Saccharomycetaceae: delineation of fungal genera based on phylogenomic analyses, genomic relatedness indices and genomics-based synapomorphies.

A correct classification of fungi, including yeasts, is of prime importance to understand fungal biodiversity and to communicate about this diversity. Fungal genera are mainly defined based on phenotypic characteristics and the results of single or multigene-based phylogenetic analyses. However, because yeasts often have less phenotypic characters, their classification experienced a strong move towards DNA-based data, from short ribosomal sequences to multigene phylogenies and more recently to phylogenomics. Here, we explore the usefulness of various genomics-based parameters to circumscribe fungal genera more correctly taking the yeast domain as an example. Therefore, we compared the results of a phylogenomic analysis, average amino acid identity (AAI) values, the presence of conserved signature indels (CSIs), the percentage of conserved proteins (POCP) and the presence-absence patterns of orthologs (PAPO). These genome-based metrics were used to investigate their usefulness in demarcating 13 hitherto relatively well accepted genera in Saccharomycetaceae, namely Eremothecium, Grigorovia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Zygosaccharomyces and Zygotorulaspora. As a result, most of these genera are supported by the genomics-based metrics, but the genera Kazachstania, Nakaseomyces and Tetrapisispora were shown to be genetically highly diverse based on the above listed analyses. Considering the results obtained for the presently recognized genera, a range of 80-92 % POCP values and a range of 60-70 % AAI values might be valuable thresholds to discriminate genera in Saccharomycetaceae. Furthermore, the genus-specific genes identified in the PAPO analysis and the CSIs were found to be useful as synapomorphies to characterize and define genera in Saccharomycetaceae. Our results indicate that the combined monophyly-based phylogenomic analysis together with genomic relatedness indices and synapomorphies provide promising approaches to delineating yeast genera and likely those of filamentous fungi as well. The genera Kazachstania, Nakaseomyces and Tetrapisispora are revised and we propose eight new genera and 41 new combinations. Citation: Liu F, Hu Z-D, Yurkov A, et al. 2024. Saccharomycetaceae: delinaeation of fungal genera based on phylogenomic analyses, genomic relatedness indices and genomics-based synapomorphies. Persoonia 52: 1-21. https://doi.org/10.3767/persoonia.2024.52.01.

[Effect of preoperative immune checkpoint inhibitors on reducing residual lymph node metastases in patients with gastric cancer: a retrospective study].

Objective: To investigate the effect of immune checkpoint inhibitors on reducing residual lymph node metastasis in patients with gastric cancer. Methods: The cohort of this retrospective study comprised patients from Nanfang Hospital of Southern Medical University and the First Affiliated Hospital of Xiamen University who had undergone systemic treatment prior to gastrectomy with D2 lymphadenectomy and had achieved Grade 1 primary tumor regression (TRG1) from January 2014 to December 2023. After exclusion of patients who had undergone preoperative radiotherapy, data of 58 patients (Nanfang Hospital: 46; First Affiliated Hospital of Xiamen University: 12) were analyzed. These patients were allocated to preoperative chemotherapy (Chemotherapy group, N=36 cases) and preoperative immunotherapy plus chemotherapy groups (Immunotherapy group, N=22 cases). There were no significant differences between these groups in sex, age, body mass index, diabetes, tumor location, pathological type, Lauren classification, tumor differentiation, pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, mismatch repair protein status, number of preoperative treatment cycles, or duration of preoperative treatment (all P>0.05). The primary outcome measure was postoperative lymph node downstaging. Secondary outcomes included postoperative depth of invasion by tumor, number of lymph nodes examined, and factors affecting residual lymph node metastasis status. Results: Lymph node downstaging was achieved significantly more often in the Immunotherapy group than the Chemotherapy group (pN0: 90.9% [20/22] vs. 61.1% [22/36]; pN1: 4.5% [1/22] vs. 36.1% [13/36]; pN2: 4.5% [1/22) vs. 0; pN3: 0 vs. 2.8% [1/36], Z=-2.315, P=0.021). There were no significant difference between the two groups in number of lymph nodes examined (40.5±16.3 vs. 40.8±17.5, t=0.076, P=0.940) or postoperative depth of invasion by primary tumor (pT1a: 50.0% [11/22] vs. 30.6% [11/36]; pT1b: 13.6% [3/22] vs. 19.4% [7/36]; pT2: 13.6% [3/22] vs. 13.9% [5/36]; pT3: 13.6% [3/22] vs. 25.0% [9/36]; pT4a: 9.1% [2/22] vs. 11.1% [4/36], Z=-1.331, P=0.183). Univariate analysis revealed that both preoperative treatment regimens were associated with residual lymph node metastasis status in patients whose primary tumor regression was TRG1 (χ2=6.070, P=0.014). Multivariate analysis incorporated the following factors: pretreatment depth of invasion by primary tumor, pretreatment lymph node stage, pretreatment clinical stage, number of preoperative treatment cycles, and preoperative treatment duration. We found that a combination of immunotherapy and chemotherapy administered preoperatively was an independent protective factor for reducing residual lymph node metastases in study patients whose primary tumor regression was TRG1 (OR=0.147, 95%CI: 0.026-0.828, P=0.030). Conclusion: Compared with preoperative chemotherapy alone, a combination of preoperative immunotherapy and chemotherapy achieved greater reduction of residual lymph node metastases in the study patients who achieved TRG1 tumor regression in their primary lesions.

