ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

Bone Metastases from Gastric Cancer Resembling Paget's Disease: A Case Report.

Systemic osteosclerotic lesions are frequently caused by multiple bone metastases or systemic metabolic disorders. However, bone metastasis from gastric cancer is rare. Herein, we describe such a case, with radiographic and clinical findings resembling Paget's disease. The patient was an 80-year-old Japanese woman with a history of early gastric cancer, treated by partial gastrectomy 2 years prior. The patient sought medical care for chronic low back pain. On imaging, systemic sclerotic lesions were observed throughout the spine and pelvis, with an increase in bone mineral density from 0.86 g/cm3 (2 years prior) to 1.38g/cm3 (current visit) in the lumbar spine. Elevated serum levels of osteoblastic and osteolytic markers were identified. A bone biopsy was used to confirm the diagnosis of metastatic gastric cancer. The patient was treated with TS-1 and denosumab, with normalization of abnormal metabolic markers and alleviation of the back pain. Bone metastasis is reported in only 10% of cases of gastric cancer and, thus, is relatively rare. Therefore, our case of gastric cancer recurrence presenting with mixed osteoblastic and osteolytic bone lesions similar to Paget's disease is relevant to the report. Bone biopsy is necessary for an accurate diagnosis.

Isolation of SARS-CoV-2 from COVID-19 Patients and an Asymptomatic Individual.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019. Despite the recent introduction of vaccines against SARS-CoV-2, more effective vaccines and antiviral drugs must be developed. Here, we isolated five SARS-CoV-2 strains from four patients with coronavirus disease (COVID-19) and an asymptomatic individual using pharyngeal swabs, nasopharyngeal swabs, and sputum samples. Cytopathic effects in inoculated Vero cells were observed between days 3 and 7. SARS-CoV-2 infection was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing. Phylogenetic analyses of the whole genome sequences showed that the virus isolates from the clinical samples belonged to the Wuhan and European lineages. These findings and the isolated viruses may contribute to the development of diagnostic tools, vaccines, and antiviral drugs for COVID-19.

Chromosomal-scale de novo genome assemblies of Cynomolgus Macaque and Common Marmoset.

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. We assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data, which were further refined based on Hi-C contact maps and alternate de novo assemblies. The assemblies achieved scaffold N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively. The high fidelity of our assembly is also ascertained by BAC-end concordance in common marmoset. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.

Outcomes after decompression surgery without fusion for patients with lumbar spinal stenosis and substantial low back pain.

PURPOSE: The aim of this study was to evaluate the efficacy of decompression surgery alone for patients with intolerable low back pain. METHODS: We retrospectively identified 222 patients who underwent spinal decompression without fusion surgery who had substantial preoperative low back pain (preoperative numerical rating scale score ≥ 5). Their clinical outcomes were assessed using the numerical rating scale and the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) preoperatively and at 3 months and 1 year after surgery. RESULTS: At 3 months and 1 year after surgery compared with baseline, there was a significant improvement in the overall mean numerical rating scale scores for low back pain (baseline = 6.8, 3 months = 2.1, 1 year = 2.7), leg pain (6.8, 2.1, 2.7), and leg numbness (6.4, 2.9, 3.2) (P < 0.05). The efficacy rate assessed by JOABPEQ was 68.1% for pain-related disorders, 47.0% for lumbar spine dysfunction, 63.3% for walking ability, 48.2% for social life dysfunction, and 21.6% for psychological disorders. When patients were classified into three groups depending on their degree of leg pain (mild, moderate, and severe), there was no significant difference in the efficacy rate between the three groups. CONCLUSION: Decompression surgery can improve low back pain, regardless of the degree of preoperative leg pain, but the average score for LBP and leg pain slightly worsened between 3 months and 1 year after surgery. These slides can be retrieved under Electronic Supplementary Material.

Prognostic Factors for Drop Foot Due to Lumbar Degenerative Diseases: The Impact of Surgical Timing on Postoperative Recovery.

