Effects of cheese ingestion on muscle mass and strength in possible sarcopenia women: an open-label, parallel-group study.

BACKGROUND: Nutrient-rich cheese supplements were demonstrated to have improvements in markers of sarcopenia in healthy elders. However, the potential effects of cheese in individuals with possible sarcopenia remain unknown. METHOD: This 90-day randomized controlled trial (RCT) included 68 women aged 60-80 years with possible sarcopenia in China, who were randomly assigned to three groups: Control group (CG), Original cheese group (OG: 9.0 g protein; 322.8 mg calcium), and Golden cheese group (GG: 12.7 g protein; 802.1 mg calcium). OG and GG were instructed to consume their habitual diet along with 4 slices of supplied cheese, while CG was directed to maintain their usual dietary habits. Face-to-face interviews, anthropometric measurements, and blood sample collection were conducted at baseline, midway (60 days), and the end of the trial. RESULT: At the end of the trial, the primary outcome, changes of Skeletal Muscle Mass Index (SMI) were found to be higher in OG (0.18 ± 0.02 kg/m2) and GG (0.14 ± 0.02 kg/m2) compared to CG (0.09 ± 0.02 kg/m2). The secondary outcome, changes of handgrip strength were higher in GG (1.82 ± 4.16 kg) than CG (-0.61 ± 3.78 kg). There were no significant differences in makers for muscle function between three groups (P > 0.05). In the self-comparison, Creatinine/Cystatin C significantly increased in both OG and GG. In addition, OG had a significant increase in changes of free and total carnitine compared to CG. CONCLUSION: Both golden and original cheese supplementation enhanced muscle strength and mass in older women with possible sarcopenia. The mechanism behind this effect may be linked to muscle cell energy metabolism. TRIAL REGISTRATION: The present study was registered in the Chinese Clinical Trial Registry with the registration number ChiCTR2300078720 (retrospectively registered, 20231215).

Tracking the impact of perfluoroalkyl acid emissions on antibiotic resistance gene profiles in receiving water by metagenomic analysis.

The ecological risks posed by perfluoroalkyl acids (PFAAs) to the aquatic environment have recently been of great concern. However, little information was available on the impact of PFAAs on antibiotic resistance genes (ARGs) profiles. In this study, the receiving river of the largest fluoropolymer production facility in China was selected to investigate the effects of PFAAs on ARGs profiles. The highest PFAAs concentration for water samples near the industrial effluent discharge point was 310.9 µg/L, which was thousands times of higher than the average concentration collected at upstream sites. Perfluorooctanoic acid accounted for more than 67.2 % of ∑PFAAs concentration in water samples collected at the downstream sites, followed by perfluorohexanoic acid (3.6 %-15.9 %). 145 ARG subtypes including high-risk ARGs were detected by metagenomic technology. The results indicated that the discharge of PFAA-containing effluents had a significant impact on the abundance and diversity of ARGs in receiving waters, and PFAAs and water quality parameters (e.g., pH, NH3N, CODMn, TP) could largely affect ARG profiles. Specifically, short-chain PFAAs had similar impacts on ARG profiles compared to the restricted long-chain PFAAs. This study confirmed the potential effects of PFAAs on ARGs in aquatic environment and provided more insights into the ecological risk raised by PFAAs.

Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Investigation of the interaction between lactones and ketones in a Cheddar cheese matrix using Feller's additive model, σ-τ plots, U-models, and aroma addition experiments.

The objective of this study was to examine the sensory interactions between lactones and ketones in a Cheddar simulation matrix through perceptual interaction analysis. The olfactory thresholds of 6 key lactones had values ranging from 8.32 to 58.88 µg/kg, whereas those of the 4 key ketones ranged from 6.61 to 660.69 µg/kg. Both Feller's additive model and σ-τ plots demonstrated complex interactions in 24 binary mixtures composed of the 6 lactones and 4 ketones, including synergy, addition, and masking effects. Specifically, we found that 6 binary mixtures exhibited aroma synergistic effects using both methods. Moreover, the σ-τ plot showed a synergistic effect of aroma in 3 ternary mixtures. The U-model further confirmed the synergistic effects of the 6 groups of binary systems and 3 groups of ternary systems on aroma at actual cheese concentrations. In an aroma addition experiment, the combination of δ-octalactone and diacetyl in binary mixtures had the most pronounced effect on enhancing milk flavor. In ternary mixtures, 2 combinations, namely δ-octalactone/δ-dodecalactone/diacetyl and γ-dodecalactone/δ-dodecalactone/acetoin, significantly enhanced the milky and sweet aroma properties of cheese, while also enhancing the overall acceptability of the cheese aroma.

