Direct quantification of optic nerve blood flow by 3D pseudo-continuous arterial spin labeling.

BACKGROUND: Blood perfusion of the optic nerve (ON) plays a key role in many optic neuropathies. Microvascular changes precede or accompany neuronal changes, and detecting these changes at an early stage may facilitate early treatment to avoid blindness. However, the quantification of ON blood perfusion remains a challenge. This study aimed to evaluate the viability of three-dimensional pseudocontinuous arterial spin labelling (3D-pCASL) MRI for the quantification of ON blood flow (BF). NEW METHOD: The ON segmentation was performed using nnFormer on a cohort of ten participants (4 males, 6 females, 25-59 years old). Subsequently, the mean BF of each ON segment was calculated using whole brain 3D-pCASL image data. RESULTS: The average ON-BF values of the left and right intraorbital segments, left and right intracanalicular segments, left and right intracranial segments, optic chiasma, and left and right optic tract were 41.308 mL/100 g/min, 43.281 mL/100 g/min, 53.188 mL/100 g/min, 57.202 mL/100 g/min, 45.089 mL/100 g/min, 49.554 mL/100 g/min, 42. 326 mL/100 g/min, 43.831 mL/100 g/min and 45.176 mL/100 g/min, respectively. The ON-BF correlated with cerebral BF (r = 0.503, p = 0.024). COMPARISON WITH EXISTING METHOD(S): The 3D-pCASL can measure tissue microvascular blood perfusion in absolute quantitative units with good test-retest repeatability over a wide field of view and without restrictions on depth. The use of the nnFormer makes the measurement easy, objective and reproducible. CONCLUSIONS: The study showed that, 3D-pCASL may be a promising tool for detecting abnormal ON-BF. In particular, 3D-pCASL coupled with the nnFormer provides an objective, reproducible, and reliable method to quantify BF in ON.

Biomechanical analysis of fixation strength at different nailing angles for femoral neck fracture with insufficient reduction.

To analyze the fixation strength of cannulated screws fixation in the treatment of femoral neck fracture with posterior tilt due to insufficient reduction. Two sets of digital models of anatomical reduction and 15° tilting reduction were established by CT data. Each group of models was modeled with two different fixation methods. One fixation method was fixed according to the standard cannulated screws recommended by AO. Another fixation method is to tilt the screw posterior tilt 15°. The final four groups of models were obtained: AO principle nailing posterior tilt model (Group A), posterior direction nailing posterior tilt model (Group B), AO principle nailing anatomic reduction model (Group C) and posterior direction nailing anatomic reduction model (Group D). The maximum displacement of the fracture end, the maximum Von-Mises stress and the stress distribution of the internal fixation were compared among the four groups. Four groups of models were established on artificial bone by 3D printing guide plate technology. The 600 N pressure test and yield test were performed on a biomechanical machine. The finite element and biomechanical models showed that groups B and C were more stable than groups A and D. The stability of group B was not worse than that of group C. When the femoral neck fracture produces a posterior tilt, a posterior reduction is allowed. The change of AO screw to posterior tilting screw fixation has more powerful advantages. No posterior tilt or posterior reduction, AO screw placement is still required.

Predictors for post-traumatic hip osteoarthritis in patients with transverse acetabular fractures following open reduction internal fixation: a minimum of 2 years' follow-up multicenter study.