[Questionnaire survey of the application of four objective indicators necessary for standardized diagnosis of Sjögren's syndrome].

Objective: To investigate the current status and challenges of carrying out the four objective indicators which are necessary for the Sjögren's syndrome (SS) diagnosis in hospitals all over China. Methods: A questionnaire survey was conducted online by Questionstar from May to July 2023 among rheumatologists nationwide, to investigate whether unstimulated salivary flow (UWSF), Van Bijsterveld score (VBS), Schirmer test and labial gland focus score (FS) are carried out in their hospitals and the challenges that hinder their development. A cohort of patients with established SS was enrolled to verify the importance of the four objective indicators in diagnosing SS. Statistical analyses were performed using the chi-square test. Results: The questionnaire was completed by rheumatologists from 660 hospitals in 225 cities of 32 provinces, autonomous regions and municipalities all over China (one doctor from each hospital completed the questionnaire), of which 548 (83.0%) were tertiary care hospitals. The rate of carrying out the objective indicators in 660 hospitals was low: UWSF (290/660, 43.9%), FS (497/660, 75.3%) and VBS (393/660, 59.5%). The percentage of hospitals who consider it difficult to carry out UWSF, VBS, minor labial gland biopsy and Schirmer test was 92.6%(611/660), 69.4%(458/660), 59.8%(395/660) and 58.6%(387/660), respectively. All four objective indicators mentioned above could be carried out in only 139 (21.1%) hospitals. In 521 hospitals in which less than four objective indicators could be carried out, 23.2% (121/521) of rheumatologists selected clinical experience to diagnose SS. A total of 180 patients with SS diagnosed by perfecting all objective indices and meeting the 2016 the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria were included, 173 females (96%), aged (46.6±13.6) years, with the missed diagnosis rate was 17.8% (32/180) assuming their labial FS was unavailable. In 166 patients with established SS who met the classic 2002 AECG criteria, 160 females (96%), aged (47.0±13.6) years, the missed diagnosis rate was 52.4% (87/166) assuming their labial FS was unavailable; or 10.8% (18/166) assuming their UWSF was unavailable. SS diagnosis couldn't be estimated according to 2002 AECG criteria, assuming both labial FS and UWSF were unavailable in 156 (94.0%) patients with positive anti-SSA/Ro; or assuming either labial FS or UWSF was unavailable in 10 (6.0%) patients with negative anti-SSA/Ro. Conclusion: The application rates of four objective indicators necessary for SS diagnosis are low, the rate of carrying out labial gland biopsy should be increased, and the labial FS reports and UWSF test should be standardized.

[Clinical analysis of adverse reactions in patients with multidrug-resistant and rifampicin-resistant pulmonary tuberculosis treated with delamanid-containing regimen].

