ABSTRACT
Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S0) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S0 as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S0 conditions, which may allow this organism to survive in a low sulfur environment.IMPORTANCESulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis. We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone.
Subject(s)
Archaeal Proteins , Sulfur , Thermococcus , Thermococcus/genetics , Thermococcus/metabolism , Sulfur/metabolism , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Ubiquitins/metabolism , Ubiquitins/genetics , RNA, Transfer/metabolism , RNA, Transfer/geneticsABSTRACT
Translation-targeting toxic Small Alarmone Synthetases (toxSAS) are effectors of bacterial Toxin-Antitoxin systems that pyrophosphorylate the 3'-CCA end of tRNA to prevent aminoacylation. toxSAS are implicated in antiphage immunity: phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The 1st, 2nd, 4th and 5th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: while the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.
ABSTRACT
Fluorescence lymph imaging with indocyanine green (ICG) is widely utilized as diagnostic tool for lymphatic diseases. While this technique offers numerous advantages, the kinetics of ICG at the injection site can pose challenges for a detailed diagnosis. In this study, we synthesized various ICG derivatives possessing cationic, anionic, or uncharged substituents and examined their photochemical properties, binding affinity to human serum albumin, as well as their correlation to pharmacokinetics in mice. The introduction of different substituents not only affected certain physiochemical properties, but also impacted the pharmacokinetics within the lymph nodes. Immunofluorescence imaging suggested that the extent of uptake of the ICG derivatives by phagocytic cells may affect the retention of the contrast ratios in the lymph nodes. These findings can provide new insights in the pharmacokinetics in lymphatic tissues, which could be useful for the development of novel fluorescent agents for lymph imaging.
Subject(s)
Indocyanine Green , Lymph Nodes , Indocyanine Green/chemistry , Animals , Mice , Humans , Lymph Nodes/diagnostic imaging , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Optical Imaging , Molecular StructureABSTRACT
PURPOSE: The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184). MATERIALS AND METHODS: VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response. RESULTS: Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas. CONCLUSION: This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.
Subject(s)
Cancer Vaccines , Neurilemmoma , Vascular Endothelial Growth Factor Receptor-1 , Vascular Endothelial Growth Factor Receptor-2 , Humans , Male , Female , Adult , Middle Aged , Vascular Endothelial Growth Factor Receptor-2/immunology , Cancer Vaccines/therapeutic use , Cancer Vaccines/adverse effects , Cancer Vaccines/administration & dosage , Neurilemmoma/immunology , Vascular Endothelial Growth Factor Receptor-1/immunology , Skin Neoplasms/immunology , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Neurofibromatosis 2 , Young Adult , Aged , NeurofibromatosesABSTRACT
RNA molecules are modified post-transcriptionally to acquire their diverse functions. Transfer RNA (tRNA) has the widest variety and largest numbers of RNA modifications. tRNA modifications are pivotal for decoding the genetic code and stabilizing the tertiary structure of tRNA molecules. Alternation of tRNA modifications directly modulates the structure and function of tRNAs and regulates gene expression. Notably, thermophilic organisms exhibit characteristic tRNA modifications that are dynamically regulated in response to varying growth temperatures, thereby bolstering fitness in extreme environments. Here, we review the history and latest findings regarding the functions and biogenesis of several tRNA modifications that contribute to the cellular thermotolerance of thermophiles.
Subject(s)
Thermotolerance , Thermotolerance/genetics , RNA Processing, Post-Transcriptional , Genetic Code , RNA, Transfer/genetics , RNA/geneticsABSTRACT
The 40-50 RNA modifications of the epitranscriptome regulate posttranscriptional gene expression. Here we show that flaviviruses hijack the host tRNA epitranscriptome to promote expression of pro-viral proteins, with tRNA-modifying ALKBH1 acting as a host restriction factor in dengue virus infection. Early in the infection of human Huh-7 cells, ALKBH1 and its tRNA products 5-formylcytidine (f5C) and 2'-O-methyl-5-formylcytidine (f5Cm) were reduced. ALKBH1 knockdown mimicked viral infection, but caused increased viral NS3 protein levels during infection, while ALKBH1 overexpression reduced NS3 levels and viral replication, and increased f5C and f5Cm. Viral NS5, but not host FTSJ1, increased f5Cm levels late in infection. Consistent with reports of impaired decoding of leucine UUA codon by f5Cm-modified tRNALeu(CAA), ALKBH1 knockdown induced translation of UUA-deficient transcripts, most having pro-viral functions. Our findings support a dynamic ALKBH1/f5Cm axis during dengue infection, with virally-induced remodeling of the proteome by tRNA reprogramming and codon-biased translation.