BACKGROUND: The predictors of post-traumatic osteoarthritis (PTOA) in patients with transverse acetabular fractures (TAFs) following open reduction internal fixation (ORIF) remain unclear. This study aimed to investigate the risk factors for PTOA in TAFs after ORIF. METHODS: Data of TAF patients receiving ORIF were collected from January 2012 and February 2021. Patients suffered PTOA were classified as the osteoarthritis group (OG), while those without PTOA were classified as the non- osteoarthritis group (NG) with a minimum follow-up of 2 years. PTOA was diagnosed according to Tönnis OA classification during the period of follow-up. Univariate analysis, logistic regression analysis, and receiver operating characteristic (ROC) curve analyses were used to evaluate demographics, injury-related characteristics, perioperative and post-discharge information. RESULTS: Three hundred and eleven TAF patients were analyzed in this study, including 261 males and 50 females, with a mean age of 40.4 years (range 18 to 64 years). The incidence of PTOA was 29.6% (92 of 311) during the mean follow-up of 36.8 months (range 24 to 70 months). Several factors of PTOA were found using univariate analysis, including transverse fracture associated with posterior wall acetabular fracture (TPW-AF, p = 0.002), acetabular roof fracture (ARF, p = 0.001), femoral head lesion (FHL, p = 0.016), longer time from injury to surgery (TIS, p＜0.001) and physical work after surgery (PWAS, p＜0.001). Logistic regression analysis showed that TPW-AF (p = 0.007, OR = 2.610, 95%CI: 1.302-5.232), ARF (p = 0.001, OR = 2.887, 95%CI: 1.512-5.512), FHL (p = 0.005, OR = 2.302, 95%CI: 1.283-4.131), TIS (p<0.0001, OR = 1.294, 95%CI: 1.192-1.405) and PWAS (p<0.0001, 3.198, 95%CI: 1.765-5.797) were independent risk factors of PTOA. Furthermore, ROC curve analysis indicated 11.5 days as the cut-off values to predict PTOA. CONCLUSIONS: Our findings identified that TPW-AF, ARF, FHL, TIS and PWAS were independent risk factors for PTOA in patients with TAFs following ORIF. It can help orthopedic surgeons to take early individualized interventions to reduce its incidence.

Anatomic Risk Factors for Osteochondral Fracture of Acute First-Time Patellar Dislocation in Adolescents: A Retrospective Magnetic Resonance Imaging Study.

Objective: To analyze the risk factors for osteochondral fracture (OCF) of first-time acute patellar dislocation (APD) through measurements of patellofemoral anatomy in adolescents. Methods: In this prospective study, all patients were divided into two groups according to whether OCF was detected on magnetic resonance imaging (MRI): Group A (associated with OCF) and Group B (without OCF). Patellofemoral anatomy was evaluated with four aspects including trochlear/patellar dysplasia, patella location, patellofemoral matching, and morphologic classification. On MRI scans, trochlear facet asymmetry ratio (TFAR), lateral trochlear inclination (LTI), sulcus angle (SA), trochlear depth (TD), and patellar depth (PD) were measured to assess trochlear/patellar dysplasia. Insall-Salvati index (ISI), Caton-Deschamps index (CDI), Blackburne-Peel index (BPI), lateral patellofemoral angle (LPFA), patellar tilt angle (PTA), and lateral patellar displacement (LPD) were measured to show the location of patella. Patellofemoral matching was analyzed through the measurements of patellofemoral congruence angle (PFCA), patellofemoral index (PFI), and patellotrochlear index (PTI). Results: A total of ninety-four adolescents from 49 boys and 45 girls (mean age, 15 years; range, 10-18 years) with first-time APD were recruited and included in Group A (65) and Group B (29). The PFI (2.62 ± 0.51 vs. 2.10 ± 0.44) and PTI (0.28 ± 0.05 vs. 0.22 ± 0.07) were significantly higher in Group B than Group A (P < 0.05). There were no significant differences in other quantitative outcomes of the two groups (P > 0.05). The distribution of Dejour/Wiberg classification was statistically similar between the two groups (P > 0.05). Conclusions: Adolescent patients with first-time APD complicating OCF have closer morphologic features of patellofemoral dysplasia and patella location when compared to adolescents without OCF. Abnormal patellofemoral matching increases the risk of OCF after first-time APD in adolescents.

Three-dimensional mapping study of pure transverse acetabular fractures.