Large-scale generation of IL-12 secreting macrophages from human pluripotent stem cells for cancer therapy.

Genetically engineered macrophages (GEMs) have emerged as an appealing strategy to treat cancers, but they are largely impeded by the cell availability and technical challenges in gene transfer. Here, we develop an efficient approach to generate large-scale macrophages from human induced pluripotent stem cells (hiPSCs). Starting with 1 T150 dish of 106 hiPSCs, more than 109 mature macrophages (iMacs) could be generated within 1 month. The generated iMacs exhibit typical macrophage properties such as phagocytosis and polarization. We then generate hiPSCs integrated with an IL-12 expression cassette in the AAVS1 locus to produce iMacs secreting IL-12, a strong proimmunity cytokine. hiPSC-derived iMacs_IL-12 prevent cytotoxic T cell exhaustion and activate T cells to kill different cancer cells. Furthermore, iMacs_IL-12 display strong antitumor effects in a T cell-dependent manner in subcutaneously or systemically xenografted mice of human lung cancer. Therefore, we provide an off-the-shelf strategy to produce large-scale GEMs for cancer therapy.

Distributed asynchronous measurement system fusion estimation based on inverse covariance intersection algorithm.

For state estimation of multi-source asynchronous measurement systems with measurement missing phenomena, this paper proposes a distributed sequential inverse covariance intersection (DSICI) fusion algorithm based on conditional Kalman filtering method. It is mainly divided into synchronized state space module, local filtering module and fusion estimation module. The missing measurements occurring in the system are modelled and described by a set of random variables obeying a Bernoulli distribution. The synchronized state space module uses a state iteration method to synchronize the asynchronous measurement system at the moment of measurement update and it ensures the integrity of the measurement information. The local filtering module uses a conditional Kalman filtering algorithm for filter estimation. The reliability of the local filtering results is guaranteed because the local estimator designs a method to interact information with the domain sensors. The fusion estimation module designs a DSICI fusion algorithm with higher accuracy and satisfying consistency, which fuses the filtering results provided by each sensor when the relevant information between multiple sensors is unknown. Simulation examples demonstrate the excellent performance of the proposed algorithm, with a 33% improvement in accuracy over existing algorithms and an iteration time of less than 3 ms.

Autophagy is essential for human myelopoiesis.

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

Effect of UV/peroxymonosulfate pretreatment on disinfection byproduct (DBP) formation during post-chlorination of humic acid.

UV/peroxymonosulfate (UV/PMS) is a promising advanced oxidation technology in water treatment. This study aimed to investigate the impact of UV/PMS on humic acid (HA) and the influence of PMS dosage, pretreatment time, pH pretreatment, nitrate, nitrite, ammonium, and bicarbonate influencing factors on disinfection byproduct (DBP) formation during post-chlorination. With increased PMS dosage or pretreatment time, the UV/PMS treatment significantly reduced ultraviolet absorbance and increased mineralization. It altered the fractional constituent as humic substances were gradually transformed into building blocks and low-molecular-weight acids. However, most DBP formation increased initially and then decreased after subsequent chlorination. Rising nitrate or nitrite concentrations markedly promoted halonitromethane (HNM) formation. The presence of ammonia had a more significant impact on dichloroacetonitrile (DCAN) formation. Bicarbonate in UV/PMS pretreatment increased carbonated disinfection byproduct (C-DBP) formation, whereas it had a negligible impact on nitrogenous disinfection byproduct (N-DBP) formation. The present study revealed the impact of a series of influencing factors on DBP formation in UV/PMS reaction systems, providing comprehensive insights on applying UV/PMS in actual practice.

Grad-MobileNet: A Gradient-Based Unsupervised Learning Method for Laser Welding Surface Defect Classification.

Deep learning technology has advanced rapidly and has started to be applied for the detection of welding defects. In the manufacturing process of power batteries for new energy vehicles, welding defects may occur due to the high directivity, convergence, and penetration of the laser beam. The accuracy of deep learning prediction relies heavily on big data, but balanced big data of welding defects is hard to acquire at the battery production site. In this paper, the authors construct a dataset named RIAM, which consists of images captured from an industrial environment for laser welding of power battery modules. RIAM contains four types of images: Normality, Lack of fusion, Surface porosity, and Scaled surface. The characteristics of RIAM are carefully considered in the application scenarios. Moreover, this paper proposes a gradient-based unsupervised model named Grad-MobileNet, which can be trained with only a few normal images and can extract the feature gradients of the input images. Welding defects can then be classified by the gradient distribution. This model is based on MobileNetV3, which is a lightweight convolutional neural network (CNN), and achieves 99% accuracy, which is higher than the accuracy expected from supervised learning.

