Unnatural Peptide Assemblies Rapidly Deplete Cholesterol and Potently Inhibit Cancer Cells.

Cholesterol-rich membranes play a pivotal role in cancer initiation and progression, necessitating innovative approaches to target these membranes for cancer inhibition. Here we report the first case of unnatural peptide (1) assemblies capable of depleting cholesterol and inhibiting cancer cells. Peptide 1 self-assembles into micelles and is rapidly taken up by cancer cells, especially when combined with an acute cholesterol-depleting agent (MßCD). Click chemistry has confirmed that 1 depletes cell membrane cholesterol. It localizes in membrane-rich organelles, including the endoplasmic reticulum, Golgi apparatus, and lysosomes. Furthermore, 1 potently inhibits malignant cancer cells, working synergistically with cholesterol-lowering agents. Control experiments have confirmed that C-terminal capping and unnatural amino acid residues (i.e., BiP) are essential for both cholesterol depletion and potent cancer cell inhibition. This work highlights unnatural peptide assemblies as a promising platform for targeting the cell membrane in controlling cell fates.

Protective effects of syringic acid in nonalcoholic fatty liver in rats through regulation of Nrf2/HO-1 signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) is an alarming ailment that leads to severe liver damage and increases the risk of serious health conditions. The prevalence of NAFLD due to oxidative stress could be mitigated by plant-derived antioxidants. This study aims to investigate the effects of syringic acid (SA) on NAFLD in a high-fat diet (HFD) rat model. Twenty-four rats were randomly divided into four groups (n = 6): normal control, HFD, SA-administered HFD, and positive control SA on a normal diet. Rats in the normal control and positive control groups received a normal diet, and the remaining groups received an HFD for 8 weeks. SA (20 mg/kg b.w.) was orally (gavage) administered for 8 weeks. Lipid profiles were controlled by SA against HFD-fed rats (p < 0.05). SA reduced the serum aspartate aminotransferase and alanine aminotransferase levels by 70%-190%. SA also suppressed pro-inflammatory cytokines and attenuated histopathological and immunohistochemical changes against HFD-fed rats. SA reversed oxidative stress by suppressing the malondialdehyde formation by 82% and replenished the nonenzymatic and enzymatic antioxidant activities (p < 0.05). Gene expressions of nuclear factor-erythroid 2-related factor/heme oxygenase 1 (Nrf2/HO-1) were elevated in SA-treated rats. Ameliorative effects of SA on NAFLD induced by an HFD in rats were prominent through the reversal of oxidative stress and inflammation, regulated by an intrinsic mechanism of defense against oxidative stress, the Nrf2/HO-1 pathway.

Enzymatic control of intermolecular interactions for generating synthetic nanoarchitectures in cellular environment.

Nanoarchitectonics, as a technology to arrange nano-sized structural units such as molecules in a desired configuration, requires nano-organization, which usually relies on intermolecular interactions. This review briefly introduces the development of using enzymatic reactions to control intermolecular interactions for generating artificial nanoarchitectures in a cellular environment. We begin the discussion with the early examples and uniqueness of enzymatically controlled self-assembly. Then, we describe examples of generating intracellular nanostructures and their relevant applications. Subsequently, we discuss cases of forming nanostructures on the cell surface via enzymatic reactions. Following that, we highlight the use of enzymatic reactions for creating intercellular nanostructures. Finally, we provide a summary and outlook on the promises and future direction of this strategy. Our aim is to give an updated introduction to the use of enzymatic reaction in regulating intermolecular interactions, a phenomenon ubiquitous in biology but relatively less explored by chemists and materials scientists. Our goal is to stimulate new developments in this simple and versatile approach for addressing societal needs.

Enzymatic reactions in cells create precise nanoarchitectures, offering insights into cell biology through controllable nanoarchitectonics, as shown by numerous examples in this review.

Enzyme-Responsive Peptide Thioesters for Targeting Golgi Apparatus.