BACKGROUND: To describe and analyze the morphological characteristics, location and frequency of pure transverse acetabular fracture lines through fracture mapping and quantitative measurements. METHODS: Transverse fractures were retrospectively reviewed and analyzed. All computed tomography (CT) data were used for reconstruction and manual reduction. The reductive fracture fragments were graphically overlaid onto a three-dimensional (3D) right hemipelvis template. Then, the fracture lines were accurately depicted onto the surface of the 3D template. The fracture lines were overlapped onto the model to create the 3D fracture map and heatmap. All cases were subdivided into infratectal (62-B1.1), juxtatectal (62-B1.2), and transtectal (62-B1.3) types based on the AO Foundation/Orthopedic Trauma Association (AO/OTA) classification. Some anatomic parameters of the transverse fractures were also analyzed in these 3 groups. RESULTS: Our study included forty-nine transverse fractures from 32 male and 17 female patients (mean age, 42 years; range 21-74 years) and included 19 type 62-B1.1, 17 type 62-B1.2, and 13 type 62-B1.3 fractures. The average anterior rim fracture angle was 70.0° (± 11.6°), and the posterior rim fracture angle was 92.4° (± 28.5°). The anterior rim fracture angles in 40 cases (40/49, 81.6%) fell within a wide range between 63° and 80°. On the heatmap, the hot zones were located on the highest position of the cotyloid fossa and the narrowed region, and the cold zone was on the inferior third of the articular surface. For type 62-B1.3 fractures, the hot zone was located on the posterior of the acetabular dome. There were no significant differences in anterior rim fracture angle and anterior height among the three patterns (P = 0.071, P = 0.072). Post hoc tests of the posterior rim fracture angle and the posterior height revealed significant differences among fracture subtypes (P < 0.01). The posterior intra-articular fracture line was significantly longer than the anterior intra-articular fracture line in type 62-B1.1 and type 62-B1.2 fractures (P < 0.01). CONCLUSION: The fracture lines of transverse fractures through the anterior rim were concentrated on the narrowed zone, and the posterior fracture lines were diffusely distributed. The intra-articular fracture line distribution was focused on the superior and middle thirds of the joint surface. The recurrent fracture lines involving the weight-bearing dome mainly converged on the posterior region of the roof.

[The treatment of Tillaux bone block in the Ruedi-Allgower type â ¢ Pilon fractures].

Objective: To analyze the importance and effectiveness of Tillaux bone block in Ruedi-Allgower type Ⅲ of Pilon fracture surgery. Method: Twenty-one patients with Pilon fractures with Tillaux dislocation were treated between February 2010 and September 2013. There were 17 males and 4 females, aged from 28 to 68 years with an average age of 42.9 years. The causes of injury included falling from height in 13 cases, falls injury in 4 cases, crush injury in 2 cases, and sprained injury in 2 cases. X-ray film and CT examination showed that all 21 patients had fibula fracture and Tillaux bone block had obvious displacement. According to AO/OTA classification, there were 3 cases of type C1.2, 1 case of type C1.3, 10 cases of type C2, and 7 cases of type C3. The duration from injury to operation ranged from 4 to 31 days, with an average of 10 days. All cases of Pilon fracture were treated with open reduction and plate internal fixation. Steel plate or screw was used to fixation for Tillaux block; allograft bone graft was selected for compression of fracture. Results: There were 2 cases of skin necrosis at the corner of wound after operation, and the wound healed after corresponding treatment; the wound healed at first intention in the other 19 cases. The effect of surgical reduction was evaluated by Burwell-Chamley imaging scoring system within 72 hours after operation, there were 19 cases of anatomical reduction and 2 cases of general reduction. All the 21 patients were followed up 18-48 months, with an average of 24.9 months. No complication such as nerve injury, loosening of internal fixation, or periprosthetic fracture was found during follow-up. All fractures obtained bone healing, which lasted from 4 to 8 months, with an average of 6 months. The ankle and hindfoot scores of the American Orthopaedic Foot and Ankle Society (AOFAS) were used to evaluate the ankle and hindfoot function at 1 year after operation or at last follow-up, the results were excellent in 13 cases, good in 6 cases, and fair in 2 cases, with an excellent and good rate of 90.5%. Internal fixator was removed from 19 patients at 15-19 months after operation. There were 17 cases of joint hyperplasia and 4 cases of osteoarthritis. All joint mobility was restored. The functional recovery of the ankle joint was evaluated according to the Olerud-Molander ankle fracture score standard, the results were good in 6 cases, fair in 8 cases, and poor in 7 cases at postoperative full weight training; and after 1 year of full weight training, the results were excellent in 10 cases, good in 3 cases, fair in 6 cases, and poor in 2 cases. Conclusion: The Tillaux bone block is a reliable marker for the reduction of complex Pilon fractures. The reconstruction of the Tillaux can improve the effectiveness of complex Pilon fractures.

