The relationship between probiotics and retinopathy of prematurity in preterm infants: A population-based retrospective study in China.

Background: Retinopathy of prematurity (ROP) is a retinal vascular disease with a high incidence in premature infants and is a leading cause of childhood blindness worldwide. The purpose of our study was to analyze the association between the use of probiotics and retinopathy of prematurity. Methods: This study retrospectively collected clinical data of premature infants with gestational age <32 weeks and birth weight <1500 g admitted to the neonatal intensive care unit from January 1, 2019 to December 31, 2021 in Suzhou Municipal Hospital, China. Demographic and clinical data of the inclusion population were collected. The outcome was the occurrence of ROP. The chi-square test was used to compare categorical variables, while the t-test and the nonparametric Mann-Whitney U rank-sum test were used for continuous variables. Univariate and multivariate logistic regression were used to analyze the relationship between probiotics and ROP. Results: A total of 443 preterm infants met the inclusion criteria, of which 264 didn't receive probiotics and 179 were supplemented with probiotics. There were 121 newborns with ROP in the included population. The results of univariate analysis showed that the preterm infants with and without probiotics were significantly different in the gestational age, the birth weight, the one-minute Apgar score, the oxygen inhalation time, the acceptance rate of invasive mechanical ventilation, the prevalence of bronchopulmonary dysplasia, ROP and severe intraventricular hemorrhage and periventricular leukomalacia (P < 0.05). Unadjusted univariate logistic regression model result showed that probiotics (OR 0.383, 95% CI 0.240∼0.611) were the factors affecting ROP in preterm infants (P < 0.01). Multivariate logistic regression result (OR 0.575, 95% CI 0.333∼0.994) was consistent with univariate analysis (P < 0.05). Conclusion: This study showed that probiotic was associated with a reduced risk of ROP in preterm infants with gestational age of <32 weeks and birth weight of <1500 g, but more large-scale prospective studies are still needed.

Coupling surfactants with ISCO for remediating of NAPLs: Recent progress and application challenges.

Non-aqueous phase liquids (NAPLs) pose a serious risk to the soil-groundwater environment. Coupling surfactants with in situ chemical oxidation (ISCO) technology is a promising strategy, which is attributed to the enhanced desorption and solubilization efficiency of NAPL contaminants. However, the complex interactions among surfactants, oxidation systems, and NAPL contaminants have not been fully revealed. This review provides a comprehensive overview on the development of surfactant-coupled ISCO technology focusing on the effects of surfactants on oxidation systems and NAPLs degradation behavior. Specifically, we discussed the compatibility between surfactants and oxidation systems, including the non-productive consumption of oxidants by surfactants, the role of surfactants in catalytic oxidation systems, and the loss of surfactants solubilization capacity during oxidation process. The effect of surfactants on the degradation behavior of NAPL contaminants is then thoroughly summarized in terms of degradation kinetics, byproducts and degradation mechanisms. This review demonstrates that it is crucial to minimize the negative effects of surfactants on NAPL contaminants oxidation process by fully understanding the interaction between surfactants and oxidation systems, which would promote the successful implementation of surfactant-coupled ISCO technology in remediation of NAPLs-contaminated sites.

The combined effect of Diversispora versiformis and sodium bentonite contributes on the colonization of Phragmites in cadmium-contaminated soil.

To promote the colonization of Phragmites in Cd polluted, nutrient deprived and structural damaged soil, the combined remediation using chemical and microbial modifiers were carried out in potting experiments. The co-application of Diversispora versiformis and sodium bentonite significantly improved the soil structure and phosphorus utilization of the plant, while decreasing the content of cadmium bound by diethylenetriaminepentaacetic acid by 77.72%. As a result, the Phragmites height, tillers, and photosynthetic capacity were increased by 71.60%, 38.37%, and 17.54%, respectively. Further analysis suggested the co-application increased the abundance of phosphorus-releasing microbial communities like Pseudomonassp. and Gemmatimonadetes. Results of rhizosphere metabolites also proved that the signal molecule of lysophosphatidylcholine regulated the phosphorus fixation and utilization by the plant. This work finds composite modifiers are effective in the colonization of Phragmites in Cd contaminated soil by decreasing the bioavailable Cd, increasing the abundance of functional microbial communities and regulating the phosphorus fixation.