The Golgi apparatus (GA) is the hub of intracellular trafficking, but selectively targeting GA remains a challenge. We show an unconventional types of peptide thioesters, consisting of an aminoethyl thioester and acting as substrates of thioesterases, for instantly targeting the GA of cells. The peptide thioesters, above or below their critical micelle concentrations, enter cells mainly via caveolin-mediated endocytosis or macropinocytosis, respectively. After being hydrolyzed by GA-associated thioesterases, the resulting thiopeptides form dimers and accumulate in the GA. After saturating the GA, the thiopeptides are enriched in the endoplasmic reticulum (ER). Their buildup in ER and GA disrupts protein trafficking, thus leading to cell death via multiple pathways. The peptide thioesters target the GA of a wide variety of cells, including human, murine, and Drosophila cells. Changing d-diphenylalanine to l-diphenylalanine in the peptide maintains the GA-targeting ability. In addition, targeting GA redirects protein (e.g., NRAS) distribution. This work illustrates a thioesterase-responsive and redox-active molecular platform for targeting the GA and controlling cell fates.

Chlorosis seedling lethality 1 encoding a MAP3K protein is essential for chloroplast development in rice.

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules in eukaryotic organisms and play essential roles in immunity and stress responses. However, the role of MAPKs in chloroplast development remains to be evidently established. RESULTS: In this study, a rice chlorosis seedling lethality 1 (csl1) mutant with a Zhonghua11 (ZH11, japonica) background was isolated. Seedlings of the mutant were characterized by chlorotic leaves and death after the trefoil stage, and chloroplasts were observed to contain accumulated starch granules. Molecular cloning revealed that OsCSL1 encoded a MAPK kinase kinase22 (MKKK22) targeted to the endoplasmic reticulum (ER), and functional complementation of OsCSL1 was found to restore the normal phenotype in csl1 plants. The CRISPR/Cas9 technology was used for targeted disruption of OsCSL1, and the OsCSL1-Cas9 lines obtained therein exhibited yellow seedlings which phenocopied the csl1 mutant. CSL1/MKKK22 was observed to establish direct interaction with MKK4, and altered expression of MKK1 and MKK4 was detected in the csl1 mutant. Additionally, disruption of OsCSL1 led to reduced expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded RNA polymerases, nuclear-encoded RNA polymerase, and nuclear-encoded chloroplast genes. CONCLUSIONS: The findings of this study revealed that OsCSL1 played roles in regulating the expression of multiple chloroplast synthesis-related genes, thereby affecting their functions, and leading to wide-ranging defects, including chlorotic seedlings and severely disrupted chloroplasts containing accumulated starch granules.

A Self-Assembling Probe for Imaging the States of Golgi Apparatus in Live Single Cells.

Despite the enormous progress in genomics and proteomics, it is still challenging to assess the states of organelles in living cells with high spatiotemporal resolution. Based on our recent finding of enzyme-instructed self-assembly of a thiophosphopeptide that targets the Golgi Apparatus (GA) instantly, we use the thiophosphopeptide, which is enzymatically responsive and redox active, as an integrative probe for revealing the state of the GA of live cells at the single cell level. By imaging the probe in the GA of live cells over time, our results show that the accumulation of the probe at the GA depends on cell types. By comparison to a conventional Golgi probe, this self-assembling probe accumulates at the GA much faster and are sensitive to the expression of alkaline phosphatases. In addition, subtle changes of the fluorophore results in slightly different GA responses. This work illustrates a novel class of active molecular probes that combine enzyme-instructed self-assembly and redox reaction for high-resolution imaging of the states of subcellular organelles over a large area and extended times.

RIP2 interacts with REL1 to control leaf architecture by modulating brassinosteroid signaling in rice.

KEY MESSAGE: RIP2 serves as a negative regulator of leaf inclination through the coordination of BR signaling in rice. Leaf angle is considered as an important morphological trait in rice. Appropriate leaf angle increases the efficiency of sunlight capture and maintains a high level of photosynthesis, ultimately improving crop yield. Our present study demonstrates that RIP2 encodes a RING finger E3 ligase protein that directly binds to ROLLED AND ERECT LEAF 1 (REL1), a key regulator of leaf morphogenesis. Further studies reveal that RIP2 is extensively involved in leaf inclination through the coordination of BR signaling. Repression of RIP2 led to altered phenotypes, including enlarged leaf inclination and fewer tillers. Conversely, rice overexpressing RIP2 exhibited erect leaves. The double mutant rel1 rip2 displayed phenotypes similar to those of rel1, characterized by rolled leaves. Transcriptome profiling of WT, rel1, rip2, and rel1 rip2 mutants revealed that BR and IAA signaling pathways were impaired in rip2. Moreover, rel1, rip2, and rel1 rip2 were insensitive to BR treatment. In summary, these findings demonstrate that RIP2 serves as a negative regulator of leaf inclination, and therefore, provides an approach for the optimization of an ideal plant type.

