Squamous cell carcinoma initially occurring on the tongue dorsum: a case series report with molecular analysis.

BACKGROUND: Squamous cell carcinoma (SCC) of the dorsum of the tongue is extremely rare, and it clinically resembles various benign lesions. Somatic mutations in TP53 and some driver genes were implicated in the development of SCC; however, the somatic genetic characteristics of dorsal tongue SCC remain unknown. With a detailed analysis of gene mutations in dorsal tongue SCC, we aimed to better understand its biology. METHODS: Four cases of SCC initially occurring on the tongue dorsum were evaluated for clinical and histological findings and immunohistochemical expression of p53 and p16. Gene mutations were analyzed using next-generation sequencing with a custom panel of driver genes. RESULTS: We retrospectively investigated 557 cases of tongue SCC, and only four cases of SCC initially occurred on the tongue dorsum. The four patients (cases 1-4) were one woman and three men with a mean age of 53.75 years (range: 15-74 years). Histological analysis revealed well-differentiated SCC. Through molecular analysis, we identified pathogenic somatic mutations, namely, TP53 p.C176F (c.527G > T) in case 3 and TP53 p.R282W (c.844 C > T) in case 4. No pathogenic variants were identified in the PI3K/AKT or RAS/RAF pathways. The p53 immunohistochemical examination revealed a wild-type expression pattern in cases 1-3 and strong expression in case 4. The results of p16 immunostaining were negative in all cases. CONCLUSIONS: We described four previously unreported genetic characteristics of dorsal tongue SCC. Somatic TP53 mutations may contribute to the development of a subset of dorsal tongue SCC; however, more cases with genetic analysis need to be accumulated.

Establishment of repeated liver biopsy technique in experimental mice.

Biopsy is a commonly used method for determining pathological diagnoses by directly using human tissues and cells. Biopsies are widely used to determine disease progression and treatment efficacy. Although organs and tissues are usually obtained by sacrifice during animal experiments, it is theoretically possible to use the same biopsy techniques in humans. In the present study, we examined the feasibility of performing four repeated liver biopsies in a spontaneous metabolic syndrome mouse model. Even though a small number of mice died accidently, most mice were able to undergo four liver biopsies without significant adverse events. We also performed three liver biopsies in mouse liver tumor carcinogen models at 4, 8, and 12 weeks of age. In addition to the sample collected at 16 weeks of age during sacrifice, we successfully collected four liver samples from the same mice at different stages of disease progression. The application of this liver biopsy technique might make it possible for direct evaluation of pathological conditions in the same individual over time, thereby reducing the number of experimental animals.

Generation of equatorial plasma bubble after the 2022 Tonga volcanic eruption.

Equatorial plasma bubbles are a phenomenon of plasma density depletion with small-scale density irregularities, normally observed in the equatorial ionosphere. This phenomenon, which impacts satellite-based communications, was observed in the Asia-Pacific region after the largest-on-record January 15, 2022 eruption of the Tonga volcano. We used satellite and ground-based ionospheric observations to demonstrate that an air pressure wave triggered by the Tonga volcanic eruption could cause the emergence of an equatorial plasma bubble. The most prominent observation result shows a sudden increase of electron density and height of the ionosphere several ten minutes to hours before the initial arrival of the air pressure wave in the lower atmosphere. The propagation speed of ionospheric electron density variations was ~ 480-540 m/s, whose speed was higher than that of a Lamb wave (~315 m/s) in the troposphere. The electron density variations started larger in the Northern Hemisphere than in the Southern Hemisphere. The fast response of the ionosphere could be caused by an instantaneous transmission of the electric field to the magnetic conjugate ionosphere along the magnetic field lines. After the ionospheric perturbations, electron density depletion appeared in the equatorial and low-latitude ionosphere and extended at least up to ±25° in geomagnetic latitude.

Diagnostic accuracy of frozen section biopsy for early gastric cancer extent during endoscopic submucosal dissection: a prospective study.