Single particle ICP-MS combined with filtration membrane for accurate determination of silver nanoparticles in the real aqueous environment.

This work presents the role of commercial microfiltration membranes combined with single particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) in removing environmental matrix interference for model silver nanoparticles (AgNPs) determination. The filters with different pore sizes (0.22 µm, 0.45 µm, 0.8 µm) and materials (mixed cellulose ester, polyether sulfone, and nylon) were investigated to acquire the recovery of particle concentration and size of AgNPs spiked into different real aqueous solutions, including ultrapure water, tap water, surface water, and sewage effluent. The maximum recovery of nanoparticle concentration was 70.2% through the 0.8 µm polyether sulfone membrane. The heated filters were able to improve the recovery of AgNPs particle concentration in the real aqueous environment. Hence, the pretreatment method by SP-ICP-MS combined with filtration membrane was simple, fast, and low-cost to quantify AgNPs in natural water environments.

Occurrence and treatment effect assessment of organophosphorus flame retardants in source and drinking water, Jinan, China.

The occurrence and removal efficiencies of organophosphorus flame retardants (OPFRs) by traditional treatment processes (pre-flocculation, sand filtration, and post-chlorination processes) and advanced treatment processes (i.e., ozone and granular activated carbon (GAC), ultraviolet/hydrogen peroxide (UV/H2O2), GAC alone, ultrafiltration membrane, nanofiltration membrane) were examined in two municipal plants and a pilot plant in Jinan, China. The concentrations of six OPFRs in raw waters were at levels of 16.8-100.0 ng/L, and three OPFRs were below the detection limits. The traditional treatment processes could not effectively remove the OPFRs (the removal efficiency was - 12.0-15.4%). The advanced oxidation with ozone and GAC (the removal efficiency was 35.6-60.3%) or UV/H2O2 and GAC processes (the removal efficiency was 68.0-86.7%) were more effective than the traditional water treatment processes. The removal efficiencies of ultrafiltration process for the OPFRs was 11.2-69.8% which were positively correlated with the logKow values of OPFRs. The nanofiltration membrane process with ultrafiltration membrane process as the pretreatment was the most effective process (the removal efficiencies were almost to 100%). These results imply that the combination of ultrafiltration membrane and nanofiltration membrane is an effective measure in the treatment of OPFRs in municipal drinking water plants.

Cloning and expression study of a high-affinity nitrate transporter gene from Zea mays L.

A nitrate transporter gene, named B46NRT2.1, from salt-tolerant Zea mays L. B46 has been cloned. B46NRT2.1 contained the same domain belonging to the major facilitator superfamily (PLN00028). The results of the phylogenetic tree indicated that B46NRT2.1 exhibits sequence similarity and the closest relationship with those known nitrate transporters of the NRT2 family. Through RT-qPCR, we found that the expression of B46NRT2.1 mainly happens in the root and leaf. Moreover, the treatment with NaCl, Na2CO3, and NaHCO3 could significantly increase the expression of B46NRT2.1. B46NRT2.1 was located in the plasma membrane. Through the study of yeast and plant salt response brought by B46NRT2.1 overexpression, we have preliminary knowledge that the expression of B46NRT2.1 makes yeast and plants respond to salt shock. There are 10 different kinds of cis-acting regulatory elements (CRES) in the promotor sequences of B46NRT2.1 gene using the PlantCARE web server to analyze. It mainly includes hormone response, abscisic acid, salicylic acid, gibberellin, methyl jasmonate, and auxin. The B46NRT2.1 gene's co-expression network showed that it was co-expressed with a number of other genes in several biological pathways, including regulation of NO3 long-distance transit, modulation of nitrate sensing and metabolism, nitrate assimilation, and transduction of Jasmonic acid-independent wound signal. The results of this work should serve as a good scientific foundation for further research on the functions of the NRT2 gene family in plants (inbred line B46), and this research adds to our understanding of the molecular mechanisms under salt tolerance.

Experimental and theoretical investigation on degradation of dimethyl trisulfide by ultraviolet/peroxymonosulfate: Reaction mechanism and influencing factors.

