Potassium transporter OsHAK17 may contribute to saline-alkaline tolerant mechanisms in rice (Oryza sativa).

Rice production is seriously affected by saline-alkaline stress worldwide. To elucidate the saline-alkaline tolerance mechanisms in a novel tolerant rice variety, Shwe Nang Gyi (SNG), we investigated ion accumulation in SNG and Koshihikari (KSH), which is a saline-alkaline sensitive rice variety, and the candidates for saline-alkaline inducible genes in SNG using RNA-seq. SNG had superior ion accumulation capacity, such as K and Zn, compared to KSH. In contrast, SNG accumulated the same level of Na content in its leaf blades as KSH despite the higher dry weight of the SNG leaf blades. We further found that the expression of numerous genes, including several K+ transporter/high-affinity K+ transporter/K+ uptake protein/K+ transporter (HAK/KUP/KT) family members, were upregulated in SNG, and that OsHAK17 and OsHAK21 expression levels in the roots were significantly higher in SNG than in KSH. Moreover, yeast complementation analysis revealed that OsHAK17 was involved in K+ uptake under high-Na conditions. These results suggested that SNG has an effective K+ acquisition system supported by OsHAK17 functioning in saline-alkaline environments.

Role of Potassium-Dependent Alternative Splicing of MYB59 in the Maintenance of Potassium Concentration in Shoots of Arabidopsis thaliana.

Potassium (K) is a major plant nutrient. K+ is taken up by channel and transporter proteins in roots and translocated from roots to shoots via the xylem. In Arabidopsis thaliana, the K+ transporter NPF7.3 mediates K+ loading into the xylem and the transcription factor MYB59 is responsible for NPF7.3 expression. Here, we demonstrate that MYB59 is regulated by alternative splicing in response to K availability. Three splicing isoforms of MYB59 are detected in roots: an isoform with the first intron spliced out encodes a protein with the full DNA-binding motif (MYB59α), and two isoforms with the first intron retained partially or completely encode a protein missing part of the DNA-binding motif (MYB59ß). Functional analysis showed that only MYB59α is capable of inducing the expression of NPF7.3. The abundance of the MYB59α isoform increased under low K, but the total abundance of MYB59 transcripts did not change, indicating that MYB59α is increased by modification of the splicing pattern in response to low K. Although MYB59α is increased by low K, NPF7.3 expression remained constant independent of K. In addition, there was no significant difference in NPF7.3 expression between an MYB59 knockout mutant and the wild type under normal K. These results suggest that an unknown mechanism is involved in NPF7.3 expression under normal K and switches roles with MYB59 under low K. We propose that the regulation of MYB59 by alternative splicing is required for the maintenance of shoot K concentration in adaptation to low K.

Glucosinolate Catabolism Maintains Glucosinolate Profiles and Transport in Sulfur-Starved Arabidopsis.

Glucosinolates (GSLs) are sulfur (S)-rich specialized metabolites present in Brassicales order plants. Our previous study found that GSL can function as a S source in Arabidopsis seedlings via its catabolism catalyzed by two ß-glucosidases (BGLUs), BGLU28 and BGLU30. However, as GSL profiles in plants vary among growth stages and organs, the potential contribution of BGLU28/30-dependent GSL catabolism at the reproductive growth stage needs verification. Thus, in this study, we assessed growth, metabolic and transcriptional phenotypes of mature bglu28/30 double mutants grown under different S conditions. Our results showed that compared to wild-type plants grown under -S, mature bglu28/30 mutants displayed impaired growth and accumulated increased levels of GSL in their reproductive organs and rosette leaves of before-bolting plants. In contrast, the levels of primary S-containing metabolites, glutathione and cysteine decreased in their mature seeds. Furthermore, the transport of GSL from rosette leaves to the reproductive organs was stimulated in the bglu28/30 mutants under -S. Transcriptome analysis revealed that genes related to other biological processes, such as ethylene response, defense response and plant response to heat, responded differentially to -S in the bglu28/30 mutants. Altogether, these findings broadened our understanding of the roles of BGLU28/30-dependent GSL catabolism in plant adaptation to nutrient stress.