Objective: To explore the characteristics of adverse drug reactions during the 24-week therapy with delamanid-containing regimen for patients with multidrug-resistant and rifampicin-resistant pulmonary tuberculosis (MDR/RR-PTB). Methods: The prospective multicenter study was conducted from June 2020 to June 2023. A total of 608 eligible patients with MDR/RR-PTB were enrolled in 26 tuberculosis medical institutions in China including 364 males and 79 females, aged 39.6(19.0-68.0) years. Patients were treated with chemotherapy regimens containing delamanid. Patients were closely supervised during treatment of medication, and all adverse reactions occurring during treatment were monitored and recorded. The clinical characteristics of adverse reactions were evaluated by descriptive analysis. Chi-square test and multivariate logistic regression were used to analyze the related factors of QTcF interval prolongation (QT corrected with Fridericia's formula). Results: Of the 608 patients enrolled in this study, 325 patients (53.5%) reported 710 adverse events within 24 weeks of treatment. The top 6 most common complications were hematological abnormalities (143 patients, 23.5%), QT prolongation (114 patients, 18.8%), liver toxicity (85 patients, 14.0%), gastrointestinal reaction (41 patients, 6.7%), peripheral neuropathy (25 patients, 4.1%) and mental disorders (21 patients, 3.5%). The prolongation of QT interval mostly occurred in the 12th week after the first dose of medication. Serious adverse reactions occurred in 21 patients (3.5%). There were 7 patients (1.2%) with mental disorders, including 2 patients (0.3%) with severe mental disorders. Conclusions: The safety of dalamanid-based regimen in the staged treatment of MDR/RR-PTB patients was generally good, and the incidence of adverse reactions was similar to that reported in foreign studies. This study found that the incidence of QT interval prolongation in Chinese patients was higher than that reported overseas, suggesting that the monitoring of electrocardiogram should be strengthened when using drugs containing delamanid that may cause QT interval prolongation.

[Determination of two perfluorinated compounds in urine by liquid chromatography-tandem mass spectrometry].

Objective: To establish a method for the determination of two perfluorinated compounds in urine by liquid chromatography-tandem mass spectrometry. Methods: In November 2022, urine samples were extracted by acidic methanol, purified by WAX solid phase extraction column, and eluted with methanol water, then Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm) was used with 1.0 mmol/L ammonium acetate solution and methanol as mobile phase. The gradient elution was carried out, the detection was carried out by electrospray negative ion multiple response monitoring (MRM) mode, and the quantitative method was internal standard method. Results: Perfluorooctanoic acid and perfluorooctane sulfonic acid had a good linear relationship in the concentration range of 0.5-50.0 µg/L, and the correlation coefficient was >0.999. The limit of detection was 0.017 µg/L, and the limit of quantitation was 0.005 µg/L. The average recoveries were 96.3% and 101.8%, respectively. Days of precision were 3.5%-6.2% and 3.1%-7.4%, respectively, daytime precision were 4.3%-6.8% and 4.7%-8.1%, respectively. Conclusion: The established method of liquid chromatography-tandem mass spectrometry is high sensitivity and accuracy, and is suitable for the determination of perfluorooctanoic acid and perfluorooctane sulfonic acid in human urine.

[Diagnostic value of identifying location and amount of free gas in the abdominal cavity by multidetector computed tomography in patients with acute gastrointestinal perforation].

