When given a long list of items to remember, people typically prioritize the memorization of the most valuable items. Prior neuroimaging studies have found that cues denoting the presence of high value items can lead to increased activation of the mesolimbic dopaminergic reward circuit, including the nucleus accumbens (NAcc) and ventral tegmental area (VTA), which in turn results in up-regulation of medial temporal lobe encoding processes and better memory for the high value items. Value cues may also trigger the use of elaborative semantic encoding strategies which depend on interactions between frontal and temporal lobe structures. We used diffusion tensor imaging (DTI) to examine whether individual differences in anatomical connectivity within these circuits are associated with value-induced modulation of memory. DTI data were collected from 19 adults who also participated in an functional magnetic resonanceimaging (fMRI) study involving a value-directed memory task. In this task, subjects encoded words with arbitrarily assigned point values and completed free recall tests after each list, showing improved recall performance for high value items. Motivated by our prior fMRI finding of increased recruitment of left-lateralized semantic network regions during the encoding of high value words (Cohen et al., 2014), we predicted that the robustness of the white matter pathways connecting the ventrolateral prefrontal cortex (VLPFC) with the temporal lobe might be a determinant of recall performance for high value items. We found that the mean fractional anisotropy (FA) of each subject's left uncinate fasciculus (UF), a fronto-temporal fiber bundle thought to play a critical role in semantic processing, correlated with the mean number of high value, but not low value, words that subjects recalled. Given prior findings on reward-induced modulation of memory, we also used probabilistic tractography to examine the white matter pathway that links the NAcc to the VTA. We found that the number of fibers projecting from left NAcc to VTA was reliably correlated with subjects' selectivity index, a behavioral measure reflecting the degree to which recall performance was impacted by item value. Together, these findings help to elucidate the neuroanatomical pathways that support verbal memory encoding and its modulation by value.

Background: Little information exists on whether gender or asthma status modifies the effects of secondhand smoke (SHS) exposure on lung function. Objective: To evaluate whether gender or asthma status modifies the association of SHS exposure with lung function. Methods: A total of 6,740 children (average 11.6 years) were recruited from 24 districts of 7 cities in northeast China in 2012. SHS exposure included exposure to environmental and maternal smoking both in utero and during early childhood (postnatal). Lung function was measured using electronic spirometers. Two-step regressions were used to analyze the association between SHS and lung function. Results: In utero and postnatal exposure to SHS was independently associated with decreased lung function in both genders; however, this association was greater among males. For example, when exposed to maternal smoking during pregnancy, the adjusted odds ratio (aOR) for decreased forced vital capacity (FVC) was 6.46 (95% confidence interval [CI]: 2.58-16.17) among males, while only 2.16 (95% CI: 0.96-4.88) among females. More positive associations between SHS exposure and decreased lung function were detected among nonasthmatic compared with asthmatic children. Nonasthmatics had significantly larger deficits from in utero exposure to maternal smoking, which concerned decreased lung FVC function (aOR = 2.58, 95% CI: 1.28-5.21) and decreased lung forced expiratory volume in 1 s (FEV1) function (aOR = 2.32, 95% CI: 1.01-5.33). A similar pattern was also observed for the associations between SHS exposure and continuous pulmonary function test measurements. Conclusions: SHS exposure was associated with decreased lung function. Males and nonasthmatics seem to be more susceptible than their respective counterparts.