Creatinine reduction ratio on postoperative day 2 predicts long-term outcomes after living donor kidney transplantation.

OBJECTIVES: To evaluate the relationship between the creatinine reduction ratio between postoperative days 1 and 2 and post-transplantation clinical outcomes after living donor kidney transplantation. METHODS: Clinical data of patients who underwent living donor kidney transplantation at Jichi Medical University Hospital, Tochigi, Japan, between 2006 and 2019 were retrieved. The creatinine reduction ratio between postoperative days 1 and 2 was calculated based on the formula: (Cre1 - Cre2) × 100/Cre1; patients were then classified into either the slow graft function (creatinine reduction ratio between postoperative days 1 and 2 ≤30%) or immediate graft function (creatinine reduction ratio between postoperative days 1 and 2 >30%) group. We carried out the log-rank test and multivariate Cox proportional hazards regression analyses to assess graft survival and rejection-free survival, and the unpaired t-test and multivariate linear regression to assess post-transplantation estimated glomerular filtration rates. Multivariate analyses used age, sex, dialysis duration, ABO compatibility, donor-specific antibody positivity and medically complex living donors as explanatory variables. RESULTS: Of the 272 patients, 30 and 242 were in the slow graft function and immediate graft function groups, respectively. Multivariate Cox proportional hazards regression analyses showed a significantly higher incidence of overall and death-censored graft loss in the slow graft function group than the immediate graft function group. The frequency of rejection after 1 week post-transplantation did not differ within the groups. Post-transplantation estimated glomerular filtration rates tended to decline earlier in the slow graft function group than in the immediate graft function group; however, the difference was not statistically significant. CONCLUSIONS: The creatinine reduction ratio between postoperative days 1 and 2 could potentially predict long-term outcomes after living donor kidney transplantation. Using the creatinine reduction ratio between postoperative days 1 and 2 and other conventional indicators might allow accurate risk classification and appropriate therapeutic interventions.

Long-term impact of baseline serum uric acid levels on living kidney donors: a retrospective study.

BACKGROUND: Preoperative characteristics of living kidney donors are commonly considered during donor selection and postoperative follow-up. However, the impact of preoperative uric acid (UA) levels is poorly documented. The aim of this study was to evaluate the association between preoperative serum UA levels and post-donation long-term events and renal function. METHODS: This was a single-center retrospective analysis of 183 living kidney donors. The donors were divided into high (≥5.5 mg/dl) and low (< 5.5 mg/dl) UA groups. We analyzed the relationship between preoperative UA levels and postoperative estimated glomerular filtration rate (eGFR), as well as adverse events (cardiovascular events and additional prescriptions for hypertension, gout, dyslipidemia, and diabetes mellitus), over 5 years after donation. RESULTS: In total, 44 donors experienced 52 adverse events over 5 years. The incidence of adverse events within 5 years was significantly higher in the high UA group than in the low UA group (50% vs. 24%, p = 0.003); this was true even after the exclusion of hyperuricemia-related events (p = 0.047). UA emerged as an independent risk factor for adverse events (p = 0.012). Donors with higher UA levels had lower eGFRs after donation, whereas body mass index, hemoglobin A1c, blood pressure, and low-density lipoprotein cholesterol did not have any impact on the eGFR. CONCLUSIONS: The findings suggest that preoperative UA levels should be considered during donor selection and postoperative follow-up.

Roles of Non-Coding RNAs in Response to Nitrogen Availability in Plants.

Nitrogen (N) is an essential nutrient for plant growth and development; therefore, N deficiency is a major limiting factor in crop production. Plants have evolved mechanisms to cope with N deficiency, and the role of protein-coding genes in these mechanisms has been well studied. In the last decades, regulatory non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and long ncRNAs (lncRNAs), have emerged as important regulators of gene expression in diverse biological processes. Recent advances in technologies for transcriptome analysis have enabled identification of N-responsive ncRNAs on a genome-wide scale. Characterization of these ncRNAs is expected to improve our understanding of the gene regulatory mechanisms of N response. In this review, we highlight recent progress in identification and characterization of N-responsive ncRNAs in Arabidopsis thaliana and several other plant species including maize, rice, and Populus.