Machine Learning-Based Multiparametric Magnetic Resonance Imaging Radiomic Model for Discrimination of Pathological Subtypes of Craniopharyngioma.

BACKGROUND: Preoperative, noninvasive discrimination of the craniopharyngioma subtypes is important because it influences the treatment strategy. PURPOSE: To develop a radiomic model based on multiparametric magnetic resonance imaging for noninvasive discrimination of pathological subtypes of craniopharyngioma. STUDY TYPE: Retrospective. POPULATION: A total of 164 patients from two medical centers were enrolled in this study. Patients from the first medical center were divided into a training cohort (N = 99) and an internal validation cohort (N = 33). Patients from the second medical center were used as the external independent validation cohort (N = 32). FIELD STRENGTH/SEQUENCE: Axial T1 -weighted (T1 -w), T2 -weighted (T2 -w), contrast-enhanced T1 -weighted (CET1 -w) on 3.0 T or 1.5 T magnetic resonance scanners. ASSESSMENT: Pathological subtypes (squamous papillary craniopharyngioma and adamantinomatous craniopharyngioma) were confirmed by surgery and hematoxylin and eosin staining. Optimal radiomic feature selection was performed by SelectKBest, the least absolute shrinkage and selection operator algorithm, and support vector machine (SVM) with a recursive feature elimination algorithm. Models based on each sequence or combinations of sequences were built using a SVM classifier and used to differentiate pathological subtypes of craniopharyngioma in the training cohort, internal validation, and external validation cohorts. STATISTICAL TESTS: The area under the receiver operating characteristic curve (AUC) was used to assess the diagnostic performance of the radiomic models. RESULTS: Seven texture features, three from T1 -w, two from T2 -w, and two from CET1 -w, were selected and used to construct the radiomic model. The AUC values of the radiomic model were 0.899, 0.810, and 0.920 in the training cohort, internal and external validation cohorts, respectively. The AUC values of the clinicoradiological model were 0.677, 0.655, and 0.671 in the training cohort, internal and external validation cohorts, respectively. DATA CONCLUSION: The model based on radiomic features from T1 -w, T2 -w, and CET1 -w has a high discriminatory ability for pathological subtypes of craniopharyngioma. LEVEL OF EVIDENCE: 4 TECHNICAL EFFICACY: 2.

An alternative splicing signature model for predicting hepatocellular carcinoma-specific survival.

BACKGROUND: Alternative splicing (AS) is a transcriptional regulation mechanism, which can expand the coding ability of genome and contribute to the occurrence and development of cancer. A systematic analysis of AS in hepatocellular carcinoma (HCC) is lacking and urgently needed. METHODS: Univariate and multivariate Cox regression analyses were used to distinguish survival-related AS events and to calculate the risk score. Kaplan-Meier analysis and receiver operating characteristic (ROC) curves were used to evaluate the AS events' clinical significance to build a risk model in HCC. RESULTS: Data of AS events was obtained from the Splice-Seq database. The corresponding clinical information of HCC was downloaded from The Cancer Genome Atlas (TCGA) data portal. We analyzed 78,878 AS events from 13,045 genes in HCC patients. A total of 2,440 and 2,888 AS events were significantly related to HCC patients' disease-free survival (DFS) and overall survival (OS). The two prognostic models (DFS and OS) were constructed based on a total of seven AS types from survival-related AS events above. The area under the curve (AUC) of the ROC curves was 0.769 in the DFS cohort and 0.886 in the OS cohort. CONCLUSIONS: The prognostic model constructed by AS events can be used to predict the prognosis of HCC patients and provide potential therapeutic targets for further validation.

The effects of biochar and dredged sediments on soil structure and fertility promote the growth, photosynthetic and rhizosphere microbial diversity of Phragmites communis (Cav.) Trin. ex Steud.

