A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38ß, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.

Development and Validity of an Intrapartum Self-Assessment Scale Aimed at Instilling Midwife-Led Care Competencies Used at Freestanding Midwifery Units.

Building experience in midwife-led care at freestanding midwifery units is needed to enhance assessment, technical, and care competencies specific to midwives. This study aimed to develop a self-assessment scale for midwifery practice competency based on the characteristics of midwife-led care practices in freestanding midwifery units. This study was conducted at 65 childbirth facilities in Japan between September 2017 and March 2018. The items on the scale were developed based on a literature review, discussion at a professional meeting, and a preliminary survey conducted at two timepoints. The validity and reproducibility of the scale were evaluated based on item analysis, compositional concept validity, internal consistency, stability, and criterion-related validity using data from 401 midwives. The final version of the scale consisted of 40 items. Cronbach's α for the overall scale was 0.982. The results for compositional concept validity, internal validity, and criterion-related validity demonstrated that this scale is capable of evaluating a midwife's practice competencies in intrapartum care. Repeated self-assessment using this scale could improve the competencies of midwives from an early stage, maximize the roles of physicians and midwives, and create an environment that provides high-quality assistance to women.

Visual prognosis of submacular hemorrhage secondary to age-related macular degeneration: A retrospective multicenter survey.

PURPOSE: To investigate the clinical features, treatment options, and visual outcomes of submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (nAMD). DESIGN: A retrospective, observational case series. METHODS: Setting: Multicenter institutional setting. Patient Population: A total of 127 patients (127 eyes; 88 men, 39 women; (mean age, 74.2 years)) diagnosed with AMD-associated SMHs exceeding 2 disc diameters involving the fovea. Observation: The AMD types, previous treatments, treatment options, anatomic findings, and best-corrected visual acuity (BCVA) were assessed. Main Outcome Measures: Clinical features, treatment options, and visual outcomes of SMHs secondary to nAMD. RESULTS: Thirty-two eyes had typical AMD, 94 eyes polypoidal choroidal vasculopathy (PCV), and one eye retinal angiomatous proliferation. Eighty-five eyes were treatment-naïve; 42 eyes were treated previously: anti-vascular endothelial growth factor (VEGF) therapy (n = 26), photodynamic therapy (n = 3), and combined therapy (n = 13). Treatment of SMHs included vitrectomy (36 eyes), pneumatic displacement (49 eyes), and anti-VEGF monotherapy (42 eyes). The final BCVA improved significantly in treatment-naïve cases from 0.86 to 0.62 logarithm of the minimal angle of resolution (logMAR) unit (Snellen equivalent from 20/145 to 20/83) and from 0.80 to 0.56 (Snellen equivalent from 20/126 to 20/73) in PCV cases. Meanwhile, the BCVA logMAR values improved from 1.15 to 0.75 (Snellen equivalent from 20/283 to 20/112) and from 0.87 to 0.63 (Snellen equivalent from 20/148 to 20/85) in eyes that underwent vitrectomy or pneumatic displacement, respectively. In eyes with BCVAs between 20/133 to 20/40 at SMH onset, the final VA in the pneumatic displacement group was better than in the anti-VEGF monotherapy group. One eye had a retinal detachment and 1 eye had a macular hole in the vitrectomy group, and 5 eyes had a vitreous hemorrhage in the pneumatic displacement group. CONCLUSIONS: The recommended treatment for SMHs secondary to nAMD exceeding 2 disc area and with BCVA below 20/40 is vitrectomy or pneumatic displacement for visual improvement.

Paclitaxel-carboplatin and bevacizumab combination with maintenance bevacizumab therapy for metastatic, recurrent, and persistent uterine cervical cancer: An open-label multicenter phase II trial (JGOG1079).

OBJECTIVE: This multicenter, open-label, phase II study aimed to evaluate the efficacy and safety of paclitaxel-carboplatin, bevacizumab, and bevacizumab-based maintenance therapy for metastatic, recurrent, and persistent uterine cervical cancer. METHODS: Patients with measurable diseases that were not adapted to regional therapies, such as surgery or radiotherapy, and were systematic chemotherapy-naïve were eligible. The participants received paclitaxel (175 mg/m2), carboplatin (AUC 5), and bevacizumab (15 mg/m2) every three weeks until disease progression or unacceptable adverse events occurred. The primary endpoint was progression-free survival (PFS). The secondary endpoints were overall response rate (ORR), overall survival (OS), safety, and time to treatment failure. RESULTS: Sixty-nine patients were analyzed using our protocol. The median paclitaxel- carboplatin therapy duration was six cycles; 40% of patients received bevacizumab maintenance therapy. The median PFS was 11.3 months. The median OS was not reached; the median time to treatment failure was 5.9 months. The ORR was 79.7% [95% confidence interval (CI) 63.8-88.4]; 16 patients (23.2%) showed complete response (CR) and 39 patients (56.5%) showed partial response (PR). The median PFS was 14.3 months (95% CI 7.3-17 months) for the 25 patients who received maintenance therapy and 7.4 months (95% CI 6.1-11 months) for nonrecipients (p = 0.0449). Gastrointestinal perforation/fistulas occurred in four patients (5.6%), all of whom had a history of radiation therapy. CONCLUSIONS: Paclitaxel-carboplatin and bevacizumab therapy is an acceptable and tolerable treatment for advanced or recurrent cervical cancer.