Genome-Wide Analysis of Long Intergenic Noncoding RNAs Responding to Low-Nutrient Conditions in Arabidopsis thaliana: Possible Involvement of Trans-Acting siRNA3 in Response to Low Nitrogen.

Long intergenic noncoding RNAs (lincRNAs) play critical roles in transcriptional and post-transcriptional regulation of gene expression in a wide variety of organisms. Thousands of lincRNAs have been identified in plant genomes, although their functions remain mostly uncharacterized. Here, we report a genome-wide survey of lincRNAs involved in the response to low-nutrient conditions in Arabidopsis thaliana. We used RNA sequencing data derived from A. thaliana roots exposed to low levels of 12 different nutrients. Using bioinformatics approaches, 60 differentially expressed lincRNAs were identified that were significantly upregulated or downregulated under deficiency of at least one nutrient. To clarify their roles in nutrient response, correlations of expression patterns between lincRNAs and reference genes were examined across the 13 conditions (12 low-nutrient conditions and control). This analysis allowed us to identify lincRNA-RNA pairs with highly positive or negative correlations. In addition, calculating interaction energies of those pairs showed lincRNAs that may act as regulatory interactors; e.g. small interfering RNAs (siRNAs). Among them, trans-acting siRNA3 (TAS3), which is known to promote lateral root development by producing siRNA against Auxin response factor 2, 3, and 4, was revealed as a nitrogen (N)-responsive lincRNA. Furthermore, nitrate transporter 2 was identified as a potential target of TAS3-derived siRNA, suggesting that TAS3 participates in multiple pathways by regulating N transport and root development under low-N conditions. This study provides the first resource for candidate lincRNAs involved in multiple nutrient responses in plants.

Factors related to suboptimal recovery of renal function after living donor nephrectomy: a retrospective study.

BACKGROUND: The renal function of the remaining kidney in living donors recovers up to 60~70% of pre-donation estimated-glomerular filtration rate (eGFR) by compensatory hypertrophy. However, the degree of this hypertrophy varies from donor to donor and the factors related to it are scarcely known. METHODS: We analyzed 103 living renal transplantations in our institution and divided them into two groups: compensatory hypertrophy group [optimal group, 1-year eGFR ≥60% of pre-donation, n = 63] and suboptimal compensatory hypertrophy group (suboptimal group, 1-year eGFR < 60% of pre-donation, n = 40). We retrospectively analyzed the factors related to suboptimal compensatory hypertrophy. RESULTS: Baseline eGFRs were the same in the two groups (optimal versus suboptimal: 82.0 ± 13.1 ml/min/1.73m2 versus 83.5 ± 14.8 ml/min/1.73m2, p = 0.588). Donor age (optimal versus suboptimal: 56.0 ± 10.4 years old versus 60.7 ± 8.7 years old, p = 0.018) and uric acid (optimal versus suboptimal: 4.8 ± 1.2 mg/dl versus 5.5 ± 1.3 mg/dl, p = 0.007) were significantly higher in the suboptimal group. The rate of pathological chronicity finding on 1-h biopsy (ah≧1 ∩ ct + ci≧1) was much higher in the suboptimal group (optimal versus suboptimal: 6.4% versus 25.0%, p = 0.007). After the multivariate analysis, the pathological chronicity finding [odds ratio (OR): 4.8, 95% confidence interval (CI): 1.3-17.8, p = 0.021] and uric acid (per 1.0 mg/dl, OR: 1.5, 95% CI: 1.1-2.2, p = 0.022) were found to be independent risk factors for suboptimal compensatory hypertrophy. CONCLUSION: Chronicity findings on baseline biopsy and higher uric acid were associated with insufficient recovery of the post-donated renal function.

Genome-wide analysis of specific alterations in transcript structure and accumulation caused by nutrient deficiencies in Arabidopsis thaliana.