Molecular Insights into Salinity Responsiveness in Contrasting Genotypes of Rice at the Seedling Stage.

Salinity is one of the most common unfavorable environmental conditions that limits plant growth and development, ultimately reducing crop productivity. To investigate the underlying molecular mechanism involved in the salinity response in rice, we initially screened 238 rice cultivars after salt treatment at the seedling stage and identified two highly salt-tolerant cultivars determined by the relative damage rate parameter. The majority of cultivars (94.1%) were ranked as salt-sensitive and highly salt-sensitive. Transcriptome profiling was completed in highly salt-tolerant, moderately salt-tolerant, and salt-sensitive under water and salinity treatments at the seedling stage. Principal component analysis displayed a clear distinction among the three cultivars under control and salinity stress conditions. Several starch and sucrose metabolism-related genes were induced after salt treatment in all genotypes at the seedling stage. The results from the present study enable the identification of the ascorbate glutathione pathway, potentially participating in the process of plant response to salinity in the early growth stage. Our findings also highlight the significance of high-affinity K+ uptake transporters (HAKs) and high-affinity K+ transporters (HKTs) during salt stress responses in rice seedlings. Collectively, the cultivar-specific stress-responsive genes and pathways identified in the present study act as a useful resource for researchers interested in plant responses to salinity at the seedling stage.

Intranuclear Nanoribbons for Selective Killing of Osteosarcoma Cells.

Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L- or D-phosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance.

Synthesis and bioactivity of pyrrole-conjugated phosphopeptides.

Here we report the synthesis and effect on the cell viability of pyrrole-conjugated phosphopeptides. Encouraged by the selective inhibition of cancer cells by a naphthyl-capped phosphopeptide (Nap-ffpy, 1), we conjugated the heteroaromatic dipyrrole or tripyrrole motif at the N-terminal of short peptides containing phosphotyrosine or phosphoserine and examined the bioactivity of the resulting phosphopeptides (2-10). Although most of the phosphopeptides exhibit comparable activities with that of 1 against HeLa cells at 200 µM, they, differing from 1, are largely compatible with HeLa cells at 400 µM. Enzymatic dephosphorylation of 2-10, at 400 µM is unable to induce a dramatic morphological transition of the peptide assemblies observed in the case of 1. These results suggest that a heteroaromatic motif at the N-terminal of peptides likely disfavors the formation of extensive nanofibers or morphological changes during enzymatic self-assembly, thus provide useful insights for the development of phosphopeptides as substrates of phosphatases for controlling cell fate.

Enzymatically Forming Intranuclear Peptide Assemblies for Selectively Killing Human Induced Pluripotent Stem Cells.

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Experimental Determination of PT-Symmetric Exceptional Points in a Single Trapped Ion.

Exceptional points (EPs) of a non-Hermitian Hamiltonian with parity-time-reversal (PT) symmetry have the potential to drastically enhance the capabilities of metrology and sensing through their power-law growing sensitivity to external perturbation. With the ability of generating and tuning dissipation in a single trapped ion system, we observe rich dynamics and detailed quantum phase transitions from the PT-symmetric phase to the symmetry-breaking phase. In this single qubit full quantum system, we develop a method to precisely determine the location of EP without any fitting parameter, and extract the eigenvalues in a unified way through all parameter regions. We can also obtain the full density matrix by quantum state tomography. Finally, we suggest from theoretical analysis that the periodically driving PT-symmetric non-Hermitian system can be used to measure the magnitude, frequency, and phase of time-dependent perturbation with EP enhancement.

Enzymatic Assemblies of Thiophosphopeptides Instantly Target Golgi Apparatus and Selectively Kill Cancer Cells*.

Changing an oxygen atom of the phosphoester bond in phosphopeptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophosphopeptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi by themselves and with Golgi proteins. Moreover, the thiophosphopeptide potently and selectively inhibits cancer cells (HeLa) with the IC50 (about 3 µM), which is an order of magnitude more potent than that of the parent phosphopeptide.

Plasma Membrane Ca2+ Permeable Mechanosensitive Channel OsDMT1 Is Involved in Regulation of Plant Architecture and Ion Homeostasis in Rice.