Prospective feasibility study of neoadjuvant dose-dense paclitaxel plus carboplatin with bevacizumab therapy followed by interval debulking surgery for advanced ovarian, fallopian tube, and primary peritoneal cancer patients.

BACKGROUND: This study aimed to investigate the clinical benefit of dose-dense paclitaxel plus carboplatin (TC) with bevacizumab therapy for advanced ovarian, fallopian tube, and primary peritoneal cancer patients in the neoadjuvant setting. METHODS: Ovarian, fallopian tube or primary peritoneal cancer patients with stage III-IV disease received neoadjuvant chemotherapy (NAC) every 3 weeks consisting of paclitaxel (80 mg/m2) on days 1, 8, and 15; carboplatin (AUC 6.0 mg/mL × min.) on day 1; and bevacizumab (15 mg/kg) on day 1. Interval debulking surgery (IDS) was performed after 3 cycles of dose-dense TC-bevacizumab therapy. The primary endpoint was the rate of complete resection by IDS. Secondary endpoints were treatment completion rate, treatment exposure, response rate to NAC, adverse events, and perioperative complications. RESULTS: Twenty-four patients were included in this study. The median age was 55.5 years (37-80 years), and most patients had high-grade serous carcinoma accounted (n = 18). IDS was performed in all patients with complete resection achieved in 75% (95% confidence interval: 57.7-92.3%). The lower limit exceeded the preset threshold rate of 55%. The response rate to NAC was 79%, and serum CA125 levels were in the normal range after NAC in 57% of patients. Grade 4 hematological toxicities and grade 3/4 non-hematological toxicities occurred in 29% and 17% of patients during NAC, respectively. Grade 3/4 perioperative complications were seen in 29% of patients, but no gastrointestinal perforations or treatment-related deaths occurred. CONCLUSIONS: Neoadjuvant dose-dense TC-bevacizumab therapy was well tolerated, and a satisfactory rate of complete resection by IDS was achieved.

Association between Regular Exercise and Self-Rated Health and Sleep Quality among Adults in Japan during the COVID-19 Pandemic.

Regular exercise may be associated with better self-rated health and sleep status. However, this correlation among various age groups, such as young, middle-aged, and older people, as well as during the COVID-19 pandemic, has not been examined. This study examined the correlation between regular exercise and self-rated health and sleep quality among adults in Japan during the COVID-19 pandemic. Data were collected using an online survey conducted between February 26 and 27, 2021. A total of 1410 adults in Japan (age range, 20-86 years) completed the online survey. Regular exercise was divided into: (1) more than 30 min of moderate exercise a day, (2) more than 2 days per week, and (3) continuous for 1 year or longer. Self-rated health and sleep quality were assessed using the Likert scale. After adjusting for multiple confounders, regular exercise was correlated with decreased poor self-rated health and poor sleep quality in middle-aged adults; however, no significant correlation was observed among young and older adults. The promotion of regular exercise among middle-aged people during the COVID-19 pandemic may contribute to better self-rated health and sleep quality status.

Remote screening of diabetic retinopathy using ultra-widefield retinal imaging.