The alteration of transcript structure contributes to transcriptome plasticity. In this study, we analyzed the genome-wide response of exon combination patterns to deficiencies in 12 different nutrients in Arabidopsis thaliana roots. RNA sequencing analysis and bioinformatics using a simulation survey revealed more than 600 genes showing varying exon combinations. The overlap between genes showing differential expression (DE) and genes showing differential exon combination (DC) was notably low. Additionally, gene ontology analysis showed that gene functions were not shared between the DE and DC genes, suggesting that the genes showing DC had different roles than those showing DE. Most of the DC genes were nutrient specific. For example, two homologs of the MYB transcription factor genes MYB48 and MYB59 showed differential alternative splicing only in response to low levels of potassium. Alternative splicing of those MYB genes modulated DNA-binding motifs, and MYB59 is reportedly involved in the inhibition of root elongation. Therefore, the increased abundance of MYB isoforms with an intact DNA-binding motif under low potassium may be involved in the active inhibition of root elongation. Overall, we provide global and comprehensive data for DC genes affected by nutritional deficiencies, which contribute to elucidating an unknown mechanism involved in adaptation to nutrient deficiency.

[A Case of Foramen Magnum Meningioma Manifesting as Hypoglossal Nerve Palsy].

We report a case of foramen magnum meningioma manifesting as hypoglossal nerve palsy. A 72-year-old woman presented with progressive hypoglossal nerve palsy and lingual atrophy on the left side. Gadolinium-enhanced T1-weighted magnetic resonance imaging revealed a heterogeneously enhanced mass lesion with dural tail sign partially extending into the hypoglossal canal. The transcondylar approach was performed to expose the hypoglossal canal and resect the tumor completely. Histological examination revealed a transitional meningioma. The postoperative course was uneventful. Hypoglossal nerve palsy improved gradually after the operation.

[Primary Intracranial Malignant Lymphoma Associated with Acquired Immunodeficiency Syndrome(AIDS):A Case Report].

The spread of human immunodeficiency virus(HIV)infection may result in an increased likelihood of surgery in patients with HIV infection. We treated a patient with intracranial malignant lymphoma associated with acquired immunodeficiency syndrome(AIDS)caused by HIV infection. The recommendations of the countermeasure manual for AIDS were followed. Only surgical staff without finger injury or inflammation were permitted to be involved in the operation. All staff were dressed in a waterproof, full-body surgical gown, and wore double gloves, double foot covers, and an N95 mask. The surgery could be performed safely with such infection control measures. Histological examination revealed a diffuse large B-cell lymphoma. The patient was referred to the Division of Infectious Diseases and Respiratory Medicine for chemotherapy.

Two distinct families of protein kinases are required for plant growth under high external Mg2+ concentrations in Arabidopsis.

Protein phosphorylation events play key roles in maintaining cellular ion homeostasis in higher plants, and the regulatory roles of these events in Na(+) and K(+) transport have been studied extensively. However, the regulatory mechanisms governing Mg(2+) transport and homeostasis in higher plants remain poorly understood, despite the vital roles of Mg(2+) in cellular function. A member of subclass III sucrose nonfermenting-1-related protein kinase2 (SnRK2), SRK2D/SnRK2.2, functions as a key positive regulator of abscisic acid (ABA)-mediated signaling in response to water deficit stresses in Arabidopsis (Arabidopsis thaliana). Here, we used immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry analyses to identify Calcineurin B-like-interacting protein kinase26 (CIPK26) as a novel protein that physically interacts with SRK2D. In addition to CIPK26, three additional CIPKs (CIPK3, CIPK9, and CIPK23) can physically interact with SRK2D in planta. The srk2d/e/i triple mutant lacking all three members of subclass III SnRK2 and the cipk26/3/9/23 quadruple mutant lacking CIPK26, CIPK3, CIPK9, and CIPK23 showed reduced shoot growth under high external Mg(2+) concentrations. Similarly, several ABA biosynthesis-deficient mutants, including aba2-1, were susceptible to high external Mg(2+) concentrations. Taken together, our findings provided genetic evidence that SRK2D/E/I and CIPK26/3/9/23 are required for plant growth under high external Mg(2+) concentrations in Arabidopsis. Furthermore, we showed that ABA, a key molecule in water deficit stress signaling, also serves as a signaling molecule in plant growth under high external Mg(2+) concentrations. These results suggested that SRK2D/E/I- and CIPK26/3/9/23-mediated phosphorylation signaling pathways maintain cellular Mg(2+) homeostasis.