Objective: To evaluate the relationships between the location and extent of diffusion of free intraperitoneal air by multi-slice spiral CT (MSCT) and between the location and size of acute gastrointestinal perforation. Methods: This was a descriptive case series. We examined abdominal CT images of 33 patients who were treated for intraoperatively confirmed gastrointestinal perforation (excluding appendiceal perforation) in the Department of General Surgery, Nanfang Hospital between January and September 2022. We identified five locations of intraperitoneal air: the subphrenic space, hepatic portal space, mid-abdominal wall, mesenteric space, and pelvic cavity. We allocated the 33 patients to an upper gastrointestinal perforation (n=23) and lower gastrointestinal perforation group (n=10) base on intraoperative findings and analyzed the relationships between the locations of free gas and of gastrointestinal perforation. Additionally, we established two models for analyzing the extent of diffusion of free gas in the abdominal cavity and constructed receiver operating characteristic (ROC) curves to analyze the relationships between the two models and the size of the gastrointestinal perforation. Results: In the upper gastrointestinal perforation group, free gas was located around the hepatic portal area in 91.3% (21/23) of patients: this is a significantly greater proportion than that found in the lower gastrointestinal perforation group (5/10) (P=0.016). In contrast, free gas was located in the mesenteric interspace in 8/10 patients in the lower gastrointestinal perforation group; this is a significantly greater proportion than was found in the upper gastrointestinal perforation group (8.7%, 2/23) (P<0.010). The sensitivity of diagnosis of upper gastrointestinal perforation base on the presence of hepatic portal free gas was 84.8% and the specificity 71.4%. Further, the sensitivity of diagnosis of lower gastrointestinal perforation base on the presence of mesenteric interspace free gas was 80.0% and the specificity 91.3%. The rates of presence of free gas in the subdiaphragmatic area, mid-abdominal wall, and pelvic cavity did not differ significantly between the two groups (all P>0.05). Receiver operating characteristic curves showed that when free gas was present in four or more of the studied locations in the abdominal cavity, the optimal cutoff for perforation diameter was 2 cm, the corresponding sensitivity 66.7%, and the specificity 100%, suggesting that abdominal free gas diffuses extensively when the diameter of the perforation is >2 cm. Another model revealed that when free gas is present in three or more of the studied locations, the optimal cutoff for perforation diameter is 1 cm, corresponding to a sensitivity of 91.7% and specificity of 76.2%; suggesting that free gas is relatively confined in the abdominal cavity when the diameter of the perforation is <1 cm. Conclusion: Identifying which of five locations in the abdominal cavity contains free intraperitoneal air by examining MSCT images can be used to assist in the diagnosis of the location and size of acute gastrointestinal perforations.

[Determination of triclocarban and triclosan in urine by QuEChERS extraction and ultra-performance liquid chromatography-tandem mass spectrometry].

Objective: To establish a method for the determination of triclocarban (TCC) and triclosan (TCS) in urine by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after purification by QuEChERS. Methods: In May 2022, urine samples were extracted by acetonitrile, purified by QuEChERS, separated by Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm), and eluated with water-acetonitrile as mobile phase gradient at a flow rate of 0.3 ml/min. The detection was conducted in negative ion mode (ESI(-)) and multiple reaction monitoring (MRM) scanning, it was quantified with a internal standard method, and the methodology was verified. Results: The linear ranges of TCC and TCS were 0.5-100.0 µg/L and 1.0-100.0 µg/L, and the correlation coefficients were 0.9997 and 0.9991, respectively. The limits of detection and quantitation of TCC and TCS were 0.17 and 0.33 µg/L, and 0.5 and 1.0 µg/L, respectively. The recoveries of TCC and TCS were 100.1%-102.8% and 96.7%-108.6%, and the relative standard deviations were 4.9%-6.7% and 4.1%-8.3%, respectively, at 2.0, 10.0 and 80.0 µg/L. Conclusion: QuEChERS-UPLC-MS/MS method is simple, rapid, sensitive and reproducible, and can be used for rapid and accurate simultaneous detection of TCC and TCS exposure levels in occupational population.

[Application of intraoperative endoscopic in laparoscopic gastric cancer surgery].

The application of intraoperative endoscopic in laparoscopic gastric cancer surgery can compensate for the limitations of a single laparoscopic mode that only relies only on serous vision, and work as the Chinese saying "four ounces can move a thousand pounds". Intraoperative endoscopy not only effectively helps to accurately locate the tumor boundary in real-time and achieve precise resection, but also enables real time and interactive inspection of the quality of esophagojejunal anastomosis, reduces postoperative complications such as anastomotic leakage and bleeding, and accelerates postoperative recovery. However, most centers in China do not emphasize the application of intraoperative endoscopy in laparoscopic gastric cancer surgery due to the lack of a good cooperation mechanism between the gastrointestinal surgery team and the digestive endoscopy team. Therefore, based on clinical practice experience, the author briefly discusses the application of intraoperative endoscopic in routine laparoscopic gastric cancer surgery, including pre-examination preparation operator position, endoscopic techniques, postoperative management, etc., covering application scenarios such as early tumor localization, confirmation of upper esophageal margin confirmation, anastomotic examination, biopsy of primary lesion specimens in neoadjuvant/conversion treatment cases. We hope that it will help gastroenterologists to better apply intraoperative endoscopy in laparoscopic gastric cancer surgery, and assist in precise resection and safe anastomosis of the operation.