AIMS: To study the possibility of constructing a remote interpretation system for retinal images. METHODS: An ultra-widefield (UWF) retinal imaging device was installed in the internal medicine department specializing in diabetes to obtain fundus images of patients with diabetes. Remote interpretation was conducted at Nagoya City University using a cloud server. The medical data, severity of retinopathy, and frequency of ophthalmologic visits were analyzed. RESULTS: Four hundred ninety-nine patients (mean age, 62.5 ± 13.4 years) were included. The duration of diabetes in 240 (48.1%) patients was less than 10 years and 433 (86.7%) patients had a hemoglobin (Hb) A1c below 8%. Regarding the retinopathy severity, 360 (72.1%) patients had no diabetic retinopathy (NDR), 63 (12.6%) mild nonproliferative retinopathy (NPDR), 38 (7.64%) moderate NPDR, 13 (2.6%) severe NPDR, and 25 (5.0%) PDR. Two hundred forty-one (48.3%) patients had an ophthalmologic consultation within 1 year, 104 (20.8%) had no history of an ophthalmologic consultation. DR was not present in 86 (82.7%) patients who had never had an ophthalmologic examination, 30 (78.9%) patients with severe NPDR or PDR had had an ophthalmologic visit within 1 year. The frequency of ophthalmic visits was correlated negatively with age, diabetes duration, HbA1c, and severity of retinopathy. CONCLUSION: Remote interpretation of DR using UWF retinal imaging was useful for retinopathy screening. During the COVID-19 pandemic, a remote screening system that can ensure compulsory social distancing and reduce the number of ophthalmic visits can be a safe system for patients and clinicians.

The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants.

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

GC-MS/MS analysis of metabolites derived from a single human blastocyst.

INTRODUCTION: The field of assisted reproductive technology (ART) has significantly advanced; however, morphological evaluation remains as the chosen method of assessment of embryo quality. OBJECTIVE: We aimed to examine metabolic changes in embryo culture medium to develop a non-invasive method for evaluation of embryo quality. METHODS: We performed metabolic analysis of culture medium obtained from a single blastocyst cultured for freezing. RESULTS: In total, 187 (39.8%) of the 469 detectable organic acid metabolites were identified. A significant change (p < 0.05) was observed in eight metabolites between the good-quality and poor-quality embryo groups. Differences were observed in several metabolic pathways between the good-quality and poor-quality embryo groups. Metabolites that showed significant changes were primarily involved in the metabolism of branched-chain amino acids. CONCLUSION: The quantification of metabolism in human embryos may assist in identification and selection of good-quality embryos with high rates of survival before freezing and implantation in conjunction with morphological classification. This may help to identify embryos with high rates of survival.

Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System.

DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal instability and eventually cause cell death. Quantitative markers, such as phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1) foci in mammalian cells, are not available for the detection of DSBs in prokaryotes. Therefore, as an alternative method, pulsed-field gel electrophoresis (PFGE) is widely used to analyze broken DNA molecules by separating them from intact DNA. Here, we examined the accumulation of bleomycin (BLM)-induced DSBs by PFGE, using a rotating gel electrophoresis (RGE) system. We defined two sets of parameters with distinct advantages; the first one focuses on the analysis of the size of the broken DNA fragments, whereas the second allows for the direct comparison of the accumulation of DSBs among strains and treatments. This method represents a powerful tool for the study of genomic integrity and the characterization of genotoxic substances.

What advice should we give our patients to preserve their fertility and avoid needing oocyte donation in the future? - A Social Fertility Preservation program.

OBJECTIVE: To describe our fertility preservation program focusing on the number of oocytes vitrified by age. METHODS: From January 2015 to December 2016, 686 oocyte vitrification cycles were performed in our units for the social fertility preservation program. In total, 288 were donors who donated their oocytes for our oocyte-banking program, and 398 were patients who underwent elective fertility preservation. RESULTS: The mean numbers of COCs retrieved and vitrified oocytes were similar among the donor cycles (women under 30 years). In those patients over 36 years of age the mean numbers of COCs retrieved and vitrified oocytes were significantly lower. We also estimated the association between age and cancelation rates. Odd ratios (OR) for total cancelation was calculated between patients of 31-35 years and 41-45 years; the OR was 5.17 (95% CI 1.89 - 14.17) and increased up to 25.67 (95% CI 5.01 - 131.42) between patients 31-35 y and those older than 45 years. No differences were found between patients of 31-35 years and 36-40 years. The OR for total cancellation increased 3.83 (95% CI 2.06 - 7.11) and 19.00 (95% CI 4.56 - 79.11) between women 36-40 years and 41-45 years, and those older than 45 years, respectively. Finally, the oocyte survival rate in patients under 36 years of age was similar to that of our donor program (94% vs. 95%). CONCLUSIONS: Based on this study, we encouraged our patients under than 36 years of age to preserve their fertility for the future.

The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos).