With a large amount of domestic sewage and industrial wastewater discharged into the water bodies, sulfur-containing organic matter in wastewater produced volatile organic sulfide, such as dimethyl trisulfide (DMTS) through microorganisms, caused the potential danger of drinking water safety and human health. At present, there is still a lack of technology on the removal of DMTS. In this study, the ultraviolet/peroxymonosulfate (UV/PMS) advanced oxidation processes was used to explore the degradation of DMTS. More than 90% of DMTS (30 µg/L) was removed under the conditions of the concentration ratio of DMTS to PMS was 3:40, the temperature (T) was 25 ± 2℃, and 10 min of irradiation by a 200 W mercury lamp (365 nm). The kinetics rate constant k of DMTS reacting with hydroxyl radical (HO·) was determined to be 0.2477 min-1. Mn2+, Cu2+ and NO3- promoted the degradation of DMTS, whereas humic acid and Cl- in high concentrations inhibited the degradation process. Gas chromatography-mass spectrometry was used to analyze the degradation products and the degradation intermediates were dimethyl disulfide and methanethiol. Density functional theory was used to predict the possible degradation mechanism according to the frontier orbital theory and the bond breaking mechanism of organic compounds. The results showed that the SS, CS and CH bonds in DMTS molecular structure were prone to fracture in the presence of free radicals, resulting in the formation of alkyl radicals and sulfur-containing radicals, which randomly combined to generate a variety of degradation products.

Comparison of UV-based advanced oxidation processes for the removal of different fractions of NOM from drinking water.

This study examined the effectiveness for degradation of hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions of natural organic matter (NOM) during UV/H2O2, UV/TiO2 and UV/K2S2O8 (UV/PS) advanced oxidation processes (AOPs). The changing characteristics of NOM were evaluated by dissolved organic carbon (DOC), the specific UV absorbance (SUVA), trihalomethanes formation potential (THMFP), organic halogen adsorbable on activated carbon formation potential (AOXFP) and parallel factor analysis of excitation-emission matrices (PARAFAC-EEMs). In the three UV-based AOPs, HPI fraction with low molecular weight and aromaticity was more likely to degradate than HPO and TPI, and the removal efficiency of SUVA for HPO was much higher than TPI and HPI fraction. In terms of the specific THMFP of HPO, TPI and HPI, a reduction was achieved in the UV/H2O2 process, and the higest removal rate even reached to 83%. UV/TiO2 and UV/PS processes can only decrease the specific THMFP of HPI. The specific AOXFP of HPO, TPI and HPI fractions were all able to be degraded by the three UV-based AOPs, and HPO content is more susceptible to decompose than TPI and HPI content. UV/H2O2 was found to be the most effective treatment for the removal of THMFP and AOXFP under given conditions. C1 (microbial or marine derived humic-like substances), C2 (terrestrially derived humic-like substances) and C3 (tryptophan-like proteins) fluorescent components of HPO fraction were fairly labile across the UV-based AOPs treatment. C3 of each fraction of NOM was the most resistant to degrade upon the UV-based AOPs. Results from this study may provide the prediction about the consequence of UV-based AOPs for the degradation of different fractions of NOM with varied characteristics.

Generation of a homozygous FIS1 knockout human embryonic stem cell line GIBHe015-B by CRISPR/Cas9 system.

The mitochondrial fission protein 1 (FIS1) is essential for mitochondrial division or fission and has been determined to mediate mitochondrial and peroxisomal fission. Other studies also found that FIS1 functions as an essential component of the mitophagy and apoptosis pathways in mammalian cells, suggesting that FIS1 has multiple important roles. Here, we generated homozygous FIS1 knockout human embryonic stem cells (hESCs) using the CRISPR/Cas9 system. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the functions of FIS1 and their implications in human health and diseases.

Facile Fabrication of Starch-Based Microrods by Shear-Assisted Antisolvent-Induced Nanoprecipitation and Solidification.

Rod-like particles have attracted increasing attention because of their unique shape-dependent properties, which enable their superior performance compared to their isotropic counterparts. Thus, rod-like particles have potential applications in many fields, especially in biomedicine. However, the fabrication of uniform rod-like particles is challenging because of the principle of interfacial energy minimization. Herein, we present a facile, rapid, and cost-effective strategy for preparing starch-based microrods with tunable aspect ratios via shear-assisted antisolvent-induced nanoprecipitation and solidification. The preformed spherical particles swollen by the mixed solvent were elongated by the shear force and solidified in rod-like shape by antisolvent induction. The resulting starch-based microrods can encapsulate hydrophobic active substances and be modified with functional groups, indicating their potential applications as drug carriers and biologically active materials. The formation mechanism of the starch-based microrods discovered in this study provides a new perspective on the fabrication of rod-like polymer particles.

Facile synthesis of mesoporous ZSM-5 aided by sonication and its application for VOCs capture.

