Recent advances in liquid biopsy of central nervous system lymphomas: case presentations and review of the literature.

Surgical biopsy is the gold standard for diagnosing central nervous system (CNS) lymphomas. However, reliable liquid biopsy methods for diagnosing CNS lymphomas have quickly developed and have been implicated in clinical decision-making. In the current report, we introduce two patients for whom liquid biopsy was essential for diagnosing CNS lymphomas and discuss the rapidly growing applications of this technology.

Missense mutation of NRAS is associated with malignant progression in neurocutaneous melanosis.

Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated, and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal (VP) shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via VP shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three mutations, namely GNAQ R183Q, S1PR3 G89S and NRAS G12V, considered pathogenic, were found, although S1PR3 mutations have not been previously reported in melanocytic tumors. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites, with copy-neutral loss-of-heterozygosity (CN-LOH) occurring in tumor. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discovering novel driver mutations associated with tumorigenesis and targeted therapy.

Successful Treatment of Acute Uric Acid Nephropathy with Rasburicase in a Primary Central Nervous System Lymphoma Patient Showing a Dramatic Response to Methotrexate-Case Report.

BACKGROUND: Primary central nervous system lymphomas (PCNSLs) are sensitive to chemotherapy. The standard treatment is high-dose methotrexate (MTX)-based chemotherapy. There are no reports of successful treatment of acute uric acid nephropathy with rasburicase after MTX administration in PCNSLs. CASE PRESENTATION: A 54-year-old man with a history of gout presented with a change in character and cognitive dysfunction. MRI showed a large enhancing mass spanning the bilateral frontal lobes and the right temporal lobe. After endoscopic biopsy, an MTX, procarbazine, and vincristine (MPV) regimen was initiated for the treatment of the PCNSL. After the initiation of chemotherapy, the patient experienced a gout attack, and blood examination revealed acute renal failure (ARF) and hyperuricemia. The considered causes of ARF included MTX toxicity and acute uric acid nephropathy. As the dramatic effect of MTX was observed, treatment was continued despite ARF, most probably due to acute hyperuricemia due to tumor lysis, which was treated in parallel. After an improvement in renal function, MTX was resumed, and rasburicase was initiated to control hyperuricemia. A complete response was obtained after induction chemotherapy. Hyperuricemia was controlled with rasburicase, and renal function was preserved. CONCLUSIONS: Acute uric acid nephropathy should be considered when ARF occurs after the initiation of MTX in PCNSLs, especially in newly diagnosed PCNSL patients with large tumors or hyperuricemia.

Efficacy of Emergency Room Skip Strategy in Patients Transferred for Mechanical Thrombectomy.

Objective: Time to recanalization is directly linked to cerebral infarction prognosis. However, patients transferred from another hospital take longer to arrive than those transported directly. To minimize time to recanalization, the emergency room (ER) skip strategy for hospital transfers was executed and reviewed. Methods: From April 2019, patients transferred from another hospital for mechanical thrombectomy were carried into the angio-suite using emergency service stretchers. Results for these patients (ER skip group) were compared with those for patients transported directly to our hospital (Direct group). Results: Among 108 cases in 32 months, 99 patients (91.7%) had major cerebral artery occlusion and underwent endovascular treatment. No differences in age, baseline National Institutes of Health Stroke Scale score, effective recanalization rate, or proportion of posterior circulation cases were seen between groups. The ER skip group (26 patients) showed significantly longer median time from onset to arrival (240 vs. 120 min; p = 0.0001) and significantly shorter median time from arrival to groin puncture (11 vs. 69 min; p = 0.0000). No significant differences were evident in time from groin puncture to recanalization (39 vs. 45 min), time from onset to recanalization (298 vs. 244 min), or rate of modified Rankin Scale score 0-2 after 90 days (42.3% vs. 32.9%). Median time from alarm to recanalization (266 vs. 176 min; p = 0.0001) was significantly longer in the ER skip group. Door-to-puncture (DTP) time for the Direct group gradually fell as the number of cases increased, reaching 40 min by the end of study period. In contrast, DTP time for the ER skip group remained extremely short and did not change further. The proportion of patients who underwent both CT and MRI before endovascular treatment was significantly lower in the Direct group (30.1%) than in the ER skip group (57.7%). In the ER skip group, median length of stay in the primary hospital was 119 min, and the median duration of interhospital transfer was 16 min. Conclusion: The ER skip strategy for patients transferred with large vessel occlusion achieved favorable outcomes comparable to that for direct transport cases. Direct transport to a thrombectomy-capable stroke center remains ideal, however, because the time to intervention is improving for direct transport cases each year.

Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensisIMPORTANCEActinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

Regulation of sporangium formation by the orphan response regulator TcrA in the rare actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n4 -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.

Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD 3',5'-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5'-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3' as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation.IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis.

UNLABELLED: Actinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of the fliQ gene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption, fliQ, fliC, fliK, and lytA Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared from A. missouriensis grown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5' ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15-17)GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously. IMPORTANCE: The biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes in A. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs. We identified a large flagellar gene cluster and an orphan flagellar gene (fliQ). These findings indicate that the zoospore flagellar components are typical of Gram-positive bacteria. However, the transcriptional analysis revealed that all flagellar genes are transcribed simultaneously during sporangium formation, a pattern differing from the orderly, regulated expression of flagellar genes in other bacteria, such as Salmonella and Escherichia coli These results suggest a novel regulatory mechanism for flagellar formation in A. missouriensis.

Proteomic analysis of the Streptomyces griseus ribosomal fraction.