The improvement of urban river revetment soil is conducive to promote the growth of pioneer plants which can accelerate the restoration of ecosystems. How to effectively amend soil structure and composition to provide a suitable soil rhizosphere for rapid plant expansion is essential to be solved in the study. Biochar and lake dredged sediments were used to amend an urban river bank soil, where compaction and lack of mineral nutrition hindered the growth of Phragmites. The study found that the addition of 50% mass of dredged sediments combined with 5% mass of straw biochar increased the plant height maximum growth rate, tiller number per unit area, and root biomass by 32.93%, 29.62%, and 41.39%, respectively. The reason for these positive effects on plant growth mainly involved the improvement of rhizosphere soil properties. Addition of biochar increased porosity and available phosphorus content while dredged sediments increased soil organic carbon, thereby increasing the underground unit total phosphorus content of Phragmites by 18.18%. An increase of the Alpha diversity index of rhizosphere microorganisms (8.18%) and the decrease in infection rate of arbuscular mycorrhizal fungi (23.61%) also proved that the rapid expansion of Phragmites was improved owing to changes of the soil physicochemical properties. The combination of biochar and dredged sediments realized synergistic improvement of soil physical structure and increase of nutrient content, which helped promote the growth and expansion of the underground part of Phragmites. This cost-effective method can be feasible used for improvement of urban river revetment ecosystem.

Using non-ionic surfactant as an accelerator to increase extracellular lipid production by oleaginous yeast Cryptococcus curvatus MUCL 29819.

The aim of this work was to study the effects of non-ionic surfactant on the accumulation of total microbial lipids and extracellular lipid by Cryptococcus curvatus MUCL 29819 with acetic acid as carbon source. Compared with Brij 58 and Triton X-100, Brij 58 most increased the total lipids, with a yield up to 2.84 g/L (extracellular lipid up to 47%). Brij 58 also increased the metabolic flow of acetic acid to lipid accumulation (maximum conversion of 0.54 g/g at 1.0 g/L Brij 58) and limited its conversion to non-lipid biomass (minimum conversion 0.12 g/g at 0.5 g/L Brij 58). The improvement in the proportion of extracellular lipid by tea saponin and Brij 58 was due to changes in cell membrane permeability and improvement of cell membrane fluidity. Triton X-100, having weaker surface activity, promoted release of extracellular lipid and also increased the proportion of polyunsaturated fatty acid (C22:6, docosahexaenoic acid).

Early-life gut microbial colonization shapes Th1/Th2 balance in asthma model in BALB/c mice.

BACKGROUND: We aimed to investigate the effect of early-life diverse microbial exposures on gut microbial colonization in an OVA-induced asthma model in BALB/c mice. METHODS: BALB/c mice were divided into 4 groups: A, offsprings were kept in a SPF environment during fetal, lactation, and childhood periods; B, offsprings were kept in the SPF environment during fetal and lactation periods, and kept in the general environment during childhood; C, offsprings were kept in the SPF environment only during fetal period, and then kept in the general environment; and D, offsprings were kept in the general environment during whole periods. The diversity of intestinal flora was analyzed using denaturing gradient gel electrophoresis. Mice were sensitized with OVA to establish an animal model of asthma. Then asthma-related inflammatory cytokines and histological analysis were performed. RESULTS: The diversity of intestinal microflora in group D was significantly higher than groups A, B and C at three days and three weeks after birth, and the diversity of intestinal microflora in groups C and D were significantly higher than groups A and B at five weeks after birth. The pathologic scores of OVA-induced asthmatic mice in group D were significantly lower than group A, and serum IFN-γ levels and the IFN-γ/IL-4 ratio in group D were significantly higher than group A. CONCLUSIONS: Exposure to diverse microbial environments in early life affects gut microbial colonization in BALB/c mice. The diversity of the intestinal flora in early life may prevent airway inflammation in asthma via regulating the Th1/Th2 balance.

[Kinetic cluster and α-divergence-based dynamic myocardial factorial analysis of positron-emission computed tomography images].

OBJECTIVE: We purpose a novel factor analysis method based on kinetic cluster and α-divergence measure for extracting the blood input function and the time-activity curve of the regional tissue from dynamic myocardial positron emission computed tomography(PET) images. METHODS: Dynamic PET images were decomposed into initial factors and factor images by minimizing the α-divergence between the factor model and actual image data. The kinetic clustering as a priori constraint was then incorporated into the model to solve the nonuniqueness problem, and the tissue time-activity curves and the tissue space distributions with physiological significance were generated. RESULTS: The model was applied to the 82RbPET myocardial perfusion simulation data and compared with the traditional model-based least squares measure and the minimal spatial overlap constraint. The experimental results showed that the proposed model performed better than the traditional model in terms of both accuracy and sensitivity. CONCLUSION: This method can select the optimal measure by α value, and incorporate the prior information of the kinetic clustering of PET image pixels to obtain the accurate time-activity curves of the tissue, which has shown good performance in visual evaluation and quantitative evaluation.

