Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.

OBJECTIVE: Abnormal joint instability contributes to cartilage damage and osteophyte formation. We investigated whether controlling joint instability inhibited chronic synovial membrane inflammation and delayed osteophyte formation and examined the role of transforming growth factor-beta (TGF-ß) signaling in the associated mechanism. DESIGN: Rats (n = 94) underwent anterior cruciate ligament (ACL) transection. Anterior tibial instability was either controlled (CAM group) or allowed to continue (SHAM group). At 2, 4, and 8 weeks after surgery, radiologic, histopathologic, immunohistochemical, immunofluorescent, and enzyme-linked immunosorbent assay examinations were performed to evaluate osteophyte formation and TGF-ß signaling. RESULTS: Joint instability increased cartilage degeneration score and osteophyte formation, and cell hyperplasia and proliferation and synovial thickening were observed in the synovial membrane. Major findings were increased TGF-ß expression and Smad2/3 following TGF-ß phosphorylation in synovial membarene, articular cartilage, and the posterior tibial growth plate (TGF-ß expression using ELISA: 4 weeks; P = 0.009, 95% CI [260.1-1340.0]) (p-Smad2/3 expression density: 4 weeks; P = 0.024, 95% CI [1.67-18.27], 8 weeks; P = 0.034, 95% CI [1.25-25.34]). However, bone morphogenetic protein (BMP)-2 and Smad1/5/8 levels were not difference between the SHAM model and the CAM model. CONCLUSIONS: This study showed that the difference between anterior tibial instability caused a change in the expression level of TGF in the posterior tibia and synovial membrane, and the reaction might be consequently involved in osteophyte formation.

Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing.

Background: The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA), against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. Methods: S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs) were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA), and 2.0% alkaline-buffered glutaraldehyde (GA). Biofilms were exposed to these agents for 1-60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student's t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. Results: PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively (p < 0.01). Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Conclusions: Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma.

This corrects the article DOI: 10.1038/onc.2016.35.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

A radiographic evaluation of facet sagittal angle in cervical spinal cord injury without major fracture or dislocation.

STUDY DESIGN: A retrospective radiographic study with a minimum 2-year follow-up. OBJECTIVE: To evaluate the relationships between the cervical articular facets' morphology and the incidence of traumatic cervical spinal cord injury (CSCI) without major fracture or dislocation. SETTING: Spinal Injuries Center, Japan. METHODS: This study included 113 patients with traumatic CSCI without major fracture or dislocation. Eighty-four healthy volunteers without neurological deficits or cervical cord pathology on magnetic resonance imaging (MRI) were defined as control subjects. We used a plain sagittal radiograph to measure the facet sagittal angles (FSA) at four cervical segments in all the CSCI patients and controls. We defined the FSA as the angle between the inferior margin of the superior cervical spinal body and the inferior articular process of the superior vertebra. RESULTS: Most frequent incidence of CSCI was seen at C3-4 segment (54%). With respect to CSCI at C3-4 segment, 55.7% of the subjects showed smallest FSA at C3-4 segment. CONCLUSION: Most of the traumatic CSCI at C3-4 segment showed raised cervical articular facets at C3-4 segment. On the basis of our results, we hypothesized that the raised cervical articular facets might have an important role in the etiology of traumatic CSCI. The cervical spinal cord at the C3-4 segment might receive the highest load during acute hyperextension of the cervical spine because of the C3-4 articular facets' morphology.

Controlling joint instability delays the degeneration of articular cartilage in a rat model.