[Prediction of protection probability against Omicron BA.1, BA.4 and BA.5 variants in symptomatic infections with prototype strain based on neutralization antibody levels].

Objective: To predict the protection probability of different clinical outcomes after reinfection with Omicron variant in symptomatic and unvaccinated COVID-19 patients who infected with prototype strain. Methods: The data used in this study were derived from a systematic review and meta-analysis which systematically searched PubMed, Embase, Web of Science, and Europe PMC databases, included published and uploaded studies of dynamic changes of neutralizing antibodies in symptomatic COVID-19 patients from 1 January 2020 to 2 October 2022 and extracted the literature information, study design, serological experiment information and antibody results. According to the scatter distribution characteristics of antibody titer data, a generalized additive model based on Gaussian distribution was used to fit the titer value of neutralizing antibody based on logarithmic conversion and the dynamic change pattern of neutralizing antibody in symptomatic and unvaccinated COVID-19 patients infected with prototype strain over time was obtained. In this study, the fitted antibody titers of patients on the 28th, 51st, and 261st day after symptom onset was selected to predict the protection probability. Results: Neutralizing antibodies produced in symptomatic and unvaccinated patients infected with prototype strain could provide protection against Omicron reinfection, and the probability of protection gradually decreased with time. Neutralizing antibody level on day 28 after symptom onset provided protection probability of 30.3% (95%CI: 20.0%-45.5%) against reinfection, 51.5% (95%CI: 33.4%-75.9%) against symptomatic reinfection, and 91.2% (95%CI: 77.1%-97.7%) against severe reinfection caused by Omicron BA.5. The protection probability against Omicron BA.1, BA.4 and BA.5 reinfections decreased significantly 261 days after symptom onset, showing 9.6%-12.9%, 18.4%-23.9% and 63.1%-70.3% against three clinical outcomes, respectively. At the same time point and against the same clinical outcome, the protection probability of BA.1 was the highest, followed by BA.4 and BA.5. Conclusions: Neutralizing antibodies induced in symptomatic and unvaccinated COVID-19 patients previously infected with the prototype strain have limited protection probability against Omicron BA.5 reinfections and symptomatic reinfections. The protection probability against Omicron BA.5 reinfections is 30.3% 28 days after symptom onset and decreases to about 10% after 261 days. However, the protection probability against severe reinfections is considerable, with over 90% 28 days after symptom onset and still exceeding 60% after 261 days.

["Graded early warning system" of RET germline mutation carriers in MEN2A/MEN2B families and total thyroidectomy (report of 7 cases)].

Objective: To explore the reasonable time of prophylactic thyroidectomy for RET gene carriers in multiple endocrine neoplasia(MEN) 2A/2B families. Methods: From May 2015 to August 2021, RET gene carriers in MEN2A/MEN2B families were dynamically followed up at the Department of Thyroid Head and Neck Surgery, Beijing Tongren Hospital of Capital Medical University. The high-risk patients were encouraged to undergo prophylacitc total thyroidectomy according to the principle of "graded early warning system", namely the evaluation of gene detection, calcitonin value and ultrasound examination successively. Seven cases underwent the surgery, including 3 males and 4 females, aged from 7 to 29 years. According to the risk stratification listed in the guidelines of the American Thyroid Association in 2015, there were 2 cases of the highest risk, 2 cases of the high risk and 3 cases of the modest risk. Calcitonin index remained within the normal range in 3 cases and elevated in 4 cases before operation. All 7 patients underwent thyroidectomy with lymph node dissection of the level Ⅵ performed in 4 patients. Results: The time from suggestion to operation was 2 to 37 months, with an average of 15.1 months. The 6 patients were medullary thyroid carcinoma and 1 case with C-cell hyperplasia. The follow-up time was 2 to 82 months, with an average of 38.4 months. Postoperative serum calcitonin levels of all cases decreased to normal level, with biochemical cure. There was no sign of recurrence on ultrasound examination. All 7 patients had no serious complications, no obvious thyroid dysfunction. Their height, weight and other indicators of pediatric patients were similar to those of their peers, with normal growth and development. Conclusion: For healthy people with MEN2A/MEN2B family history, prophylactic thyroidectomy can be carried out selectively based on the comprehensive evaluation of "graded early warning system" with strict screening and close monitoring.