STUDY DESIGN: Retrospective clinical series. OBJECTIVE: To investigate the prognostic factors for the improvement of drop foot caused by degenerative lumbar disease after surgery. SUMMARY OF BACKGROUND DATA: Drop foot may be caused by lumbar spinal diseases and may affect daily life. There are limited data regarding predictors of drop foot, especially surgical timing. METHODS: We retrospectively reviewed data from 87 patients with drop foot. Prognostic factors and precise timing of surgery affecting drop foot improvement were examined by multivariate logistic regression analysis. RESULTS: Forty patients (46.0%) recovered from drop foot after surgery. Age [odds ratio (OR)=0.93; 95% confidence interval (CI), 0.87-0.98; P=0.01], preoperative anterior tibialis muscle strength (OR=12.0; 95% CI, 2.41-59.9; P=0.002), and duration of drop foot before surgery (OR=3.59; 95% CI, 1.09-11.8; P=0.04) were significant prognostic factors. Two months was the most appropriate cutoff for surgery after appearance of symptoms. CONCLUSIONS: Age, preoperative tibialis anterior muscle strength, and duration of drop foot were statistically significant prognostic factors of recovery from drop foot. Surgery within 2 months after the onset of drop foot may improve postoperative outcome.

[A Case of Metastatic Mucinous Adenocarcinoma Derived from the Right Ovary to the Ileal Ureter].

A 76-year-old woman presented with temporary right back pain and renal dysfunction. She had undergone ileal ureter interposition for right ureteral resection due to retroperitoneal liposarcoma about forty years ago. Abdominal computed tomographic (CT) scan showed right hydronephrosis and a stenosis in the middle of the ileal ureter. The symptoms were relieved, and the patient was followed. Six months later, however, a contrast enhanced lesion was newly observed at the stenosis of the ileal ureter, and nineteen months later, right ovarian tumor and peritoneal dissemination were subsequently observed. Aiming at diagnosis and cytoreduction, right nephroureterectomy and bilateral oophorectomy were performed. Histopathological findings of the resected tumors showed common characteristics of infiltrating and proliferating adenocarcinoma. Immunohistochemistry findings showed that Cytokeratin 7 and Cytokeratin 20 stained positive. On the other hand, Mucin 2 and Special AT-rich sequence-binding protein 2 were not found. The histopathological diagnosis was metastatic mucinous adenocarcinoma of the ovary with ileal ureter and a wide peritoneal dissemination. The patient rejected adjuvant chemotherapy and a new lesion was found in the pelvic fourmonths afterthe surgery.

Calcaneal insufficiency fractures following total knee arthroplasty: Classification and clinical findings.

BACKGROUND: Calcaneal insufficiency fracture (IF) following total knee arthroplasty (TKA) is a rare disorder. This study aimed to examine the prevalence and clinical findings of calcaneal IF following TKA. METHODS: We retrospectively reviewed 3,585 consecutive patients undergoing primary TKA between 2012 and 2017 in four hospitals. Calcaneal IF following TKA was diagnosed by plain radiography or magnetic resonance imaging. First, we investigated the prevalence and clinical findings of calcaneal IF following TKA. Second, we classified calcaneal IF into three types based on its location: type 1, fracture by traction force around the Achilles tendon insertion; type 2, compression fracture around the posterior subtalar joint; and type 3, fracture by ground reaction force at the bottom of the calcaneus. Finally, we compared the clinical findings between calcaneal IF with and without TKA. RESULTS: Calcaneal IF following TKA was seen in 17 (0.5%) of the 3,585 patients undergoing primary TKA. All patients were female, with a mean age of 76.5 ± 5.9 years, relatively high body mass index (BMI), and osteoporosis. All fractures achieved bone union with conservative treatment. Type 1 fractures were the most common. Calcaneal IFs following TKA were significantly shorter in height and the patients had higher BMI than those without TKA. The locations of calcaneal IF following TKA varied, while only type 1 calcaneal IFs were seen in cases without TKA. However, there were no significant differences with regard to the bone union period or malunion between the two groups. CONCLUSIONS: Calcaneal IF should be suspected in patients presenting with ipsilateral foot pain following TKA, particularly in female patients with a relatively high BMI and osteoporosis. Calcaneal IF can be classified into three types based on the fracture location. These variations in calcaneal IF may be due to differences in conditions and changes in mechanical loading of the lower extremity and bone quality following TKA.

Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells.

Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off-target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off-target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off-target effects at the cellular level is not clear. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for the assessment of off-target effects of gapmer ASOs.

[A Case of Malignant Lymphoma with an Elevated Serum IgG4].