Induction of Nickel Accumulation in Response to Zinc Deficiency in Arabidopsis thaliana.

Excessive accumulation of nickel (Ni) can be toxic to plants. In Arabidopsis thaliana, the Fe²âº transporter, iron (Fe)-regulated transporter1 (IRT1), mediates Fe uptake and also implicates in Ni²âº uptake at roots; however, the underlying mechanism of Ni²âº uptake and accumulation remains unelucidated. In the present study, we found that zinc (Zn) deficient conditions resulted in increased accumulation of Ni in plants, particularly in roots, in A. thaliana. In order to elucidate the underlying mechanisms of Ni uptake correlating zinc condition, we traced 63Ni isotope in response to Zn and found that (i) Zn deficiency induces short-term Ni²âº absorption and (ii) Zn²âº inhibits Ni²âº uptake, suggesting competitive uptake between Ni and Zn. Furthermore, the Zrt/Irt-like protein 3 (ZIP3)-defective mutant with an elevated Zn-deficient response exhibited higher Ni accumulation than the wild type, further supporting that the response to Zn deficiency induces Ni accumulation. Previously, expression profile study demonstrated that IRT1 expression is not inducible by Zn deficiency. In the present study, we found increased Ni accumulation in IRT1-null mutant under Zn deficiency in agar culture. These suggest that Zn deficiency induces Ni accumulation in an IRT1-independen manner. The present study revealed that Ni accumulation is inducible in response to Zn deficiency, which may be attributable to a Zn uptake transporter induced by Zn deficiency.

The effect of fertilization on cesium concentration of rice grown in a paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 nuclear power plant in March 2011, radioactive cesium was released and paddy field in a wide area of Fukushima Prefecture was contaminated. To reduce radioactive Cs uptake by rice, it is important to understand factors that affect Cs uptake in rice. Here we describe our study in 2011 and 2012 to investigate Cs concentration in two rice cultivars, Koshihikari and Hitomebore, the top two cultivars in Fukushima prefecture, grown under different fertilizer conditions in the contaminated paddy field. Our study demonstrated that high nitrogen and low potassium conditions increase Cs concentrations both in straw and brown rice.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.

[Usefulness of extradural optic canal unroofing and decompression of the optic nerve for improvement of visual acuity in traumatic optic neuropathy].

An 81-year-old man presented with poor visual acuity of the left eye, swelling of the left eyelid, and elevation of the left intraocular pressure after contusion of the left palpebral portion. CT revealed left ocular proptosis and left intraorbital hematoma. Traumatic optic neuropathy was suspected, and emergent optic nerve decompression was performed through extradural anterior clinoidectomy followed by optic canal release. Postoperatively, his left visual acuity was markedly improved and the elevated intraocular pressure decreased. Postoperative CT demonstrated improvement of the left ocular proptosis and decompression of the optic nerve. Emergent optic canal release has been recommended in patients who have suffered visual dysfunction caused by optic canal fracture or intraorbital hematoma. The advantages of extradural anterior clinoidectomy followed by optic canal release include a shorter surgical route and easy identification of the optic nerve, resulting in fewer surgical complications. In addition, this procedure can achieve intraorbital decompression. We recommend extradural anterior clinoidectomy followed by optic canal release as a safe and reliable procedure for optic nerve decompression in patients with traumatic optic neuropathy.

[A case of primary intraosseous cavernous hemangioma extending from the orbital rim to the sphenoid wing: a case report].

A primary intraosseous cavernous hemangioma located at the sphenoid bone with extensive involvement of the orbital roof and the lateral wall of the orbit is very rare. A 48-year-old woman presented with progressive right exophthalmos and diplopia. CT showed a bony mass lesion in the right sphenoid bone extending to the orbital bone. MRI showed an abnormal lesion in the sphenoid bone, which was heterogeneously enhanced with gadolinium. All of the abnormal bone was surgically removed, and histological examination confirmed a cavernous angioma. We also present a brief clinical and radiological review of seven previously reported cases.