Plant architecture is an important factor for crop production. Plant height, tiller pattern, and panicle morphology are decisive factors for high grain yield in rice. Here, we isolated and characterized a T-DNA insertion rice mutant Osdmt1 (Oryza sativa dwarf and multi-tillering1) that exhibited a severe dwarf phenotype and multi-tillering. Molecular cloning revealed that DMT1 encodes a plasma membrane protein that was identified as a putative Ca2+ permeable mechanosensitive channel. The transcript expression level was significantly higher in the dmt1 mutant compared to wild type (WT). Additionally, the dmt1 homozygous mutant displayed a stronger phenotype than that of the WT and heterozygous seedlings after gibberellic acid (GA) treatment. RNA-seq and iTRAQ-based proteome analyses were performed between the dmt1 mutant and WT. The transcriptome profile revealed that several genes involved in GA and strigolactone (SL) biosyntheses were altered in the dmt1 mutant. Ca2+ and other ion concentrations were significantly enhanced in the dmt1 mutant, suggesting that DMT1 contributes to the accumulation of several ions in rice. Moreover, several EF-hand Ca2+ sensors, including CMLs (CaM-like proteins) and CDPKs (calcium-dependent protein kinases), displayed markedly altered transcript expression and protein levels in the dmt1 mutant. Overall, these findings aid in the elucidation of the multiply regulatory roles of OsDMT1/OsMCA1 in rice.

Therapeutic Peptide Amphiphile as a Drug Carrier with ATP-Triggered Release for Synergistic Effect, Improved Therapeutic Index, and Penetration of 3D Cancer Cell Spheroids.

Despite the great progress in the field of drug delivery systems for cancer treatment over the last decade, many challenges still lie ahead, such as low drug loading, deep penetration of tumors, side effects, and the development of drug resistance. A class of cationic membrane lytic peptides has shown potential as an anticancer agent by inducing cancer cell death via membrane disruption; meanwhile, their intrinsic selectivity renders them as having low cytotoxicity towards noncancerous cells. Here, we report the use of a cationic peptide amphiphile (PA), named PAH6, to load doxorubicin (Dox) that is intercalated in an ATP-binding aptamer-incorporated DNA scaffold. The PA contains a cationic lytic sequence, (KLAKLAK)2, a polyhistidine segment for the "proton sponge" effect, and a hydrophobic alkyl tail to drive the self-assembly. Dox-loaded DNA was found to form a spherical nanocomplex (NC) with PAH6 with particle sizes below 100 nm at various ratios. Since the carrier PAH6 is also a therapeutic agent, the drug loadings of the NC reached up to ~86% within the ratios we tested, and Dox was released from the NC in an ATP-rich environment. In vitro studies indicate that the presence of PAH6 could permeabilize cell membranes and kill cells through fast membrane disruption and depolarization of mitochondrial membranes. The cytotoxicity tests were conducted using A549 nonsmall cell lung cancer cells and NIH-3T3 fibroblast cells. PAH6 showed selectivity towards A549 cells. Significantly, the Dox-DNA/PAH6 NC exhibited a synergistic effect against A549 cells, with the IC50 decreased up to ~90% for Dox and ~69% for PAH6 when compared to the IC50 values of the two components, respectively. Furthermore, the selectivity of PAH6 conferred to the complex an improved therapeutic index between A549 and NIH-3T3 cells. A 3D-cultured A549 spheroid model was adopted to test the capability of Dox-DNA/PAH6 for tumor penetration. The PAH6 or Dox-DNA/PAH6 complex was found to break the spheroids into pieces, while Dox-treated spheroids maintained their shapes. In summary, this work provides a new strategy for constructing nanomedicines using therapeutic agents to meet the features required by anticancer treatment.

Intranuclear assembly of leucine-rich peptides for selective death of osteosarcoma cells.

Herein, we show a pair of leucine-rich L- and D-phosphopeptides which self-assemble into twisting nanofibers, whose secondary structures contain a strong ß-sheet component after being dephosphorylated by alkaline phosphatase (ALP). While being incubated with ALP overexpressing osteosarcoma cells, both of the peptides self-assemble in the nuclei and induce cell death. The cell death involves multiple cell death modalities and occurs along with the disruption of cell membranes. Enzyme-instructed self-assembly (EISA) inhibits osteosarcoma cells and shows no side effect to other cells. In addition, the cancer cells hardly gain drug resistance after repeated treatment. This work reports a pair of EISA-based nanofibers to target cell nuclei, and also provides a novel chemotherapeutic agent to inhibit osteosarcoma cells without side effects and drug resistance.

Green transformation of CO2 into γ-amino alcohols with continuous stereocenters.