Microbial conversion of mixed volatile fatty acids into microbial lipids by sequencing batch culture strategy.

Four mixed volatile fatty acids (VFAs) were used as sole carbon source to culture oleaginous yeast Cryptococcus curvatus by sequencing batch culture strategy. The highest lipid content (42.7%) and concentration (1.77g/L) were achieved when the ratio of VFAs (acetic, propionic, and butyric acids) was 6:3:1. The oleaginous yeast favored to use VFAs for lipid biosynthesis rather than cell proliferation. With regard to the utilization ratio of VFAs, acetic acid reached over 99%, whereas propionic acid was barely 35%. The produced lipids contained nearly 45% of monounsaturated fatty acids, which can be the ideal raw materials for biodiesel production. Additionally, the produced odd-numbered fatty acid content reached 23.6% when the propionate acid content of VFAs was 50%. Further analysis showed that increasing the ratio of acetic acid was most beneficial to cell mass and lipid production, whereas propionic acid and butyric acid were more conducive to lipid and cell mass synthesis, respectively.

Bioconversion of volatile fatty acids derived from waste activated sludge into lipids by Cryptococcus curvatus.

Pure volatile fatty acid (VFA) solution derived from waste activated sludge (WAS) was used to produce microbial lipids as culture medium in this study, which aimed to realize the resource recovery of WAS and provide low-cost feedstock for biodiesel production simultaneously. Cryptococcus curvatus was selected among three oleaginous yeast to produce lipids with VFAs derived from WAS. In batch cultivation, lipid contents increased from 10.2% to 16.8% when carbon to nitrogen ratio increased from about 3.5 to 165 after removal of ammonia nitrogen by struvite precipitation. The lipid content further increased to 39.6% and the biomass increased from 1.56g/L to 4.53g/L after cultivation for five cycles using sequencing batch culture (SBC) strategy. The lipids produced from WAS-derived VFA solution contained nearly 50% of monounsaturated fatty acids, including palmitic acid, heptadecanoic acid, ginkgolic acid, stearic acid, oleic acid, and linoleic acid, which showed the adequacy of biodiesel production.

Culture strategies for lipid production using acetic acid as sole carbon source by Rhodosporidium toruloides.

Rhodosporidium toruloides AS 2.1389 was tested using different concentrations of acetic acid as a low-cost carbon source for the production of microbial lipids, which are good raw materials for biodiesel production. It grew and had higher lipid contents in media containing 4-20 g/L acetic acid as the sole carbon source, compared with that in glucose-containing media under the same culture conditions. At acetic acid concentrations as high as 20 g/L and the optimal carbon-to-nitrogen ratio (C/N) of 200 in a batch culture, the highest biomass production was 4.35 g/L, with a lipid content of 48.2%. At acetic acid concentrations as low as 4 g/L, a sequencing batch culture (SBC) with a C/N of 100 increased biomass production to 4.21 g/L, with a lipid content of 38.6%. These results provide usable culture strategies for lipid production by R. toruloides AS 2.1389 when using diverse waste-derived volatile fatty acids.

[Identification of A Novel HLA Allele DRB1 * 16:36 by Sequencing-Based Typing].

OBJECTIVE: To identify a novel HLA allele DRB1 * 16:36 from a Uygur woman. METHODS: PCR-SBT technology was applied to the extracted DNA for genotyping, and a possible new gene was sequenced by using sequence specific primers and single stranded SBT. This novel allele was compared with known most homologous gene sequences and their difference was analyzed. RESULTS: This novel allele was different from HLA alle DRB1 * 16:23, and had highest similarity in 2 nucleotides at position 227 A→T and 236 T→C in exon 2, resulting in 3 amino acid changes from Tyr to Phe at codon 47 and Val to Ala at codon 50. The sequence of this novel allele had been submitted to GenBank. CONCLUSION: This HLA allele DRB1 * 16:23 has been confirmed to be a novel allele, and has been officially named DRB1 * 16:36 by the World Health Organization (WHO) Nomenclature Committee in May 2015.

[Reconstruction of dynamic positron emission tomographic images by exploiting low rank and sparse penalty].