To understand the role of aryl hydrocarbon receptor (AHR) isoforms in avian species, we investigated the functional characteristics of two AHR isoforms (designated as jcAHR1 and jcAHR2) of the jungle crow (Corvus macrorhynchos). Two amino acid residues corresponding to Ile324 and Ser380 (high sensitive type) in chicken AHR1 that are known to determine dioxin sensitivity were Ile325 and Ala381 (moderate sensitive type) in jcAHR1 and Val306 and Ala362 (low sensitive type) in jcAHR2. The quantitative comparison of the two jcAHR mRNA expression levels in a Tokyo jungle crow population showed that jcAHR2 accounted for 92.4% in the liver, while jcAHR1 accounted for only 7.6%. Both in vitro-expressed jcAHR1 and jcAHR2 proteins exhibited a specific binding to [3H]-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transactivation potencies for jcAHR1 and jcAHR2 in in vitro reporter gene assays were measured in jcAHR-expressed cells exposed to 16 dioxins and related compounds (DRCs). Both jcAHR1 and jcAHR2 were activated in a congener- and an isoform-specific manner. EC50 value of TCDD for jcAHR2 (0.61 nM) was six-fold higher than that for jcAHR1 (0.098 nM), but jcAHR2 had higher transactivation efficacy than jcAHR1 in terms of the magnitude of response. The high transactivation efficacy of jcAHR2 in DRCs is in contrast to that of AHR2s in other avian species with low transactivation efficacy. Molecular docking simulations of TCDD with in silico jcAHR1 and jcAHR2 homology models showed that the two sensitivity-decisive amino acids indirectly controlled TCDD-binding modes through their surrounding amino acids. Deletion assays of jcAHR2 revealed that 736-805 amino acid residues in the C-terminal region were critical for its transactivation. We suggest that jcAHR2 plays a critical role in regulating the AHR signaling pathway, at least in its highly expressed organs.

Open-label study of cognitive behavioural therapy for individuals with at-risk mental state: Feasibility in the Japanese clinical setting.

AIM: To date, most cognitive behavioural therapy (CBT) trials for individuals with at-risk metal state (ARMS) have been conducted in few Western countries and its feasibility in other regions, including Japan, has not been established. METHODS: We designed an open-label pilot study. Fourteen ARMS participants received CBT over 6 months and were followed-up for 6 months. RESULTS: Thirteen individuals completed the CBT intervention and assessments. The mean total score on the Positive and Negative Syndrome Scale improved from 60.2 to 46.0 after the intervention (Cohen's d = 1.1). The effects were maintained at the follow-up assessment. One participant transitioned to psychosis after the CBT intervention, and was the only patient who received antipsychotics. CONCLUSIONS: We confirmed the feasibility of the provision of CBT for ARMS in Japan. Since overprescription of antipsychotics is a matter of great concern in Japan, CBT could be a valuable alternative treatment strategy.

Pathophysiology of Diabetic Retinopathy: The Old and the New.

Vision loss in diabetic retinopathy (DR) is ascribed primarily to retinal vascular abnormalities-including hyperpermeability, hypoperfusion, and neoangiogenesis-that eventually lead to anatomical and functional alterations in retinal neurons and glial cells. Recent advances in retinal imaging systems using optical coherence tomography technologies and pharmacological treatments using anti-vascular endothelial growth factor drugs and corticosteroids have revolutionized the clinical management of DR. However, the cellular and molecular mechanisms underlying the pathophysiology of DR are not fully determined, largely because hyperglycemic animal models only reproduce limited aspects of subclinical and early DR. Conversely, non-diabetic mouse models that represent the hallmark vascular disorders in DR, such as pericyte deficiency and retinal ischemia, have provided clues toward an understanding of the sequential events that are responsible for vision-impairing conditions. In this review, we summarize the clinical manifestations and treatment modalities of DR, discuss current and emerging concepts with regard to the pathophysiology of DR, and introduce perspectives on the development of new drugs, emphasizing the breakdown of the blood-retina barrier and retinal neovascularization.

A healthy HLA-matched baby born by using a combination of aCGH and Karyomapping: the first latin american case.

PGD for HLA typing is a procedure that can be performed when an affected child requires a transplant to treat a non-hereditary disorder related to the hematopoietic and/or immune system. Hematopoietic stem cell transplantation from an HLA-identical donor provides the best treatment option. Three conventional ovarian stimulation procedures for IVF were performed in a couple with a 10-year-old child diagnosed with T-cell acute lymphoblastic leukemia of high risk. Trophectoderm biopsy and aCGH examination were performed on 15 blastocysts, three on the first IVF procedure, four on the second cycle, and eight on the third. Three euploid blastocysts HLA-compatible with the genome of the affected child were identified. One euploid blastocyst HLA-compatible with the affected child was warmed and transferred, resulting in an HLA-matched live birth. In conclusion, combined aCGH for aneuploidy screening and Karyomapping may be performed in a single biopsy procedure.

Mosaic embryo transfer after oocyte in vitro maturation in combination with non-invasive prenatal testing (NIPT)-first report of a euploid live birth.