Metabolomic Profiling of Mice with Tacrolimus-Induced Nephrotoxicity: Carnitine Deficiency in Renal Tissue.

Tacrolimus (TAC)-induced chronic nephrotoxicity (TAC nephrotoxicity) has a detrimental effect on long-term kidney graft survival. However, the pathogenesis of TAC nephrotoxicity remains largely unknown. We explored it by focusing on metabolic changes in renal tissues. In this study, mice were separated into TAC and control groups (n = 5/group). TAC was administered to the TAC group (1 mg/kg/d for 28 days) subcutaneously. The control group was similarly treated with normal saline. Renal tissue metabolomes were evaluated. Renal fibrosis was observed only in the TAC group. Metabolomic analysis showed that carnitine and related metabolites were substantially lower in the TAC group than in the control group, presumably due to impaired biosynthesis and reabsorption. Low carnitine levels impair antioxidation in renal tissues and ß-oxidation in mitochondria, which may lead to renal tissue damage. This metabolomic analysis revealed that carnitine deficiency in renal tissue appears to explain TAC nephrotoxicity.

Epigenetic regulation of organic anion transporting polypeptide 1B3 in cancer cell lines.

PURPOSE: The expression of a multispecific organic anion transporter, OATP1B3/SLCO1B3, is associated with clinical prognosis and survival of cancer cells. The aims of present study were to investigate the involvement of epigenetic regulation in mRNA expression of a cancer-type variant of OATP1B3 (Ct-OATP1B3) in cancer cell lines. METHODS: The membrane localization and transport functions of Ct-OATP1B3 were investigated in HEK293 cells transiently expressing Ct-OATP1B3. DNA methylation profiles around the transcriptional start site of Ct-OATP1B3 in cancer cell lines were determined. The effects of a DNA methyltransferase inhibitor and siRNA knockdown of methyl-DNA binding proteins (MBDs) on the expression of Ct-OATP1B3 mRNA were investigated. RESULTS: 5'-RACE identified the TSS of Ct-OATP1B3 in PK-8 cells. Ct-OATP1B3 was localized on the plasma membrane, and showed the transport activities of E217ßG, fluvastatin, rifampicin, and Gd-EOB-DTPA. The CpG dinucleotides were hypomethylated in Ct-OATP1B3-positive cell lines (DLD-1, TFK-1, PK-8, and PK-45P) but were hypermethylated in Ct-OATP1B3-negative cell lines (HepG2 and Caco-2). Treatment with a DNA methyltransferase inhibitor and siRNA knockdown of MBD2 significantly increased the expression of Ct-OATP1B3 mRNA in HepG2 and Caco-2. CONCLUSIONS: Ct-OATP1B3 is capable of transporting its substrates into cancer cells. Its mRNA expression is regulated by DNA methylation-dependent gene silencing involving MBD2.

Retroperitoneoscopic Donor Nephrectomy on the Right Side Provides Equivalent Outcomes and Donor Safety to That on the Left Side.

BACKGROUND: The left kidney is typically selected for laparoscopic donor nephrectomy. In contrast, right kidney donation raises concerns for donor safety, and venous anastomosis may be difficult to achieve due to the short renal vein. We investigated the safety and operative outcomes of right donor nephrectomy compared with those of the left. METHODS: We retrospectively analyzed the clinical records of living donor-kidney transplant donors and evaluated operative outcomes such as operative time, ischemic time, blood loss, and surgical complications in the donor. RESULTS: We identified 79 donors (left:right = 62:17 cases) between May 2020 and March 2023. There were no significant differences between the 2 groups regarding age, sex, body mass index, and number of renal arteries. Although the operative time (left and right: 190 and 225 minutes, excluding waiting time; P = .009) and warm ischemic time (left and right: 143 and 193 seconds, P = .021) were significantly longer on the right side, the total ischemic time (82 and 86 minutes, P = .463) and blood loss (left and right: 35 and 25 mL, P = .159) were comparable between the groups. There were no significant differences between the groups regarding the surgical complications. CONCLUSIONS: Operative outcomes were similar in both donor sides in the retroperitoneoscopic donor nephrectomies. The right side should be considered for donation in this operative procedure.