OBJECTIVE: To propose a new method for dynamic positron emission tomographic (PET) image reconstruction using low rank and sparse penalty (L&S). METHODS: The L&S reconstruction model was established and the split Bregman method was used to solve the optimal cost function. The one-tissue compartment model was used to simulate a set of PET 82Rb myocardial perfusion image. The L&S reconstruction method was compared with maximum likelihood expectation maximization (MLEM) method, low-rank penalty method and sparse penalty method. RESULTS: The L&S reconstruction method had the smallest MSE and well maintained the feature information. The polar map created by L&S method was the most similar with the reference actual polar map. CONCLUSION: L&S reconstruction method is better than the other three methods in both visual and quantitative analysis of the PET images.

[Incorporation of TV regularization in deconvolution for partial volume correction in PET imaging].

OBJECTIVE: We propose a method using total variation (TV) regularization in deconvolution for partial volume correction in PET imaging. In the degraded image model, we used TV regularization procedure in Van Cittert (VC) and Richardson-Lucy (RL) deconvolution algorithms. These methods were tested in simulated NCAT images and images of NEMA NU4-2008 IQ phantom and tumor-bearing mouse scanned by Simens Invoen microPET. The simulated experiment and tumor-bearing mouse experiment showed that the algorithms using TV regularization provided superior qualitative and quantitative appearance compared with traditional VC and RL algorithms. When the mean intensity of the tumor increased by (10±1.8)%, the SD increase percentage was decreased from 49.98% to 14.26% and from 42.76% to 4.70%, suggesting the efficiency of the proposed algorithms for reducing PVEs in PET.

[Laser Raman spectra study on Co-Mo/Al2O3 hydrodesulphurization catalysts].

Due to the implementation of more stringent specifications in sulfur content for gasoline , a deep understanding of the active phase of Co-Mo/Al2O3 catalysts is necessary to the development of hydrodesulphurization (HDS) catalysts. A series of Co-Mo/Al2O3 HDS catalysts with different metal loading were studied by laser Raman spectra. The existence form and the content of the active component of the catalyst were obtained by Raman spectra. The result shows that the percentage of characteristic Raman bands 940 cm(-1) correlates linearly with the HDS selectivity, which can be used as an experimental evidence for developing industrial selective HDS catalysts. Raman spectra of sulfided catalysts show that the bands of oxidic catalysts at 839 and 940 cm(-1) disappeared, and simultaneously, the bands of Mo-S at 372 and 408 cm(-1) emerged, which indicate that the oxidic sample is sulfided completely.

[Extraction of surface active substance and analysis of demulsifying characteristics for the demulsifying strain Alcaligenes sp. S-XJ-1].

Extraction and identification of surface active substance of Alcaligenes sp. S-XJ-1, as well as description of its emulsion breaking process were conducted to reveal the demulsifying characteristics of this demulsifying strain. Alkali solvent was adopted in the extraction process with conditions optimized as 35 degrees C, 0.08 mol x L(-1) of alkali concentration, 12 g x L(-1) of sample to solution ratio, and 4 h of extraction time by launching both single-factor and orthogonal tests. Under this optimal condition, the extracted surface active substance (the extraction ratio was 36.1%) achieved 77% emulsion breaking ratio for 500 mg x L(-1) within 48 h. FT-IR showed the existence of glycolipids, lipids and proteins in the surface active substance, the molecular weight of which mainly scattered between 55 and 61 256. Saccharides, lipids and proteins were identified as the three chief components in surface active substance with the content of 22.2%, 7.5% and 13.4%, respectively. The proteins were further proved to take the most responsibility for the emulsion breaking ability. Moreover, obvious difference in the emulsion breaking process was demonstrated between the original demulsifying strain S-XJ-1 and the extracted surface active substance by real time observation of Turbiscan Lab Expert. The results suggested that the demulsifying efficiency of the strain was jointly contributed by its surface active substance and demulsifying cell morphology, and the former possessed higher functional priority than the latter.

[Influence of yeast extract on the fermentation of glucose by the demulsifying strain Alcaligenes sp. S-XJ-1].