PURPOSES: The purpose of this study is to describe a healthy life birth after a mosaic embryo transfer in oocyte in vitro maturation (IVM). METHODS: Patient received minimal stimulation, starting on day 3 after menstrual period. No hCG trigger was administered. Oocyte retrieval was performed and oocytes were matured for 30 h. After denuding, mature oocytes were inseminated by ICSI. Embryos were cultured until blastocyst stage and biopsied. RESULTS: One euploid embryo after array comprehensive genome hybridization (aCGH) was diagnostic. However, the next-generation sequencing (NGS) re-analysis showed that embryo was a mosaic for chromosome 13 and 21. Nevertheless, pregnancy ultrasound scans and non-invasive prenatal test (NIPT-Verifi-Illumina) indicated a normal fetus development. Finally, a healthy baby was born after 38 weeks. Its weight was 4480 g, head circumference 36 cm, and total length of 51 cm. To confirm that the baby was chromosomically normal, an NGS test was performed in buccal cells, a normal profile was obtained. CONCLUSIONS: Our finding confirmed that mosaic embryo transfer would bring a healthy offspring.

Maduración in vitro de ovocitos con cultivo extendido hasta estadio de blastocisto: Primer nacimiento reportado en el Perú / In vitro oocyte maturation combined with extended blastocyst embryo culture: Report of the first baby born in Peru

La maduración in vitro de ovocitos (MIV) es una técnica de reproducción asistida muy poco difundida entre los centros de reproducción asistida, debido al bajo éxito en obtener embarazos. Sin embargo, en los últimos años, diferentes estrategias empleadas han demostrado tasas de embarazo similares a las técnicas convencionales de fecundación in vitro (FIV). En el presente reporte, describimos el caso clínico del primer nacido vivo usando MIV en combinación del cultivo extendido hasta estadio de blastocisto.

In vitro oocyte maturation is not yet considered a well-established technique in in vitro fertilization (IVF) laboratories. This is due to a lower pregnancy rates. However in the last few years, reports have shown similar pregnancy rates compared to the conventional IVF techniques. The current report describes the first baby born after an IVM treatment in combination with extended blastocyst culture in Peru.

Effects of reversible deactivation of mossy fibers in the dentate-CA3 system on geometric center detection task in mice: Functional separation of spatial learning and its generalization to new environment.

Using diethyldithiocarbamate (DEDTC), a zinc chelator, we deactivated the mossy fibers that project from the dentate gyrus (DG) to the CA3 during acquisition and testing of a center detection task in mice. The mice were trained to find a food pellet at the center of four objects in a circular area. DEDTC injection just before the training sessions impaired this learning, whereas DEDTC injection before the probe test did not impair recall of the memory. DEDTC injection before a pattern completion test in which only one of the four objects was presented did not cause deficits in this test. DEDTC injection did, however, cause severe deficits in an array shift test in which all four objects were moved to new positions. These results demonstrated that 1) the DG-CA3 system plays a crucial role in the learning of geometric center detection task but not in its recall or pattern completion, and 2) the DG-CA3 system is involved in generalization to a new environment but is not crucial for pattern completion.

Mice recognize the center of an enclosure.

In recent years, it has been shown that animals can localize the geometric center of an area by reference to the shape of the environment. We trained a group of mice (experimental group) to search for a pellet hidden under sand in the center of a square-shaped dry maze. Three weeks later, they were tested in a triangular enclosure half the size of the training area and a circular enclosure double the size of the training area to see transfer to these enclosures. We compared their searching behavior with that of subjects that had received no training. The results show that the experimental group searched the geometric center of each enclosure in both transfer tests, while the untrained control group walked along the walls. This indicates that the experimental group localized the center not by reference to the absolute distance from the corners but by equal distances from all walls (geometric center).

Perivertebral B-cell lymphoma in a Queensland koala (Phascolarctos cinereus adustus) with paralytic symptoms in the hind limbs.

A male Queensland koala (Phascolarctos cinereus adustus) at Kanazawa Zoological Gardens (Kanagawa, Japan) exhibited paralytic symptoms in the hind limbs. Computed tomography and magnetic resonance imaging revealed a mass on the left ventral side of the 11th to 13th thoracic vertebrae, and the presence of myelitis or edema in the spinal cord. The koala was under anesthesia during the examination and suddenly developed ventricular fibrillation and died. Necropsy revealed a firm flat ovoid hemorrhagic mass on the vertebrae. Following a microscopic examination including immunohistochemistry, the perivertebral mass was diagnosed as B cell lymphoma. Therefore, neoplastic cell infiltration into the spinal cord may cause paralytic symptoms in the hind limbs.