Study on the metabolites of isoalantolactone in vivo and in vitro by ultra performance liquid chromatography combined with Triple TOF mass spectrometry.

Isoalantolactone (IAL) is an active sesquiterpene naturally present in many vegetables, condiments and medicinal plants. In this study, an efficient strategy was developed for the identification of metabolites following the in vivo metabolism and in vitro biotransformation of IAL with rat intestinal bacteria utilizing ultra performance liquid chromatography combined with Triple TOF mass spectrometry (UPLC-Triple TOF-MS/MS). As a result, 46 metabolites including 34 novel sulfur-containing products were identified. The results demonstrated that IAL could undergo general metabolic reactions, including oxidation, hydration, hydrogenation, demethylation, cysteine conjugation and N-acetylcysteine conjugation, but the most noticeable characteristic of IAL metabolism was the H2S addition to the double bond between C-11 and C-13 and subsequent reactions to produce a series of novel sulfur-containing dimers. This is the first study of IAL metabolism in vivo, whose results provide novel and useful data for a better understanding of the safety and efficacy of IAL.

Identification of in vitro and in vivo metabolites of alantolactone by UPLC-TOF-MS/MS.

Alantolactone (AL), an active sesquiterpene originating from Inula helenium, is a potential anticancer and anti-inflammatory agent. However so far, studies on AL metabolism have not been reported. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AL using ultra performance liquid chromatography combined with time of flight mass spectrometry (UPLC-TOF-MS/MS). A unique on-line information-dependent acquisition (IDA) method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was applied to trace all of the probable metabolites of AL. Five MMDF templates were set according to the core structure of AL and the general metabolite biotransformation patterns, and other five sulfur-containing dimer filter templates were first established on the basis of structural elucidation of AL metabolites obtained from rat intestinal bacteria biotransformation. As a result, 44 metabolites were characterized: 41 metabolites from rat urine, bile and feces after oral administration of AL, and 13 metabolites from AL biotransformation by rat intestinal bacteria. Particularly, 26 metabolites were identified as novel sulfur-containing products. The results indicated that addition of double bond at Δ((11,13)) and oxidization were the main metabolic reactions of AL. A new metabolism pathway to produce addition products of H2S to AL and further generate a series of sulfur-containing dimers of AL was revealed. This study significantly enriched our knowledge about AL metabolism, which will lead to a better understanding of the safety and efficacy of AL. At the same time, the established methodology can be widely applied for the structural determination of the metabolites of other sesquiterpene containing α-methylene-γ-lactone moiety.

Treatments for the Fifth Metacarpal Neck Fractures: A Network Meta-analysis of Randomized Controlled Trials.