OBJECTIVE: Joint instability induced by anterior cruciate ligament (ACL) transection is commonly considered as a predisposing factor for osteoarthritis (OA) of the knee; however, the influence of re-stabilization on the protection of articular cartilage is unclear. The aim of this study was to evaluate the effect of joint re-stabilization on articular cartilage using an instability and re-stabilization ACL transection model. DESIGN: To induce different models of joint instability, our laboratory created a controlled abnormal joint movement (CAJM) group and an anterior cruciate ligament transection group (ACL-T). Seventy-five Wistar male rats were randomly assigned to the CAJM (n = 30), ACL-T (n = 30), or no treatment (INTACT) group (n = 15). Cartilage changes were assessed with soft X-ray analysis, histological and immunohistochemistry analysis, and real-time polymerase chain reaction (PCR) analysis at 2, 4, and 12 weeks. RESULTS: Joint instability, as indicated by the difference in anterior displacement between the CAJM and ACL-T groups (P < 0.001), and cartilage degeneration, as evaluated according to the Osteoarthritis Research Society International (OARSI) score, were significantly higher in the ACL-T group than the CAJM group at 12 weeks (P < 0.001). Moreover, joint re-stabilization maintained cartilage structure (thickness [P < 0.001], surface roughness [P < 0.001], and glycosaminoglycan stainability [P < 0.001]) and suppressed tumor necrosis factor-alpha (TNF-α) and caspase-3 at 4 weeks after surgery. CONCLUSION: Re-stabilization of joint instability may suppress inflammatory cytokines, thereby delaying the progression of OA. Joint instability is a substantial contributor to cartilage degeneration.

Low-dose thymoglobulin as second-line treatment for steroid-resistant acute GvHD: an analysis of the JSHCT.

A nationwide retrospective study for the clinical outcomes of 99 patients who had received thymoglobulin at a median total dose of 2.5 mg/kg (range, 0.5-18.5 mg/kg) as a second-line treatment for steroid-resistant acute GvHD was conducted. Of the 92 evaluable patients, improvement (complete or partial response) was observed in 55 patients (60%). Multivariate analysis demonstrated that male sex and grade III and IV acute GvHD were associated with a lower improvement rate, whereas thymoglobulin dose (<2.0, 2.0-3.9 and ⩾4.0 mg/kg) was NS. Factors associated with significantly higher nonrelapse mortality included higher patient age (⩾50 years), grade IV acute GvHD, no improvement of GvHD and higher dose of thymoglobulin (hazard ratio, 2.55; 95% confidence interval, 1.34-4.85; P=0.004 for 2.0-3.9 mg/kg group and 1.79; 0.91-3.55; P=0.093 for ⩾4.0 mg/kg group). Higher dose of thymoglobulin was associated with a higher incidence of bacterial infections, CMV antigenemia and any additional infection. Taken together, low-dose thymoglobulin at a median total dose of 2.5 mg/kg provides a comparable response rate to standard-dose thymoglobulin reported previously, and <2.0 mg/kg thymoglobulin is recommended in terms of the balance between efficacy and adverse effects.

RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma.

RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-ß in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-ß repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-ß. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.

TGF-ß1-siRNA delivery with nanoparticles inhibits peritoneal fibrosis.

Gene therapies may be promising for the treatment of peritoneal fibrosis (PF) in subjects undergoing peritoneal dialysis (PD). However, a method of delivery of treatment genes to the peritoneum is lacking. We attempted to develop an in vivo small interfering RNA (siRNA) delivery system with liposome-based nanoparticles (NPs) to the peritoneum to inhibit PF. Transforming growth factor (TGF)-ß1-siRNAs encapsulated in NPs (TGF-ß1-siRNAs-NPs) dissolved in PD fluid were injected into the peritoneum of mice with PF three times a week for 2 weeks. TGF-ß1-siRNAs-NPs knocked down TGF-ß1 expression significantly in the peritoneum and inhibited peritoneal thickening with fibrous changes. TGF-ß1-siRNAs-NPs also inhibited the increase of expression of α-smooth muscle actin-positive myofibroblasts. These results suggest that the TGF-ß1-siRNA delivery system with NPs described here could be an effective therapeutic option for PF in subjects undergoing PD.

Comparison of the NMIJ and the ARPANSA standards for absorbed dose to water in high-energy photon beams.