Intracranial plaque characteristics on high-resolution MRI and high-sensitivity C-reactive protein levels: association and clinical relevance in acute cerebral infarction.

AIM: To investigate the association between intracranial plaque characteristics and high-sensitivity C-reactive protein (hs-CRP) levels, and their combined effects on the occurrence of acute cerebral infarction (ACI). MATERIALS AND METHODS: One hundred and forty-three patients with recent ischaemic events in the territory of middle cerebral artery or basilar artery were enrolled and divided into the ACI group (n=93) and non-ACI group (n=50) according to clinical data and diffusion-weighting imaging (DWI) results. All recruited patients underwent high-resolution magnetic resonance imaging (MRI) to assess intracranial plaque characteristics, including plaque enhancement, standardised wall index, stenosis ratio, T1 hyperintense component, remodelling pattern, plaque area, plaque burden, and maximum wall thickness. hs-CRP levels were further grouped into the low group (<1 mg/l), the intermediate group (1-3 mg/l), and the high group (≥3 mg/l). Multivariate logistic regression and receiver operating characteristic curve were constructed to evaluate the association between intracranial plaque characteristics and hs-CRP levels, as well as their synergistic effects on determining the occurrence of ACI. RESULTS: High hs-CRP levels were associated with strong plaque enhancement (p<0.001, odds ratio [OR] = 7.497). Strong plaque enhancement (p=0.002, OR=2.109) and high hs-CRP levels (p=0.009, OR=3.893) were independently associated with the occurrence of ACI after adjustments for sex, age, and other traditional atherosclerotic risk factors. The combination of hs-CRP levels and strong plaque enhancement provided incremental information to determine ACI with an AUC of 0.823, which was significantly higher than that of strong plaque enhancement (0.711) and hs-CRP levels (0.686), respectively. CONCLUSION: High hs-CRP levels were associated with strong plaque enhancement. The synergistic effects of hs-CRP levels and strong plaque enhancement provided incremental effects on the occurrence of ACI.

Assessment of the degree of arterial stenosis in intracranial atherosclerosis using 3D high-resolution MRI: comparison with time-of-flight MRA, contrast-enhanced MRA, and DSA.

AIM: To compare the accuracy of three-dimensional (3D) high-resolution (HR) magnetic resonance imaging (MRI), time-of-flight magnetic resonance angiography (TOF-MRA), contrast-enhanced magnetic resonance angiography (CE-MRA), and digital subtraction angiography (DSA) in measuring the degree of stenosis in intracranial atherosclerosis. MATERIALS AND METHODS: All patients with intracranial artery ischaemic events underwent HR-MRI, TOF-MRA, and CE-MRA analysis, and some of these patients underwent DSA examination. The correlation between different methods for measuring the degree of lumen stenosis was analysed. The accuracy of HR-MRI, TOF-MRA, and CE-MRA was evaluated and compared with that of DSA. RESULTS: A total of 189 arterial stenoses were identified in 93 patients. Of these, 72 patients with 142 arterial stenoses underwent DSA examination. A very strong correlation between HR-MRI and CE-MRA measurements was shown (r=0.839, p<0.0001). The correlation between HR-MRI and TOF-MRA measurements was strong (r=0.720, p<0.0001). A very strong correlation between HR-MRI and DSA measurements was found (r=0.864, p<0.0001), and a similar correlation was observed between CE-MRA, and DSA measurements (r=0.843, p<0.0001). The correlation between TOF-MRA and DSA measurements was strong (r=0.686, p<0.0001). There was substantial agreement between HR-MRI and DSA measurements (K = 0.772) and between CE-MRA, and DSA measurements (K = 0.734) that was slightly higher than the agreement between TOF-MRA and DSA measurements (K = 0.636). CONCLUSION: HR-MRI can accurately measure stenosis (especially for moderate and severe stenosis) in intracranial atherosclerosis by direct visualisation of the vessel lumen and steno-occlusive plaque.