BACKGROUND: Accurate diagnosis of the lateral extent of early gastric cancer during endoscopic submucosal dissection (ESD) is crucial to achieve negative resection margins. Similar to intraoperative consultation with a frozen section in surgery, rapid frozen section diagnosis with endoscopic forceps biopsy may be useful in assessing tumor margins during ESD. This study aimed to evaluate the diagnostic accuracy of frozen section biopsy. METHODS: We prospectively enrolled 32 patients undergoing ESD for early gastric cancer. Biopsy samples for the frozen sections were randomly collected from fresh resected ESD specimens before formalin fixation. Two different pathologists independently diagnosed 130 frozen sections as "neoplasia," "negative for neoplasia," or "indefinite for neoplasia," and the frozen section diagnosis was compared with the final pathological results of the ESD specimens. RESULTS: Among the 130 frozen sections, 35 were from cancerous areas, and 95 were from non-cancerous areas. The diagnostic accuracies of the frozen section biopsies by the two pathologists were 98.5 and 94.6%, respectively. Cohen's kappa coefficient of diagnoses by the two pathologists was 0.851 (95% confidence interval: 0.837-0.864). Incorrect diagnoses resulted from freezing artifacts, a small volume of tissue, inflammation, the presence of well-differentiated adenocarcinoma with mild nuclear atypia, and/or tissue damage during ESD. CONCLUSIONS: Pathological diagnosis of frozen section biopsy is reliable and can be applied as a rapid frozen section diagnosis for evaluating the lateral margins of early gastric cancer during ESD.

An analysis of sugary endosperm in sorghum: Characterization of mutant phenotypes depending on alleles of the corresponding starch debranching enzyme.

Sorghum is the fifth most important cereal crop. Here we performed molecular genetic analyses of the 'SUGARY FETERITA' (SUF) variety, which shows typical sugary endosperm traits (e.g., wrinkled seeds, accumulation of soluble sugars, and distorted starch). Positional mapping indicated that the corresponding gene was located on the long arm of chromosome 7. Within the candidate region of 3.4 Mb, a sorghum ortholog for maize Su1 (SbSu) encoding a starch debranching enzyme ISA1 was found. Sequencing analysis of SbSu in SUF uncovered nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region, containing substitutions of highly conserved amino acids. Complementation of the rice sugary-1 (osisa1) mutant line with the SbSu gene recovered the sugary endosperm phenotype. Additionally, analyzing mutants obtained from an EMS-induced mutant panel revealed novel alleles with phenotypes showing less severe wrinkles and higher Brix scores. These results suggested that SbSu was the corresponding gene for the sugary endosperm. Expression profiles of starch synthesis genes during the grain-filling stage demonstrated that a loss-of-function of SbSu affects the expression of most starch synthesis genes and revealed the fine-tuned gene regulation in the starch synthetic pathway in sorghum. Haplotype analysis using 187 diverse accessions from a sorghum panel revealed the haplotype of SUF showing severe phenotype had not been used among the landraces and modern varieties. Thus, weak alleles (showing sweet and less severe wrinkles), such as in the abovementioned EMS-induced mutants, are more valuable for grain sorghum breeding. Our study suggests that more moderate alleles (e.g. produced by genome editing) should be beneficial for improving grain sorghum.

Multiple orthokeratinized odontogenic cysts: clinical, pathological, and genetic characteristics.

BACKGROUND: Orthokeratinized odontogenic cyst (OOC) is a rare developmental odontogenic cyst of the jaw. It was originally believed to be a variant of odontogenic keratocyst (OKC) but is now considered to be a distinct entity. OOC usually presents as a single lesion and recurs infrequently. On the other hand, OKC often presents with multiple lesions and displays locally aggressive behavior and a high recurrence rate associated with the protein patched homolog 1 (PTCH1) gene mutation. Multiple OOC cases are extremely rare and seem to be aggressive, but their pathogenesis is not fully understood. This study aimed to determine the clinical, pathological, and genetic characteristics of multiple OCC. METHODS: Three cases of multiple OOC were evaluated for clinical and histological findings, and immunohistochemical expression of Ki-67 and Bcl-2. Furthermore, PTCH1 mutations were analyzed by next-generation sequencing using a custom panel to cover the entire exon of PTCH1. RESULTS: The three cases of multiple OOC included two men and one woman with a mean age of 25.3 years old (range, 18-38 years old). Each case had two or three OOCs (total of seven OOCs), all of which were simultaneously detected. Of the seven OOCs that manifested as multiple jaw cysts, seven (100%) occurred in the posterior regions, four (57.1%) occurred in the mandible, and four (57.1%) were associated with an impacted tooth. Histological examination revealed cysts lined by orthokeratinized stratified squamous epithelium. Immunohistochemistry showed a low Ki-67 labeling index and no Bcl-2 expression in the seven OOCs. No pathogenic PTCH1 mutations were detected in any of the seven OOCs. None of the patients had any other symptoms or signs of recurrence at the last follow-up (6-60 months). CONCLUSION: Multiple OOCs appeared to occur more often in younger patients than solitary OOC. Both multiple and solitary OOCs may be related diseases within the entity of odontogenic cysts. Multiple OOCs are clinicopathologically and genetically distinct from OKC.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

Secondary Adrenal Insufficiency in a Patient with Metastatic Melanoma Treated with Nivolumab.