The authors report the results of an indirect comparison of the standards of absorbed dose to water in high-energy photon beams from a clinical linac and (60)Co radiation beam performed between the National Metrology Institute of Japan (NMIJ) and the Australian Radiation Protection and Nuclear Safety Agency (ARPANSA). Three ionisation chambers were calibrated by the NMIJ in April and June 2013 and by the ARPANSA in May 2013. The average ratios of the calibration coefficients for the three ionisation chambers obtained by the NMIJ to those obtained by the ARPANSA were 0.9994, 1.0040 and 1.0045 for 6-, 10- and 15-MV (18 MV at the ARPANSA) high-energy photon beams, respectively. The relative standard uncertainty of the value was 7.2 × 10(-3). The ratio for (60)Co radiation was 0.9986(66), which is consistent with the results published in the key comparison of BIPM.RI(I)-K4.

Rapid progressive clinical deterioration of cervical spondylotic myelopathy.

STUDY DESIGN: Retrospective clinical study. OBJECTIVE: To elucidate the pathophysiology of rapid progressive clinical deterioration following the onset of cervical myelopathy. SETTING: Spinal Injuries Center, Fukuoka, Japan. METHODS: A total of 43 cervical spondylotic myelopathy (CSM) patients were treated surgically by a senior surgeon. All patients showed intramedullary intensity changes on magnetic resonance (MR) imaging. Overall, eight patients suffered rapid progressive clinical deterioration; four of them had obvious anamnesis of minor trauma. We assessed the responsible injured segment by MR T2-weighted images. Clinical instabilities at the focal segment were evaluated using functional sagittal plain radiographs. Neurological evaluations were performed preoperatively and at 12 months postoperatively using American Spinal Injury Association (ASIA) motor scores and Japanese Orthopaedic Association (JOA) scores for cervical myelopathy. Intraoperatively, we evaluated the presence of adhesive scar tissue on the dura mater at the focal segment. RESULTS: The responsible injured segment was C3-4 in 75% of the rapid progressive (rp)-CSM and in 28.57% of the conventional CSM subjects. One with rp-CSM showed sagittal translational segmental instability. Preoperative ASIA motor scores and JOA scores in the rp-CSM were significantly lower than those in the conventional CSM subjects. Postoperative ASIA motor scores between the subjects showed no significant differences; however, postoperative JOA scores in the rp-CSM subjects were significantly lower. Moreover, an epidural membrane was observed in 62.5% of rp-CSM and 11.4% of conventional CSM subjects. CONCLUSIONS: We hypothesized that the pathophysiology of rp-CSM might be additional cervical cord disorder following the onset of cervical myelopathy. Early decompression surgery is recommended in such patients.

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

The potential for functional recovery of upper extremity function following cervical spinal cord injury without major bone injury.

STUDY DESIGN: This was a retrospective observational study. OBJECTIVES: The objectives were to describe the prognosis of upper extremity function following cervical spinal cord injury (CSCI), and to identify prognostic factors for functional recovery. SETTING: Spinal Injuries Center, Japan. METHODS: Sixty patients with C3-4 CSCI without major bone injury participated in the study. Patients were treated nonsurgically and evaluated using the American Spinal Injury Association (ASIA) scales for the upper and lower extremities, their residual cervical motor functions, the modified Frankel grade and an upper extremity function scale. We compared the findings for the upper extremity function scale at 6 months with those for the residual cervical motor functions and modified Frankel grade obtained 3 days after injury. RESULTS: Most patients with CSCI who could flex their hip and knee from a supine position (95%) or who showed some active elbow extension (86%) 3 days after their injury could use a spoon at 6 months. We compared patients who used their fingers at 6 months to those who could not, and observed significant differences in age and ASIA scores for the upper and lower extremities obtained 3 days after injury. A strong correlation was observed between the initial motor scores and the extent of functional recovery at 6 months. CONCLUSION: Hip and knee flexion from the supine position and elbow extension 3 days after injury significantly predicted a positive prognosis for upper extremity function. Younger age and higher ASIA motor scores obtained 3 days after injury were factors associated with neurological recovery.

Epithelial to mesenchymal transition in murine tracheal allotransplantation: an immunohistochemical observation.