The fifth metacarpal neck fractures (commonly termed boxer's fractures) are the most common type of metacarpal fractures. Many types of treatments are available in clinical practice, some of which have already been compared with other treatments by various researchers. However, a comprehensive treatment comparison is lacking. We estimated the comparative efficacy of different interventions for total complications, through a network meta-analysis of randomized controlled trials. We conducted a systematic search of the literature through October 2015. The outcome measurements were the total complications. We used a Bayesian network meta-analysis to combine direct and indirect evidence and to estimate the relative effects of treatment. We identified 6 RCTs registering a total of 288 patients who were eligible for our network meta-analysis. The literature's quality is relatively high. The median Structured Effectiveness for Quality Evaluation of Study score for the included trials was 33.8. The overall methodological quality was high. Of the 6 studies, all were 2-arm controlled trials comparing active intervention. Among the 4 treatments--conservative treatment (CT), antegrade intramedullary nailing (AIMN), transverse pinning (TP) with K-wires, and plate fixation (PF)--CT had the best rankings (ie, lowest risk of total complications), followed by PF, AIMN, and TP (ie, highest risk of total complications). Furthermore, we also presented the results using surface under the cumulative ranking curve. The surface under the cumulative ranking curve probabilities were 94.1%, 52.9%, 37.3%, and 15.7% for CT, PF, AIMN, and TP, respectively. In conclusion, current evidence suggested that conservative treatment is the optimum treatment for the fifth metacarpal neck fractures because of reduced total complication rates. Moreover, the TP with K-wires is the worst option with highly total complication rates. PF and AIMN therapy should be considered as the first-line choices. Larger and higher-quality randomized controlled trials are required to confirm these conclusions and better inform clinical decision-making.

Polyhydroxytriterpenoids and Phenolic Constituents from Forsythia suspensa (Thunb.) Vahl Leaves.

Forsythia suspensa (Thunb.) Vahl leaves have been consumed in China as a health-promoting functional tea for centuries. Three new polyhydroxytriterpenoid glycosides named suspensanosides A-C (1-3), seven known polyhydroxytriterpenoids (4-10), and 12 known phenolics (11-22) were identified from F. suspensa leaves. Compounds 1-10, 15-17, and 22 have not been found in the Forsythia genus previously, whereas compound 14 was first reported to be isolated from the leaves of F. suspensa. All isolates were tested for their antiproliferative activities on BGC-823 and MCF-7 human tumor cell lines, whereas all phenolics were further investigated for their antioxidant activities by a DPPH assay. The results clearly demonstrate that triterpenoids, especially ursane-type triterpenoids, and the diverse phenolic components are crucial for the beneficial effects of F. suspensa leaves.

[MODIFIED POSTEROLATERAL COUNTER-CURVED INCISION WITH DOUBLE INTERMUSCULAR APPROACH IN TREATING POSTEROLATERAL TIBIAL PLATEAU FRACTURES].

OBJECTIVE: To evaluate the effectiveness of the modified posterolateral counter-curved incision with double intermuscular approach for the treatment of posterolateral tibial plateau fractures. METHODS: A retrospective analysis was made on the clinical data of 32 patients with posterolateral tibial plateau fractures between September 2012 and October 2014. There were 22 males and 10 females, aged 19 to 55 years (mean, 40.5 years). The causes of injury included traffic accident in 17 cases, falling from height in 9 cases, and falling in 6 cases. They had fresh closed fracture; injury to hospitalization time was 3 hours to 5 days (mean, 2 days). According to Schatzker tibial plateau fracture classification criteria, 20 cases were rated as type II, and 12 cases as type III. All patients underwent a modified posterolateral counter-curved incision with double intermuscular approach to expose tibial posterolateral condyle and anterolateral condyle. After a good visual control of fracture reduction, the anterolateral and posterolateral fractures were fixed with two-dimensional buttress plate respectively. RESULTS: The incisions healed at stage I, with no major neurovascular injury. According to radiological assessment of the DeCoster score, the results were excellent in 21 cases, and fair in 11 cases. All of the 32 patients were followed up 18 to 30 months (mean, 20.5 months). The X-ray films showed that all patients obtained good fracture union, and the mean time of fracture union was 12.3 weeks (range, 10-16 weeks). No fixation failure or no obvious loss of articular surface reduction was observed during follow-up. The range of motion of the affected knees was 2-135° (mean, 120°). The mean American Hospital for Special Surgery (HSS) score was 90.05 (range, 83-96) at 18 months after operation. CONCLUSIONS: The modified posterolateral counter-curved incision with double intermuscular approach could fully expose posterolateral tibia plateau, and good fracture reduction and reliable fixation can be obtained under direct vision.