A man in his 60s was referred to our hospital for further examination of left hydronephrosis and renal dysfunction. An enhanced abdominal computed tomographic scan showed that the patient had chronic abdominal aortic dissection and a non-enhancing retroperitoneal soft tissue occupying the front of the abdominal aorta as well as the bilateral common iliac arteries. The left ureter was compressed by the soft tissue at the fourth lumbar level. No tumor markers were significantly elevated and idiopathic retroperitoneal fibrosis was suspected to be the cause. Before starting treatment, however, right hydronephrosis was newly observed. We placed bilateral ureteral stents and reviewed our diagnosis. Elevated serum IgG4 and accumulation of 18F-fluorodeoxyglucose in the soft tissue were the points at issue. To determine the diagnosis, we performed open wedge biopsy. Histopathological findings showed mainly fibrous connective tissue with lymphocytic infiltration, which was positive for CD10, CD20, and bcl-2. These findings indicated follicular lymphoma. Induction chemotherapy was performed with 6 cycles of rituximab/cyclophosphamide/vincristine/prednisolone. The soft tissue tumor shrank markedly and the patient has been free from bilateral ureteral stents.

CXCL4/PF4 is a predictive biomarker of cardiac differentiation potential of human induced pluripotent stem cells.

Selection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening. We aimed to identify biomarkers for predicting cardiac differentiation potential of hiPSC lines by comparing the gene expression profiles of six undifferentiated hiPSC lines with different cardiac differentiation capabilities. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA array, and microRNA array to efficiently screen biomarkers related to cardiac differentiation of hiPSCs. Statistical analysis revealed candidate biomarker genes with significant correlation between the gene expression levels in the undifferentiated hiPSCs and their cardiac differentiation potential. Of the candidate genes, PF4 was validated as a biomarker expressed in undifferentiated hiPSCs with high potential for cardiac differentiation in 13 additional hiPSC lines. Our observations suggest that PF4 may be a useful biomarker for selecting hiPSC lines appropriate for the generation of cardiomyocytes.

Nucleic acid bases in Titan tholins and possible genetic systems in the Titan liquidosphere.

Titan is the largest moon of Saturn and possesses a dense atmosphere composed of nitrogen and methane. Various types of organic compounds (hydrocarbons, nitriles, etc.) have been found on Titan, which were generated by reactions taking place in its atmosphere. These reactions are considered to provide crucial evidence for chemical reactions which may have occurred in the atmosphere of primitive Earth. Cassini discovered several lakes of liquid methane and ethane on Titan's surface; in addition, the presence of ammonia water in its sub-surface was implied. In order to simulate the chemical reactions in Titan's atmosphere, gas mixtures of nitrogen and methane have been exposed to plasma discharges to synthesize complex organic matters. In this study, we focused on the formation of nucleic acid bases and related compounds recovered from synthesized Titan tholins. The five nucleic acid bases that terrestrial life uses (adenine, cytosine, thymine, guanine, and uracil) have already been reported to be present in synthesized Titan tholins. Purines and pyrimidines, including the five aforementioned nucleic acid bases, were extracted from synthesized Titan tholins and analyzed by HPLC and LC/MS. As a result, the pyrimidine bases of isocytosine and 2, 4-diaminopyrimidine were detected together with the terrestrial nucleic acid bases of adenine, uracil, and cytosine. The results obtained in conjunction with those from previous studies show that some nucleic acid bases and related pyrimidine bases are found in synthesized Titan tholins, suggesting that chemical evolutions toward xenogenetic systems could occur in Titan's environment.

[Two-Stage Radical Resection of a Case of Colon Cancer with Abdominal Wall Invasion and Cholangiocarcinoma].

A woman in her 50s was admitted to our hospital with fever and lower abdominal swelling. Abdominal CT/MRI examinations revealed irregular thickening of the transverse colon wall, which was attached to a subcutaneous abscess. An abdominal wall mass, a patent urachus, and a tumor in the 5th segment of the liver were also noted. Colonoscopy revealed type 2 advanced transverse colon cancer. The solitary, sessile tumor was observed at the apex of the bladder under cystoscopy, suggesting the formation of the urachal carcinoma. Transcutaneous liver biopsy obtained from the liver tumor indicated adenocarcinoma, which was morphologically different from the existing transverse colon cancer. Right hemicolectomy with resection of the umbilicus, abdominal wall, urachus, and part of the bladder wall was performed. Diagnosis of the transverse colon cancer invading the abdominal wall and bladder was confirmed by histopathological examination. Hepatectomy was performed in the next surgery, and the tumor was histopathologically diagnosed as an intrahepatic cholangiocarcinoma. Both the transverse colon cancer and the intrahepatic cholangiocarcinoma were radically resected. Radical surgical diagnostic resection may be valuable in cases of multicentric cancers of unknown primary origin, if radical resection of each individual tumor is required.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

[A Case Report : Metastatic Carcinoma of the Collecting Ducts of Bellini in a Hemodialysis Patient Treated with Temsirolimus].