BACKGROUND: Aberrant epithelial repair is a crucial event in the airway remodeling that characterizes obliterative bronchiolitis (OB) in transplanted lungs. Recent data from experiments using epithelial cell lines and human airway tissues from lung transplant recipients suggest that epithelial to mesenchymal transition (EMT) plays an important role in OB. The aim of this study was to clarify whether EMT is involved in airway remodeling in an animal model. METHODS: We performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice with from BALC/c to BALB/c mouse grafts as controls. Five allogeneic and 3 syngeneic recipients were humanely killed at predetermined postoperative days 2-12 as well as 14 and 21. Histology was evaluated using hematoxylin-eosin (H&E) staining. We studied the expression of specific markers, including E-cadherin, an epithelial marker; α-smooth muscle actin (SMA), and S100A4, mesenchymal markers, and zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-related transcription factor. RESULTS: Histologic assessment of serial H&E stains of allogeneic grafts showed remarkable pseudostratified respiratory epithelium with subepithelial inflammatory cell infiltration, as well as denuded and flattened epithelium and subepithelial fibrosis. The dynamic epithelial changes occurred earlier than the subepithelial fibrosis. Immunohistochemical evaluation indicated the emergence of α-SMA- positive epithelial cells that were most prominent on day 7. The expression of E-cadherin was attenuated in α-SMA-positive epithelial cells. S100A4 was also expressed in epithelial cells. A few days before the intraepithelial expression of α-SMA, ZEB1 emerged in the nuclei of epithelial cells. CONCLUSIONS: We observed expression of an EMT-related transcription factor and mesenchymal markers along with the attenuation of epithelial marker expression in epithelial cells, several days before prominent subepithelial fibrosis formation, results that suggest epithelial cells to play an important fibrosis role in airway remodeling during epithelial to mesenchymal transition.

Accumulation and dissipation of positive charges induced on a PMMA build-up cap of an ionisation chamber by (60)Co gamma-ray irradiation.

The signal current from an ionisation chamber with a PMMA build-up cap decreases with irradiation time due to electric fields produced by positive charges induced on the cap. In the present study, it was confirmed that the signal current decreases faster for irradiation using narrower (60)Co gamma-ray beams. This is because the number of secondary electrons that are emitted from surrounding materials and penetrate the build-up cap is smaller in a narrower gamma-ray beam, so that fewer positive charges are neutralised. The ionisation chamber was first subjected to continuous gamma-ray irradiation for 24 h, following which it was irradiated with shorter periodic gamma-ray bursts while measuring the current signal. This allowed the coefficients of positive charge accumulation and dissipation to be determined. It was found that the dissipation coefficient has a large constant value during gamma-ray irradiation and decreases asymptotically to a small value after irradiation is stopped. From the coefficients, the minimum signal current was calculated, which is the value when accumulation and dissipation balance each other under continuous irradiation. The time required for the signal current to recover following irradiation was also calculated.

Colonization of chicken flocks by Campylobacter jejuni in multiple farms in Japan.

Campylobacter jejuni was monitored in 4 chicken farms during the period 2003 to 2006 to elucidate the mechanisms of transmission. Three farms (1 to 3), located at least 14 km from each other, belonged to an integrated poultry company, which also provided the farms with day-old chicks from several hatcheries as well as chicken feed. Another farm (4), which belonged to a different company, was located 270 m from farm 1. A total of 206 C. jejuni isolates obtained from the 4 farms were classified into 10 flaA-based RFLP types. Identical RFLP types were found in isolates obtained from chickens originating from multiple hatcheries and reared in different chicken houses on individual farms. Flocks were colonized by strains with 1 or 2 RFLP types in each production cycle, sometimes differing between cycles. Identical RFLP types were found in isolates obtained from the environment around the chicken houses. Using multilocus sequence typing, strains with different RFLP types could be distinguished from each other. Identical RFLP and multilocus sequence typing profiles were found in isolates obtained from farms 1 and 4, and from farms 1 and 2. These results suggest that C. jejuni in these farms comes from common sources external to the farms, even if the farms belong to different companies and obtain chicks from different suppliers.