The Streptomyces griseus 70S ribosome fraction was analyzed by radical-free and highly reducing two-dimensional (RFHR 2D) gel electrophoresis and mass spectrometry. Among the 60 putative ribosomal proteins that are encoded by the S. griseus genome, 48 were identified in the 70S ribosome fraction prepared from mycelia grown in liquid culture for 12, 36, and 48 h. Ribosomal protein S3 was detected at two different positions on the 2D gel, and the distribution changed completely in the course of the growth, suggesting that it was modified or processed. The SGR3624 protein was also identified in the 70S ribosome fraction, but detailed cellular fractionation analysis indicated that it localizes mainly at the membrane rather than the ribosome. An SGR3624-deleted mutant showed slow growth on solid media, indicating that SGR3624 has an important role in the growth of the substrate mycelium in solid culture.

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures.

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.

Brain-derived neurotrophic factor enhances periodontal tissue regeneration.

To address whether brain-derived neurotrophic factor (BDNF) could be involved in periodontal tissue regeneration, we examined the effects of BDNF on proliferation and the expression of bone (cementum)- related proteins (osteopontin, bone morphogenetic protein [BMP]-2, type I collagen, alkaline phosphatase [ALPase], and osteocalcin) in cultures of human periodontal ligament (HPL) cells, which are thought to be prerequisite for periodontal tissue regeneration, and on proliferation and angiogenesis in human endothelial cells. Furthermore, we examined the effect of BDNF on the regeneration of periodontal tissues in experimentally induced periodontal defects in dogs. BDNF elevated the expression of ALPase and osteocalcin mRNAs and increased the synthesis of osteopontin, BMP-2, and type I collagen DNA in HPL cells. BDNF stimulated mRNA expression of vascular endothelial growth factor-B and tenascin-X, and proliferation and angiogenesis in human endothelial cells. In vivo studies showed that BDNF stimulated the formation of new alveolar bone cementum and connective new fibers, which were inserted into the newly formed cementum and bone. BDNF also stimulated blood capillary formation. These findings suggest that the regulation of functioning of periodontal ligament cells and endothelial cells by BDNF results in the promotion of periodontal tissue regeneration.

Characterization of epithelial cells derived from periodontal ligament by gene expression patterns of bone-related and enamel proteins.

The cells of epithelial rests of Malassez (ERM) are thought to play a number of roles, such as protection against root resorption and cementoblast differentiation. However, little is known about characteristics of these cells. In the present study, we compared the expression patterns of the bone-related proteins osteopontin (OPN), osteonectin/secreted protein, acidic and rich in cysteine (SPARC), osteoprotegerin (OPG), bone morphogenetic proteins (BMP-2 and BMP-4) and the enamel matrix proteins amelogenin and tuftelin in epithelial cells derived from human periodontal ligament (ECHPL) with those of human gingival epithelial cells (HGEC) and oral mesenchymal cells (human gingival fibroblasts, human periodontal ligament fibroblasts and human pulp cells). The mRNA expression patterns were determined by the reverse-transcription polymerase chain reaction (RT-PCR). Cytokeratin 8 mRNA was expressed by oral epithelial cells but not oral mesenchymal cells. The OPN mRNA levels in ECHPL were by far the highest in the five cell types investigated. ECHPL and oral mesenchymal cells expressed OPG mRNA, whereas HGEC did not. BMP-2, SPARC and tuftelin mRNAs were detected in ECHPL and the other cells examined. The oral mesenchymal cells expressed BMP-4 mRNA much more strongly than did the oral epithelial cells. Amelogenin mRNA expression could not be detected in any of the cells. These findings suggest that cultured ERM cells are characterized by expression of the cytokeratin 8, OPG and OPN genes.

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells.

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.

Enhancement of alkaline phosphatase synthesis in pulp cells co-cultured with epithelial cells derived from lower rabbit incisors.

Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.

Differential gene expression of bone-related proteins in epithelial and fibroblastic cells derived from human periodontal ligament.

Hertwig's epithelial root sheath (HERS) is involved in the differentiation of cementoblasts. The cells of epithelial rests of Malassez (ERM) may contribute to that process. However, little is known about the role of these epithelial cells in cementum repair. In the present study, we investigated the expression of alkaline phosphatase (ALPase), osteopontin (OPN), bone morphogenetic protein (BMP)-2 and BMP-4 in epithelial cells (E cells) and fibroblastic cells (F cells) derived from the same human periodontal ligament. E cells were identified by immunoblotting with anti-cytokeratin 5 and 8 antibody. Reverse transcription-polymerase chain reaction analysis showed that E cells have lower ALPase and BMP-4 mRNA levels than F cells. On the other hand, the expression of OPN mRNA in E cells was stronger than in F cells. No significant difference was observed in BMP-2 expression between E and F cells. Thus, they have different expression patterns of ALPase, BMP-4 and OPN, suggesting that ERM and mesenchymal cells in periodontal ligament may be cooperatively involved in cementum repair. Furthermore, E cell cultures will be useful in elucidating the role of ERM.

Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells.

Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.

The effect of extracellular calcium ion on gene expression of bone-related proteins in human pulp cells.

Calcium hydroxide is often used for induction of reparative dentin formation in endodontic treatment. However, little is known about the mechanism by which calcium hydroxide works. The calcium ion (Ca2+) is an important regulator of cell functions. In this study, we examined the effect of extracellular Ca2+ on gene expression of bone-related proteins in human cultured pulp cells in serum-free conditions. A Ca2+ level elevated by 0.7 mM induced an increase in mRNA expression of osteopontin and bone morphogenetic protein (BMP)-2. However, mRNA levels of BMP-4 and alkaline phosphatase decreased under the elevated Ca2+ culture condition. The same concentration of additional magnesium ions had little effect on expressions of the examined bone-related protein mRNAs. These findings suggest that Ca2+ in Ca(OH)2 specifically modulates osteopontin and BMP-2 levels during calcification in pulp.