We report a case of secondary adrenal insufficiency due to nivolumab. An 83-year-old man with acral lentiginous types of melanoma on the right sole visited our department in March 2017. He received primary surgery at referred hospital in June 2017, and pathological stage was IIIC (pT3bN3M0) according to AJCC (American Joint Committee on Cancer) 7th edition criteria. During the follow-up period, a lot of in-transit metastases appeared on the right leg. While we were resecting in-transit metastases, we concurrently started nivolumab in September 2018. After 17 cycles of nivolumab treatment, he developed severe nausea and anorexia. At baseline, his cortisol and adrenocorticotropic hormone levels were both at normal range, but corticotropin-releasing hormone loading test revealed secondary adrenal insufficiency. We diagnosed isolated adrenal insufficiency due to nivolumab. Treatment by hydrocortisone immediately relieved nausea and anorexia, and we could have continued treatment of nivolumab.

Assessment of Ultra-Early-Stage Liver Fibrosis in Human Non-Alcoholic Fatty Liver Disease by Second-Harmonic Generation Microscopy.

Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.

Kinetics of atrial and brain natriuretic peptides during hemodialysis are regulated in association with different cardiac functional changes.

The blood levels of atrial and brain natriuretic peptides (ANP and BNP) are both increased markedly in hemodialysis patients, but the kinetics of the two are not always parallel. The present study investigated the association of changes in ANP and BNP levels before and after dialysis with changes in cardiac function in hemodialysis patients. A total of 57 patients (mean age 64 years, 47 males and 10 females) on maintenance hemodialysis with sinus rhythm were enrolled. Blood samples were taken at the beginning and end of dialysis, and plasma levels of ANP and BNP were measured. Changes in cardiac function during dialysis were examined by echocardiography performed just before and after dialysis. Both plasma ANP and BNP concentrations decreased significantly after hemodialysis, but the rate of decrease in BNP [mean ± SD, 555 ± 503 to 519 ± 477 pg/mL (- 6.4%), P = 0.011] was much smaller than that in ANP [233 ± 123 to 132 ± 83 pg/mL (- 43.4%), P < 0.001]. As for the relation to the changes in echocardiographic parameters before and after dialysis, the decrease in inferior vena cava diameter had a close correlation with the decrease in ANP (r = 0.528, P < 0.001), but not BNP. In contrast, the decrease in left ventricular end-diastolic volume index was correlated only with the decrease in BNP (r = 0.297, P = 0.035). The peak velocity ratio of early diastolic to atrial filling decreased with preload reduction by dialysis, and its decrease was more strongly correlated with the decrease in BNP (r = 0.407, P = 0.002) than that in ANP (r = 0.273, P = 0.040). These results demonstrated that in hemodialysis patients, the decrease in plasma ANP by a single dialysis was essentially caused by blood volume reduction, while BNP decrease was mainly induced by the reduction of left ventricular overload. Our findings indicate that the kinetics of both peptides during dialysis are regulated by different cardiac and hemodynamic factors.

Catheter-based ultrasound renal denervation in patients with resistant hypertension: the randomized, controlled REQUIRE trial.

Renal denervation is a promising new non-pharmacological treatment for resistant hypertension. However, there is a lack of data from Asian patients. The REQUIRE trial investigated the blood pressure-lowering efficacy of renal denervation in treated patients with resistant hypertension from Japan and South Korea. Adults with resistant hypertension (seated office blood pressure ≥150/90 mmHg and 24-hour ambulatory systolic blood pressure ≥140 mmHg) with suitable renal artery anatomy were randomized to ultrasound renal denervation or a sham procedure. The primary endpoint was change from baseline in 24-hour ambulatory systolic blood pressure at 3 months. A total of 143 patients were included (72 renal denervation, 71 sham control). Reduction from baseline in 24-hour ambulatory systolic blood pressure at 3 months was not significantly different between the renal denervation (-6.6 mmHg) and sham control (-6.5 mmHg) groups (difference: -0.1, 95% confidence interval -5.5, 5.3; p = 0.971). Reductions from baseline in home and office systolic blood pressure (differences: -1.8 mmHg [p = 0.488] and -2.0 mmHg [p = 0.511], respectively), and medication load, did not differ significantly between the two groups. The procedure-/device-related major adverse events was not seen. This study did not show a significant difference in ambulatory blood pressure reductions between renal denervation and a sham procedure in treated patients with resistant hypertension. Although blood pressure reduction after renal denervation was similar to other sham-controlled studies, the sham group in this study showed much greater reduction. This unexpected blood pressure reduction in the sham control group highlights study design issues that will be addressed in a new trial. CLINICAL TRIAL REGISTRATION: NCT02918305 ( http://www.clinicaltrials.gov ).