A standard for absorbed dose rate to water in a 60Co field using a graphite calorimeter at the National Metrology Institute of Japan.

A primary standard for the absorbed dose rate to water in a 6°Co radiation field has been newly established at the National Metrology Institute of Japan. This primary standard combines the calorimetric measurements using a graphite calorimeter with the ionometric measurements using a thick-walled graphite cavity ionisation chamber. The calorimeter is operated in the constant temperature mode using AC Wheatstone bridges. The absorbed dose rate to water was determined to be 12 mGy s⁻¹ at a point of 1 m from the radiation source and at a water depth of 5 g cm⁻². The uncertainty on the calibration coefficient in terms of the absorbed dose to water of an ionisation chamber using this standard was estimated to be 0.39 % (k=1).

The pincers effect on cervical spinal cord in the development of traumatic cervical spinal cord injury without major fracture or dislocation.

STUDY DESIGN: Retrospective radiographic study. OBJECTIVE: To investigate the pincers effect on cervical spinal cord in the development of traumatic cervical spinal cord injury (CSCI) without major fracture or dislocation. SETTING: The Japan LHWO Spinal Injuries Center. METHODS: Two hundred and twenty cases of traumatic CSCI without major fracture or dislocation were examined. The pinched diameters of the cervical spinal cord for 70 patients who complained of neck pain without neurological deficits were measured using sagittal-plane neutral and extension radiographs at 5 segments. These 70 patients were divided into 2 groups: group A patients were less than 40 years old and group B patients were 41 or more. We defined the pinched ratio of the cervical spinal cord during extension as ((sagittal diameter in the neutral image)-(sagittal diameter in the extension image))/(sagittal diameter in the neutral image)*100. RESULTS: The incidence of traumatic CSCI without major fracture or dislocation at the C3-4, C4-5, C5-6 and C6-7 was 59.5, 25, 11.4 and 4.1%, respectively. Further, the pinched ratio of the cervical spinal cord at the C3-4 segment was significantly higher than that at the other segments. CONCLUSION: We concluded that the cervical spinal cord at the C3-4 segment might receive the highest bony impingement load during acute hyperextension of the cervical spine. The extreme pincers load on the cervical spinal cord at the C3-4 segment may have one of the important roles in the development of traumatic CSCI at the C3-4 segment.

Limited sampling strategies to estimate the area under the concentration-time curve. Biases and a proposed more accurate method.

BACKGROUND: Over 100 limited sampling strategies (LSSs) have been proposed to reduce the number of blood samples necessary to estimate the area under the concentration-time curve (AUC). The conditions under which these strategies succeed or fail remain to be clarified. OBJECTIVES: We investigated the accuracy of existing LSSs both theoretically and numerically by Monte Carlo simulation. We also proposed two new methods for more accurate AUC estimations. METHODS: We evaluated the following existing methods theoretically: i) nonlinear curve fitting algorithm (NLF), ii) the trapezium rule with exponential curve approximation (TZE), and iii) multiple linear regression (MLR). Taking busulfan (BU) as a test drug, we generated a set of theoretical concentration-time curves based on the identified distribution of pharmacokinetic parameters of BU and re-evaluated the existing LSSs using these virtual validation profiles. Based on the evaluation results, we improved the TZE so that unrealistic parameter values were not used. We also proposed a new estimation method in which the most likely curve was selected from a set of pre-generated theoretical concentration-time curves. RESULTS: Our evaluation, based on clinical profiles and a virtual validation set, revealed: i) NLF sometimes overestimated the absorption rate constant Ka, ii) TZE overestimated AUC over 280% when Ka is small, and iii) MLR underestimated AUC over 30% when the elimination rate constant Ke is small. These results were consistent with our mathematical evaluations for these methods. In contrast, our two new methods had little bias and good precision. CONCLUSIONS: Our investigation revealed that existing LSSs induce different but specific biases in the estimation of AUC. Our two new LSSs, a modified TZE and one using model concentration-time curves, provided accurate and precise estimations of AUC.