Here, we present a synthetic route towards γ-amino alcohols with continuous stereocenters based on a copper-catalyzed asymmetric conjugate addition/CO2-trapping tandem reaction of α,ß-unsaturated amide, followed by a reduction of the generated α-carboxyl amide. This strategy provides a green route for the transformation of CO2 into valuable chiral organic molecules.

From Lignin to Value-Added Pharmaceutical Intermediates Based on a Benzylic Oxidation Method with O2.

We present a catalytic strategy for converting lignin into various pharmaceutical intermediates based on a highly selective lignin depolymerization method and a green benzylic oxidation method employing O2. Selective depolymerization of lignin first afforded 4-ethylphenol, which then efficiently generates several pharmaceutical intermediates with a simple 5-step process, resulting in substantial economic benefits. The study provides an innovative solution for the efficient utilization of lignin and the green acquisition of pharmaceutical intermediates.

RIP5 Interacts with REL1 and Negatively Regulates Drought Tolerance in Rice.

Improving the drought resistance of rice is of great significance for expanding the planting area and improving the stable yield of rice. In our previous work, we found that ROLLED AND ERECT LEAF1 (REL1) protein promoted enhanced tolerance to drought stress by eliminating reactive oxygen species (ROS) levels and triggering the abscisic acid (ABA) response. However, the mechanism through which REL1 regulates drought tolerance by removing ROS is unclear. In this study, we identified REL1 interacting protein 5 (RIP5) and found that it directly combines with REL1 in the chloroplast. We found that RIP5 was strongly expressed in ZH11 under drought-stress conditions, and that the rip5-ko mutants significantly improved the tolerance of rice plants to drought, whereas overexpression of RIP5 resulted in greater susceptibility to drought. Further investigation suggested that RIP5 negatively regulated drought tolerance in rice by decreasing the content of ascorbic acid (AsA), thereby reducing ROS clearance. RNA sequencing showed that the knockout of RIP5 caused differential gene expression that is chiefly associated with ascorbate and aldarate metabolism. Furthermore, multiple experimental results suggest that REL1 is involved in regulating drought tolerance by inhibiting RIP5. Collectively, our findings reveal the importance of the inhibition of RIP5 by REL1 in affecting the rice's response to drought stress. This work not only explains the drought tolerance mechanism of rice, but will also help to improve the drought tolerance of rice.

Risk factors of lymph node metastasis in patients with T1 stage colorectal cancer-a retrospective cohort study based on the Surveillance, Epidemiology, and End Results database.

Background: Patients with T1 stage early colorectal cancer (CRC) can be treated with radical surgery or endoscopic surgery. Endoscopic surgery has a number of advantages, including minimal trauma and a rapid recovery. However, it cannot remove regional lymph nodes to assess whether there is lymph node metastasis. Thus, the analysis of the risk factors of lymph node metastasis in patients with T1 stage CRC is of great significance in the selection of appropriate treatment methods. Although previous studies have explored the risk factors for lymph node metastasis in T1 stage CRC patients, the number of cases were relatively insufficient, and further exploration is necessary. Methods: A total of 2,085 patients who had been pathologically diagnosed with CRC from 2015 to 2017 from the Surveillance, Epidemiology, and End Results (SEER) database. Among the patients, 324 had lymph node metastasis. A multivariate logistic regression analysis was conducted to analyze the risk factors of lymph node metastasis in patients with T1 stage CRC. Next, we established a prediction model to predict lymph node metastasis in patients with T1 stage CRC. Results: The results of the multivariate logistic regression analysis showed that age at diagnosis, rectosigmoid cancer, poorly differentiated or undifferentiated tumor cells, and distant metastasis were independent factors of lymph node metastasis in patients with T1 stage CRC (P<0.05). This study used the R4.0.3 statistical software for the statistical analysis. The data set was randomly divided into a training set and verification set. The training set comprised 1,460 patients, and the verification set comprised 625 patients. The area under the receiver operating characteristic curve (AUC) of the training set was 0.675 [95% confidence interval (CI): 0.635-0.714], and the AUC of the verification set was 0.682 (95% CI: 0.617-0.747). In the validation set, the model was tested by the Hosmer-Lemeshow Goodness-of-Fit Test (χ2=4.018, P=0.855), and the results showed that the model was reliable at predicting lymph node metastasis in patients with T1 stage CRC. Conclusions: For CRC patients with high risk factors of lymph node metastasis, endoscopic physicians should carefully evaluate the advantages and disadvantages of the endoscopic surgery before deciding whether to perform this surgery.