Anti-proteinase 3 antineutrophil cytoplasmic antibody reflects disease activity and predicts the response to steroid therapy in ulcerative colitis.

BACKGROUND: Serum anti-proteinase 3 antineutrophil cytoplasmic antibody (PR3-ANCA) is a disease-specific antibody against granulomatosis with polyangiitis. PR3-ANCA is a useful serological marker for disease severity in ulcerative colitis (UC). The purpose of this study was to investigate whether PR3-ANCA levels could also predict the success of induction therapy and to compare its performance against other markers, including serum CRP and fecal hemoglobin. METHODS: This was a multicenter retrospective study. In total, 159 patients with active-phase UC underwent colonoscopy. Disease activity was measured using the Mayo endoscopic subscore (MES). PR3-ANCA positivity and the response to induction therapy, either 5-aminosalicylic acid or steroid, were assessed. PR3-ANCA, CRP, and fecal hemoglobin were measured during the active phase, and during clinical remission. RESULTS: Eighty-five (53.5%) of 159 patients with active UC were positive for PR3-ANCA. PR3-ANCA titers were significantly higher in the group of patients with MES 3 compared to patients with MES 1 (P = 0.002) or MES 2 (P = 0.035). Steroid therapy was administered to 56 patients with a median partial Mayo score of 7 (5-9), which is equivalent to moderate-to-severe disease activity. PR3-ANCA positivity of non-responders to steroid therapy was significantly higher than that of responders (71.9% vs, 41.7%, P = 0.030), whereas CRP and fecal hemoglobin were not predictive of steroid response. Multivariate analysis demonstrated that PR3-ANCA positivity was associated with non-response to steroid therapy (odds ratio 5.19; 95% confidence interval, 1.54-17.5; P = 0.008). Of the 37 patients treated to clinical remission who were also positive for PR3-ANCA during the active phase, 27 had an MES of ≥ 1, and 10 patients had an MES of 0. In clinical remission, the proportion of patients with MES 0 in 17 patients whose PR3-ANCA became negative was significantly higher than that in 20 patients whose PR3-ANCA remained positive (47.1% vs. 10.0%, P = 0.023). CONCLUSIONS: PR3-ANCA not only serves as a marker of disease activity, but also predicts the failure of steroid therapy in moderate-to-severe UC. TRIAL REGISTRATION: This study was retrospectively registered in the UMIN Clinical Trials Registry System (000039174) on January 16, 2020.

The dominance model for heterosis explains culm length genetics in a hybrid sorghum variety.

Heterosis helps increase the biomass of many crops; however, while models for its mechanisms have been proposed, it is not yet fully understood. Here, we use a QTL analysis of the progeny of a high-biomass sorghum F1 hybrid to examine heterosis. Five QTLs were identified for culm length and were explained using the dominance model. Five resultant homozygous dominant alleles were used to develop pyramided lines, which produced biomasses like the original F1 line. Cloning of one of the uncharacterised genes (Dw7a) revealed that it encoded a MYB transcription factor, that was not yet proactively used in modern breeding, suggesting that combining classic dw1or dw3, and new (dw7a) genes is an important breeding strategy. In conclusion, heterosis is explained in this situation by the dominance model and a combination of genes that balance the shortness and early flowering of the parents, to produce F1 seed yields.

Active auroral arc powered by accelerated electrons from very high altitudes.