© 2016. American Geophysical Union. All Rights Reserved. The Earth's surface volcanism exerts first-order controls on the composition of the atmosphere and the climate. On Earth, the majority of surface volcanism occurs at mid-ocean ridges. In this study, based on the dependence of melt fraction on temperature, pressure, and composition, we compute melt production and degassing rate at mid-ocean ridges from three-dimensional global mantle convection models with plate motion history as the surface velocity boundary condition. By incorporating melting in global mantle convection models, we connect deep mantle convection to surface volcanism, with deep and shallow mantle processes internally consistent. We compare two methods to compute melt production: a tracer method and an Eulerian method. Our results show that melt production at mid-ocean ridges is mainly controlled by surface plate motion history, and that changes in plate tectonic motion, including plate reorganizations, may lead to significant deviation of melt production from the expected scaling with seafloor production rate. We also find a good correlation between melt production and degassing rate beneath mid-ocean ridges. The calculated global melt production and CO2degassing rate at mid-ocean ridges varies by as much as a factor of 3 over the past 200 Myr. We show that mid-ocean ridge melt production and degassing rate would be much larger in the Cretaceous, and reached maximum values at ∼150–120 Ma. Our results raise the possibility that warmer climate in the Cretaceous could be due in part to high magmatic productivity and correspondingly high outgassing rates at mid-ocean ridges during that time.

Retinitis pigmentosa is one of the main causes of blindness throughout the world and its pathogenesis remains unclear. In this study, patch-clamp recording was performed from ganglion cells in retinal dystrophic Royal College of Surgeons rats and normal rats during development. The three discharge patterns of action potential in ganglion cells of Royal College of Surgeons rats were single, transient, and sustained firing. The main discharge pattern was single firing in P1-2W, then transient and sustained firing. However, during later stages of retinal degeneration, action potential was reduced in amplitude and frequency, and was even lost in Royal College of Surgeons rats. These findings differed dramatically from those of rats without retinal degeneration, suggesting there are functional ganglion cells in Royal College of Surgeons rats early, but lose part function during retinal degeneration, even though morphological differences are not apparent. Understanding the changes of electrophysiological characteristics during retinal degeneration in detail may help explore the optimal time course for treating retinitis pigmentosa.

We measured the physical imaging performance of a 41×41 cm 2 amorphous silicon flat panel detector designed for angiographic and R&F imaging applications using methods from the emerging IEC standard for the measurement of detective quantum efficiency (DQE) in digital radiographicdetectors.Measurements on 12 production detectors demonstrate consistent performance. The mean DQE at the detector center is about 0.77 at zero frequency and 0.27 at the Nyquist frequency (2.5 cycles/mm) when measured with a 7 mm of Al HVL spectrum at about 3.6 μGy. The mean MTF at the center of the detector for this spectrum is 0.24 at the Nyquist frequency. For radiographic operation all 2048×2048 detector elements are read out individually. For fluoroscopy, the detector operates in two 30 frame per second modes: either the center 1024×1024 detector elements are read out or the entire detector is read out with 2×2 pixel binning. A model was developed to predict differences in performance between the modes, and measurements demonstrate agreement with the model. Lag was measured using a quasi-equilibrium exposure method and was found to be 0.044 in the first frame and less than 0.007 after 1 s. We demonstrated that it is possible to use the lag data to correct for temporal correlation in images when measuring DQE with a fluoroscopic imaging technique. Measurements as a function of position on the detector demonstrate a high degree of uniformity. We also characterized dependences on spectrum, exposure level, and direction. Finally, we measured the DQE of a current state of the art image intensifier/CCD system using the same method as for the flat panel. We found the image intensifier system to have lower DQE than the flat panel at high exposure levels and approximately equivalent DQE at fluoroscopic levels.

Genome-wide association studies (GWAS) have been successful in identifying common genetic variation involved in susceptibility to etiologically complex disease. We conducted a GWAS to identify common genetic variation involved in susceptibility to upper aero-digestive tract (UADT) cancers. Genome-wide genotyping was carried out using the Illumina HumanHap300 beadchips in 2,091 UADT cancer cases and 3,513 controls from two large European multi-centre UADT cancer studies, as well as 4,821 generic controls. The 19 top-ranked variants were investigated further in an additional 6,514 UADT cancer cases and 7,892 controls of European descent from an additional 13 UADT cancer studies participating in the INHANCE consortium. Five common variants presented evidence for significant association in the combined analysis (p