[BIOMECHANICAL RESEARCH OF DIFFERENT INTERNAL FIXATIONS USING LOCKING RECONSTRUCTION PLATE FOR ACETABULAR TRANSVERSE FRACTURE].

OBJECTIVE: To compare the biomechanical stability of acetabular transverse fracture fixed with single column locking reconstruction plate, locking reconstruction plate combined with lag screw, and double columns lag screws. METHODS: Acetabular transverse fractures were established in 20 adult fresh semi-pelvis specimens, and divided into 5 groups randomly (n=4). The anterior columns were fixed with locking reconstruction plates in group A; the posterior columns were fixed with locking reconstruction plates in group B; the anterior columns were fixed with locking reconstruction plates and the posterior columns with lag screws in group C; the anterior columns were fixed with lag screws and the posterior columns with locking reconstruction plates in group D; the double columns were fixed with lag screws in group E. When loading three times weight, the longitudinal displacement of fracture fragment was measured, and the shear rigidity was calculated to compare the stability among groups. RESULTS: The longitudinal displacement of groups A and B were significantly greater than that of groups C, D, and E (P<0.05), and the shear rigidity of groups A and B were significantly lower than that of groups C, D, and E (P< 0.05). The longitudinal displacement of group B was significantly greater than that of group A (P< 0.05), but no significant difference was found in the shear rigidity between groups A and B (P>0.05). There was no significant difference in the longitudinal displacement and shear rigiditramong groups C, D, and E (P>0.05). CONCLUSION: Locking reconstruction plate combined with lag screw and double columns lag screws have similar stability, they have stronger stability than the single column locking reconstruction plate. The stability of anterior column locking reconstruction plate is better than that of the posterior column locking reconstruction plate.

PPARγ agonist from Chromolaena odorata.

A phytochemical investigation of Chromolaena odorata resulted in the isolation of five new compounds, 5aα,6,9,9aß,10-pentahydro-10ß-hydroxy-7-methylanthra[1,2-d][1,3]dioxol-5-one (1), 1,2-methylenedioxy-6-methylanthraquinone (2), 3-hydroxy-1,2,4-trimethoxy-6-methylanthraquinone (3), 3-hydroxy-1,2-dimethoxy-6-methylanthraquinone (4), and 7-methoxy-7-epi-medioresinol (5), together with 12 known compounds, odoratin (6), 3ß-acetyloleanolic acid (7), ursolic acid (8), ombuin (9), 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (10), (-)-pinoresinol (11), austrocortinin (12), tianshic acid (13), cleomiscosin D (14), (-)-medioresinol (15), (-)-syringaresinol (16), and cleomiscosin A (17). All the compounds were evaluated for their PPARγ transactivation activity, and compound 6 showed moderate activity with an EC(50) value of 3.10 µM.

[The recent research progress of chemistry of marine natural products].

Ocean is a unique and excellent resource that provides a diverse array of intriguing natural products. Marine natural products have demonstrated significant and extremely potent biological activities and have captured the attention of natural products chemists in the past few decades. It is increasingly recognized that a wealth of fascinating natural products and novel chemical entities will play a dominant role in the discovery of useful leads for the development of pharmaceutical agents and provide useful probes to lead to breakthroughs in a variety of life-science fields. This article focused on the research progress of chemistry of marine natural products in recent five years.

Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions.

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH(2)-terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA end-binding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of gamma-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy.

Structure elucidation and complete NMR spectral assignments of a new taxane glycoside from the needles of Taxus cuspidata.

A new taxane glycoside was isolated from a methanol extract of the needles of Taxus cuspidata. The structure was established as 2alpha,9alpha,10beta-triacetoxytaxa-4(20),11-dien-13-one-5alpha-O-beta-D-glucopyranoside (1) on the basis of 1D- and 2D NMR and high-resolution fast atom bombardment mass spectral analyses.