The demulsifying strain Alcaligenes sp. S-XJ-1, isolated from oil contaminated soil, was cultivated with glucose as the carbon source. The influences of yeast extract on the growth, demulsifying ability and the element composition of the strain were investigated. The results showed that the yeast extract could increase the biomass and enhance the glucose utilization of Alcaligenes sp. S-XJ-1. When the concentration of the yeast extract was 5 g x L(-1), the biomass was increased up to 3.0 g x L(-1), and the glucose utilization achieved 58%. The demulsifying ability of the strain was improved with increasing yeast extract concentration. When the concentration of the yeast extract was 10 g x L(-1), the demulsification ratio of the obtained cell was 76%. While the C/N ratio of the cells decreased with the increasing concentration of yeast extract. The proteins of cells were extracted and measured. The results showed that the proteins of the obtained cell increased with the increasing concentration of yeast extract, in accordance with the increased concentrations of proteins on the surface of the cells as measured by FTIR. It is estimated that the increase of the proteins leads to the improvement of the demulsifying ability of the demulsifying strain and theses proteins play essential roles in the demulsifying process.

Pretreatment methods for aquatic plant biomass as carbon sources for potential use in treating eutrophic water in subsurface-flow constructed wetlands.

Plant biomass is usually added to constructed wetlands (CW) to enhance denitrification. In this study, we investigated effects of different pretreatments on two common external plant carbon sources, cattail and reed litter. We determined the average ratio of chemical oxygen demand (COD) to total nitrogen (TN), designated as C/N, in water samples after addition of litter subjected to various pretreatments. The C/N in the water samples ranged from 4.8 to 6.4 after addition of NaOH-pretreated cattail litter, which was four to six times greater than that of water from the Yapu River and 3.84-39.15% higher than that of systems that received untreated cattail litter. The C/N of systems that received H(2)SO(4)-pretreated carbon sources varied from 1.7 to 3.6. These two methods resulted in TN and total phosphorus (TP) levels lower than those in river water. The C/N was 1.4-1.7 after addition of CH(3)COOH-pretreated reed litter, which was 34.87-53.83% higher than that of river water. The C/N was 2.5 in systems that received mild alkali/oxidation-pretreated reeds, which was 30.59% higher than that of systems that received non-pretreated reeds. The residue rates of cattail and reed litter subjected to various pretreatments were greater than 60%. Our results showed that NaOH, H(2)SO(4), and mild alkali/oxidation pretreatments were useful to rapidly improve the C/N of river water and enhance denitrification.

[Vulvar intraepithelial neoplasia: a clinicopathologic study of twenty cases].

OBJECTIVE: To investigate the clinical, pathological and immunohistochemical features of vulvar intraepithelial neoplasia (VIN). METHODS: According to the 2004 modified terminology of International Society for the Study of Vulvovaginal Diseases (ISSVD), the cases were diagnosed as VIN from patients who had performed vulvar biopsy in Beijing Wuzhou Women's Hospital from February 2009 to December 2011, which were reclassified as usual VIN and differentiated VIN. The clinical and pathological studies were conducted respectively. MaxVision immunohistochemical staining was used to detect the expression of Ki-67, p16 and p53 proteins. RESULTS: There were 20 cases of VIN in 237 patients, and the incidence of VIN was 8.4% in all of contemporary vulvar biopsy. In 17 cases of usual VIN, mean age was 29.6 years, the lesion typically presented with atypical cells involving almost all layers of the epithelium, which was equivalent to the high-grade squamous intraepithelial neoplasia of cervix. Immunohistochemistry for Ki-67 and p16 was strongly positive in usual VIN. High risk human papillomavirus (HPV) detection was also positive. The incidence of differentiated VIN was less than usual VIN, and there were only 3 cases in this study. In differentiated VIN, patients aged over 50 years, with mean of 53.7 years, and the lesion most commonly presented with lichen sclerosis background. There were epithelial thickening and extending, and parakeratosis, and atypia was strictly confined to the basal and parabasal layers of the epithelium where the cells enlarged with abundant eosinophilic cytoplasm, presented with prominent nucleoli, increased cellularity and abnormal keratinization. In differentiated VIN, p53 was strongly positive, Ki-67 and p16 immunohistochemical expression was confined to the basal layer only. CONCLUSIONS: VIN is a precursor of invasive squamous cell carcinoma of the vulva. The modified terminology of ISSVD classifies VIN as high-grade lesions. Definitive pathological diagnosis of VIN plays an important role in its timely treatment and the prevention of vulvar carcinoma.