Application of ultrasound power to the mother liquor is popular pretreatment for zeolite synthesis which offers a simple way of accelerating crystallization process and finetuning the properties of nanocrystalline zeolites. In this work, sonication-aided synthesis of mesoporous ZSM-5 at low temperature and ambient pressure was systematically studied, in an attempt to reach efficient and benign synthesis of zeolites with hierarchical pore structure, which has wide applications as catalysts and sorbents. The effects of sonication duration, power density, sonication temperature and seeding on the crystallization of ZSM-5 were investigated. The obtained samples were characterized by XRD, SEM, BET and VOCs capture. High quality mesoporous ZSM-5 can be obtained by a facile 5 d synthesis at 363 K, much faster than conventional hydrothermal synthesis. The reduced synthesis time was mainly attributed to the enhanced crystallization kinetics caused by the fragmentation of seeds and nuclei, while sonication radiation had little impact on the nucleation process. Compared with control sample, mesoporous ZSM-5 prepared by sonochemical method had higher surface area and mesoporosity which demonstrated improved adsorption performance for the capture of isopropanol.

The Involvement of Antioxidant Enzyme System, Nitrogen Metabolism and Osmoregulatory Substances in Alleviating Salt Stress in Inbred Maize Lines and Hormone Regulation Mechanisms.

Salt stress inhibited the growth of maize. B46 and NC236 were chosen as materials and NaCl concentrations (0, 55, 110, 165, and 220 mmol L-1) were set. We found the activities of SOD, POD, CAT, APX, GR, MDHAR, and DHAR decreased under NaCl stress. Compared with NC236, the contents of AsA and GSH, AsA/DHA and GSH/GSSG of B46 decreased. The content of O2-, H2O2, MDA, and EL of B46 increased. The contents of NO3- and NO2- decreased, while the content of NH4+ increased under high NaCl concentration. The activities of NR and NiR decreased, while the activities of GS and GOGAT increased first and then decreased. For B46 and NC236, the maximum of NADH-GDH and NAD-GDH appeared at 165 and 110 mmol L-1 NaCl concentration, respectively. Compared with B46, and the GOT and GPT activities of NC236 increased first and then decreased. With the increase of NaCl concentration, the contents of proline, soluble protein, and soluble sugar were increased. The Na+ content of B46 and NC236 increased, and the K+ content and K+/Na+ decreased. Compared with NC236, B46 had higher IAA content in leaf, higher Z + ZR content in leaf and root, and lower ABA content in leaf and root.

Generating functional cells through enhanced interspecies chimerism with human pluripotent stem cells.

Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extremely low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in the presence of mouse PSCs (mPSCs), while MYCN, a pro-growth factor, dramatically promotes hPSC contributions in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into pre-implantation embryos of different species, such as mice, rabbits, and pigs, and substantially contributed to mouse post-implantation chimera in embryonic and extra-embryonic tissues. Strikingly, M/B-hPSCs injected into pre-implantation Flk-1+/- mouse embryos show further enhanced chimerism that allows for obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera-derived human CD34+ cells further gave rise to various subtype blood cells in a typical colony-forming unit (CFU) assay. Thus, we provide proof of concept to obtain functional human cells through enhanced interspecies chimerism with hPSCs.

Renal puncture access using a blunt needle: proposal of the blunt puncture concept.

PURPOSE: Severe haemorrhage in percutaneous nephrolithotomy (PCNL) is an alarming event, and preventing injury to renal major vessels is a challenge. We evaluated the efficiency of a blunt needle in renal puncture procedures. METHODS: We first retrospectively reviewed the embolization images of post-PCNL patients to analyse the types of arteries injured, which were considered target arteries. Then, either a blunt needle or a conventional needle was used to directly puncture target arteries in ex vivo porcine kidneys and to establish renal access ex vivo and in vivo. The primary outcome was the incidence of target artery injuries, which were observed by digital subtraction angiography, nephroscopy and 3-dimensional endocasts. The secondary outcome was the rate of excreted fluid per access. RESULTS: The segmental and interlobar arteries were the most common types of injured arteries that needed to be embolized after PCNL. When these arteries were punctured directly, blunt needles reduced injury (1/20 vs. 16/20; OR 4.750; 95% CI 1.966-11.478; P < .001) by 76% compared to injuries induced by conventional needles. Moreover, the blunt needle group also had a significantly lower incidence of these arteries' injuries ex vivo due to renal puncture and yielded a lower rate of excreted fluid in ex vivo and in vivo renal puncture procedures. CONCLUSION: A blunt needle for renal puncture can be effective in reducing injury to renal major arteries and the accompanying haemorrhage. We propose the concept of blunt puncture, which may be a promising method for achieving safe renal access in PCNL.