Bright, discrete, thin auroral arcs are a typical form of auroras in nightside polar regions. Their light is produced by magnetospheric electrons, accelerated downward to obtain energies of several kilo electron volts by a quasi-static electric field. These electrons collide with and excite thermosphere atoms to higher energy states at altitude of ~ 100 km; relaxation from these states produces the auroral light. The electric potential accelerating the aurora-producing electrons has been reported to lie immediately above the ionosphere, at a few altitudes of thousand kilometres1. However, the highest altitude at which the precipitating electron is accelerated by the parallel potential drop is still unclear. Here, we show that active auroral arcs are powered by electrons accelerated at altitudes reaching greater than 30,000 km. We employ high-angular resolution electron observations achieved by the Arase satellite in the magnetosphere and optical observations of the aurora from a ground-based all-sky imager. Our observations of electron properties and dynamics resemble those of electron potential acceleration reported from low-altitude satellites except that the acceleration region is much higher than previously assumed. This shows that the dominant auroral acceleration region can extend far above a few thousand kilometres, well within the magnetospheric plasma proper, suggesting formation of the acceleration region by some unknown magnetospheric mechanisms.

6p21 translocation renal cell carcinoma: A case report.

6p21 translocation renal cell carcinoma (RCC) was newly classified in the WHO 2016 classification as a subtype of microphthalmia-associated transcription factor (MIT) family translocation RCC.A 42-year-old man was referred to our hospital with an asymptomatic solid mass in the right kidney identified during routine medical checkup. Computed tomography (CT) revealed a 14-mm buried-type solid mass accompanied by punctate calcification. CT-guided biopsy suggested clear-cell carcinoma. He underwent robotic-assisted partial nephrectomy. Pathological findings revealed 6p21 translocation RCC based on diffuse nuclear immunoreactivity for TFEB and TFEB gene rearrangement in tumor cells by FISH analysis.

Titanium oxide nano-radiosensitizers for hydrogen peroxide delivery into cancer cells.

Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (H2O2), which is released by PAA-TiOx NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiOx NPs with free H2O2 molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the H2O2 adsorbed onto the PAA-TiOx NPs during synthesis could be released during a treatment regimen. The H2O2 release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiOx NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiOx NP or H2O2 solution, the intracellular H2O2 levels remained higher with PAA-TiOx NP treatment compared with the H2O2 solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular H2O2 levels. These results indicate that PAA-TiOx NPs are efficient carriers of H2O2 into cancer cells and thus enhance the radiosensitivity.

Neuroendocrine carcinoma arising from Barrett's esophageal adenocarcinoma: a case report.

Neuroendocrine carcinoma in Barrett's esophagus is rare and its developmental mechanisms remain unclear. Neuroendocrine carcinoma arising in Barrett's esophagus with adenocarcinoma was detected at an early stage and resected by endoscopic submucosal dissection. Detailed pathological examination revealed that the neuroendocrine carcinoma originated via differentiation of the preexisting adenocarcinoma. A 79-year-old man presented with a flat protruding lesion in the esophagogastric junction. Esophagogastroduodenoscopy revealed a red flat 10-mm protruding lesion in the Barrett's epithelium and a shallow depression at the distal end. Narrow band imaging with magnification showed that the blood vessels in the protrusion were dilated and meandered irregularly, while those in the depression were small and did not form a network; the blood vessels were missing in some parts of the depression. Well-differentiated adenocarcinoma was diagnosed after analysis of the biopsy specimen of the protrusion, and endoscopic submucosal dissection was performed. The pathological diagnosis was neuroendocrine carcinoma with an adenocarcinoma component.

Histopathological analysis of autoimmune hepatitis with "acute" presentation: Differentiation from drug-induced liver injury.

AIM: Presently, no standardized definition or acceptable diagnostic criteria have been established for acute presentation of autoimmune hepatitis (AP-AIH), making it difficult to differentiate that condition from drug-induced liver injury (DILI). This study aimed to characterize clinical and histological features for distinguishing between AP-AIH and DILI. METHODS: Clinical, biochemical, and histological characteristics of AP-AIH and DILI in clinically well-characterized cases were compared in a standardized manner to clarify differences. RESULTS: In clinical evaluations, immunoglobulin G level and rate of anti-nuclear antibody positivity were greater in AP-AIH than DILI cases. As for diagnosis of each condition, significant (P < 0.01) differences were found for 10 features: lobular necrosis/inflammation, cobblestone appearance of hepatocytes, plasma cell infiltration in liver parenchyma, centrilobular fibrosis, hepatic rosette formation in areas with cobblestone appearance, portal inflammation, interface hepatitis, prominent plasma cells in portal areas, bile duct injury, and hepatic rosette formation in periportal areas. The area under the curve and cut-off values for the combination of these 10 features were 0.95 and 9 (sensitivity 86%, specificity 90%), respectively. CONCLUSION: Combinations of histological features were found to be helpful for differentiating AP-AIH from DILI, but we were not able to statistically identify an individual feature as definitive.