Contains fulltext : 96576.pdf (Publisher’s version ) (Open Access) Genome-wide association studies (GWAS) have been successful in identifying common genetic variation involved in susceptibility to etiologically complex disease. We conducted a GWAS to identify common genetic variation involved in susceptibility to upper aero-digestive tract (UADT) cancers. Genome-wide genotyping was carried out using the Illumina HumanHap300 beadchips in 2,091 UADT cancer cases and 3,513 controls from two large European multi-centre UADT cancer studies, as well as 4,821 generic controls. The 19 top-ranked variants were investigated further in an additional 6,514 UADT cancer cases and 7,892 controls of European descent from an additional 13 UADT cancer studies participating in the INHANCE consortium. Five common variants presented evidence for significant association in the combined analysis (p

Small-scale convection (SSC) rolls below the oceanic lithosphere have the tendency to align with the large-scale shearing direction and thus with the plate motion direction relative to the deep mantle. Understanding the timescales of and processes responsible for realignment would contribute significantly to our understanding of the unresolved phenomena in the Pacific such as gravity lineations, small-scale seismic velocity variations, and intraplate volcanism that cannot be explained by hot spots. In this study we examine the evolution of those convection rolls when this relative plate motion direction is suddenly changed, as suggested by the kink in the Hawaii-Emperor seamount chain. Using three-dimensional numerical flow models, we investigate the realignment of SSC rolls after a change in plate motion direction. From the nature of the SSC, it is expected that rheological parameters dominate the characteristics of this realignment. Our results show that this is indeed the case. We find that (1) using constraints from onset timing of SSC, realignment of rolls can occur as fast as within 20 Ma, but might also take much longer, dependent on the rheology; (2) the realignment period is strongly correlated to the sum of large-scale shear stress induced by plate motion and small-scale shear stress from the SSC itself; (3) in a mantle deforming by dislocation creep, realignment occurs faster than by diffusion creep, because dislocation creep SSC is more vigorous; and (4) activation energy has little influence on the realignment time. Possible evidence for the realignment period might come from precise age determination of intraplate volcanism or azimuthal seismic anisotropy.

The formation and shape variation of the Hawaiian plume swell is re-examined numerically. Scaling laws for the plume buoyancy flux and swell width and height help gaining new insight in relationships between swell formation and relevant model parameters, like plume temperature and size, and mantle rheology. A scaling law for the plume buoyancy F = Aη0 −1.2 R p 3.5ΔT p 2.2 exp(1.3 × 10−8 EΔT p ), with background mantle viscosity η0, plume radius R p , plume excess temperature ΔT p , and activation energy E fits numerical flux measurements within 8%. Scaling laws for the swell width and height have similar forms, and their multiplication resembles the buoyancy flux scaling law within 10%. These scaling laws suggest that the background mantle viscosity plays a significant role, and that the increased Hawaiian plume intensity ∼25 Ma ago is due to a plume excess temperature increase of 50%.

LiMn1-xCrxO2 compounds with an α-NaFeO2 layer-type crystal structure have been prepared by the Pechini method. The effects of ethylene glycol (EG) content and calcination temperature on powder characteristics and electrochemical performance are evaluated. It is found that the homogeneity of the powder is increased by increasing the molar ratio of EG to citric acid, and a trace of MnO can be detected in the powder calcined below 800°C for 4 h. Compared with the ss-LiMn1-x CrxO2 powder (made by the solid-state reaction), the p-LiMn1-xCrxO2 compound (made by the Pechini method) yielded higher specific capacity for both charge and discharge, and the rate capability has been improved due to the smaller particle size and good homogeneity. The evolution of discharge curves with cycling shows that less additional spinel-type tetrahedral sites for Li formed during cycling of the p-LiMn1-xCrxO2 compound.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1.5-Mb tandem DNA duplication in chromosome 17p11.2-p12, while hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus. The 1.5-Mb CMT1A monomer unit duplicated in CMT1A and deleted in HNPP is flanked by low-copy repeats termed CMT1A-REPs. Both diseases appear to be caused by an altered copy number of the peripheral myelin protein 22 gene (PMP22), which lies within the critical region. To identify additional genes rapidly, we used a cosmid contig of this region and reciprocal probing of arrayed chromosome 17-specific cosmid and cDNA libraries. Three cDNA clones were identified within the CMT1A duplication/HNPP deletion region and one just proximal to the critical region. The cDNA for human heme A:farnesyltransferase (COX10) mapped 10 kb centromeric to the distal CMT1A-REP. The other two cDNA clones from within the critical interval mapped to cosmid 126D1 at the mfd41 (D17S261) DNA marker, and their conceptual translation showed homology to 60S ribosomal protein L9 (RPL9) and chromosomal protein RMSA-1 (RMSA-1). A gene that is homologous to human peroxisome proliferator activated receptor alpha (hPPARA) was identified near the proximal CMT1A-REP.