Carcinoma of the collecting ductsof Bellini isa rare histological subtype of renal cell carcinoma and mostly has unfavorable prognosis. Radical nephrectomy is generally chosen for the 1st line treatment but therapeutic approaches for the metastasis/recurrence have not been established. We report a case of carcinoma of collecting ducts of Bellini in a patient receiving hemodialysis treated with temsirolimus. A 62- year-old man receiving hemodialysis was admitted to our hospital with drug-resistant anemia and high-grade cyclic fever. Computed tomography revealed the right renal tumor and multiple metastatic lung tumors. Open radical nephrectomy wasperformed. Pathological findingswere compatible with carcinoma of the collecting ducts of Bellini. He was given weekly temsirolimus treatment. The disease progressed modestly but kept the stable disease (SD) status for six months. He died of the cancer 11 months after the initial diagnosis.

Adsorption of RNA on mineral surfaces and mineral precipitates.

The prebiotic significance of laboratory experiments that study the interactions between oligomeric RNA and mineral species is difficult to know. Natural exemplars of specific minerals can differ widely depending on their provenance. While laboratory-generated samples of synthetic minerals can have controlled compositions, they are often viewed as "unnatural". Here, we show how trends in the interaction of RNA with natural mineral specimens, synthetic mineral specimens, and co-precipitated pairs of synthetic minerals, can make a persuasive case that the observed interactions reflect the composition of the minerals themselves, rather than their being simply examples of large molecules associating nonspecifically with large surfaces. Using this approach, we have discovered Periodic Table trends in the binding of oligomeric RNA to alkaline earth carbonate minerals and alkaline earth sulfate minerals, where those trends are the same when measured in natural and synthetic minerals. They are also validated by comparison of co-precipitated synthetic minerals. We also show differential binding of RNA to polymorphic forms of calcium carbonate, and the stabilization of bound RNA on aragonite. These have relevance to the prebiotic stabilization of RNA, where such carbonate minerals are expected to have been abundant, as they appear to be today on Mars.

Comment on "Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs".

Meinert et al (Reports, 8 April 2016, p. 208) reported the formation of prebiotic molecules, including ribose, in an interstellar ice analog experiment. We show that if their experimental procedure is accurately described, much or most of their products may have been formed during their analysis process, not in the parent ice.

[A Case of Triple Negative Spindle Cell Carcinoma of the Breast and Improved Quality of Life Following Irinotecan Chemotherapy].

We report a case of triple negative spindle cell carcinoma of the breast, responsive to irinotecan chemotherapy. A 49-year old woman who had a tumor in the chest wall with a skin ulcer visited our hospital. After being diagnosed with triple negative spindle cell carcinoma of the breast, she underwent surgery, adjuvant chemotherapy, and radiation at the other hospital. Fourteen months after the surgery, she developed an ipsilateral breast tumor as a result of local recurrence. Since eribulin and paclitaxel plus bevacizumab chemotherapies were not effective, she was transferred to our hospital, and we administered irinotecan as third-line chemotherapy. Skin lesions and effusion were reduced and her quality of life improved for 4 months.

[A Case of Unresectable Locally Advanced Pancreatic Cancer Resulted in R0 Surgery after Chemotherapy].

A 67-year-old man visited our hospital for jaundice. Abdominal dynamic CT showed the hypovascular tumor at the head of the pancreas that surrounded superior mesenteric artery(SMA)at an angle of 220 degree. No metastasis in lymph nodes and other organs was observed. We diagnosed the tumor unresectable locally advanced(UR-LA)pancreatic cancer. Chemotherapy was administered with gemcitabine and nab-paclitaxel(GEM+nab-PTX)and achieved partial response. Regression in size and in range around SMA to an angle of 150 was observed. We assessed it possible to resect the tumor curatively, and performed subtotal stomach preserving pancreaticoduodenectomy and dissection of the plexus around the SMA, resulted in R0 surgery. Adjuvant chemotherapy was administered, and no recurrence was observed up to present, more than a year. It is suggested that GEM+nab-PTX can be effective as the primary therapy against UR-LA pancreatic cancer.