A conductive ceramic, barium metaplumbate (BaPbO3), has been synthesized by solid-state reactions for use as an anode in lithium-ion batteries. The electrochemical characteristics of the electrode were tested in ethylene and diethyl carbonate (EC-DEC) solutions of LiPF6 versus Li metal in test cells. During cycling, the material showed voltage plateaus between 0.2 V and 0.6 V versus Li, demonstrated a gravimetric discharge capacity of about 110 mAh g-1 and a volumetric capacity of 960 mAh cm-3. Two phases, BaPbO3 and PbO, were found in compositions containing excess PbO. The free PbO in the fired bodies was found in the experiment to react with the lithium and form a new compound, probably a Pb-Li alloy, which was unstable and brought an intensive capacity fading. A single phase, BaPbO3, which was obtained by increasing the calcining temperature and the partial pressure of O2 in the ambient air, presented an unproved capacity and a stable pervoskite structure. The results from cyclic voltammograms suggest that the insertion and de-insertion of Li ions proceed in the voltage range from 0.01 to 1.2 V versus Li/ Li+.

Cordyceps sinensis (Berk.) Sacc. is one of the well-described fungi that has been used in traditional Chinese medicine for over 700 years. Fungal mycelia contain some polysaccharides that are responsible for their biological activity. C. sinensis has traditionally been cultivated on the high Tibetan plateau as a parasitic fungus growing on caterpillars. However, currently it is being cultivated on some insects and in artificial media. This article deals with the advances in the production, isolation and purification of Cordyceps polysaccharide (CP) in recent years, as well as the structure elucidation and pharmacological action. The article also aims to provide some references for further application and exploitation in the future.

These observations suggest several immediate clinical strategies. In gene therapy, approaches could be targeted to obtain cycling of hematopoietic stem cells and gene-carrying retrovirus vector integration followed by engraftment at an appropriate time interval which favors engraftment. The same type of approach can be utilized for stem cell expansion approaches. Alternatively marrow or peripheral stem cell engraftment can be obtained with minimal to no toxicity in allochimeric strategies in such diseases as sickle cell anemia or thalassemia. A similar approach could be useful in obtaining cell engraftment with minimal toxicity in therapies employing cellular immune (T-cell and NK-cell) attack against cancer. These areas of clinical application are outline in Table 3.

To establish digitized virtual Chinese No.1 female (VCH-F1) image database.A 19 years old female cadaver was scanned by CT, MRI, and perfused with red filling material through formal artery before freezing and em- bedding. The whole body was cut by JZ1500A vertical milling machine with a 0.2 mm inter-spacing. All the images was produced by Fuji FinePix S2 Pro camera.The body index of VCH-F1 was 94%. We cut 8 556 sections of the whole body, and each image was 17.5 MB in size and the whole database reached 149.7 GB. We have totally 6 versions of the database for different applications.Compared with other databases, VCH-F1 has good representation of the Chinese body shape, colorful filling material in blood vessels providing enough information for future registration and segmentation. Vertical embedding and cutting helped to retain normal human physiological posture, and the image quality and operation efficiency were improved by using various techniques such as one-time freezing and fixation, double-temperature icehouse, large-diameter milling disc and whole body cutting.