ABSTRACT
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.
Subject(s)
Bacterial Proteins , Cryoelectron Microscopy , DNA Transposable Elements , Deinococcus , Endodeoxyribonucleases , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA/ultrastructure , DNA Transposable Elements/genetics , RNA, Guide, CRISPR-Cas Systems/chemistry , RNA, Guide, CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems/metabolism , RNA, Guide, CRISPR-Cas Systems/ultrastructure , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/ultrastructure , Deinococcus/enzymology , Deinococcus/genetics , Substrate SpecificityABSTRACT
Post-transcriptional modifications have critical roles in tRNA stability and function1-4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2'-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2'-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.
Subject(s)
Archaea , RNA, Transfer , Thermotolerance , Archaea/genetics , Extreme Environments , Phosphorylation , RNA Processing, Post-Transcriptional , RNA, Archaeal/chemistry , RNA, Archaeal/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , UridineABSTRACT
N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5' splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.
Subject(s)
Gene Expression Regulation, Fungal , RNA Splicing , RNA, Fungal/metabolism , RNA, Small Nuclear/metabolism , Schizosaccharomyces/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Adenosine/analogs & derivatives , Adenosine/metabolism , Exons/genetics , Mass Spectrometry , Methyltransferases/genetics , Methyltransferases/isolation & purification , Methyltransferases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splice Sites/genetics , RNA, Fungal/genetics , RNA, Small Nuclear/genetics , RNA-Seq , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear , Ribonucleoprotein, U5 Small Nuclear , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/isolation & purification , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Delivery systems to lymph node-resident T cells around tumor tissues are essential for cancer immunotherapy, in order to boost the immune responses. We previously reported that anionic dendrimers, such as carboxyl-, sulfonyl-, and phosphate-terminal dendrimers, were efficiently accumulated in lymph nodes via the intradermal injection. Depending on the terminal structure, their cell association properties were different, and the carboxyl-terminal dendrimers did not associate with any immune cells majorly. In this study, we investigated the delivery of carboxyl-terminal dendrimers with different hydrophobicity to lymph node-resident lymphocytes. Four types of carboxyl-terminal dendrimers-succinylated (C) and 2-carboxy-cyclohexanoylated (Chex) dendrimers with and without phenylalanine (Phe)-were synthesized and named C-den, C-Phe-den, Chex-den, and Chex-Phe-den, respectively. Chex-Phe-den was well associated with lymphocytes, but others were not. Chex-Phe-den, intradermally injected at the footpads of mice, was accumulated in the lymph node, and was highly associated with the lymphocytes, including T cells. Our results suggest that Chex-Phe-den has the potential for delivery to the lymph node-resident T cells, without any specific T cell-targeted ligands.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The development of drug delivery vehicles to cancer and/or immune cells in lymph nodes is important for cancer diagnosis, therapy, and immunotherapy. We previously reported that anionic carboxyl-terminal dendrimers were accumulated in lymph nodes. In this study, three anionic dendrimers with carboxyl-, sulfonyl-, and phosphate-terminal groups were prepared to examine the lymph node targeting and the association with immune cells in the lymph nodes. These anionic dendrimers were accumulated in the lymph node by intradermal injection. Although the carboxyl- and sulfonyl-terminal dendrimers were diffused from the injection site, the phosphate-terminal dendrimers were mostly retained. The phosphate-terminal dendrimer was recognized by the macrophages, dendritic cells, and B cells in the lymph node, whereas the carboxyl- and sulfonyl-terminal dendrimers were not. Our results show that these anionic dendrimers were accumulated in the lymph node where the association with immune cells could be controlled by the terminal structure of the dendrimer. The phosphate-terminal dendrimer can be used as a nanoplatform for the delivery of some bioactive molecules to some immune cells, including B cells, in the lymph node.
Subject(s)
Dendrimers/pharmacokinetics , Drug Carriers , Fluorescent Dyes/pharmacokinetics , Lymph Nodes/metabolism , Nanoparticles , Optical Imaging , Animals , Dendrimers/administration & dosage , Dendrimers/chemical synthesis , Drug Compounding , Female , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Injections, Intradermal , Lymph Nodes/diagnostic imaging , Lymph Nodes/immunology , Mice, Inbred BALB C , Phosphorylation , Tissue DistributionABSTRACT
The anti-VEGF antibody bevacizumab has shown efficacy for the treatment of neurofibromatosis type 2 (NF2). Theoretically, vascular endothelial growth factor receptors (VEGFRs)-specific cytotoxic T lymphocytes (CTLs) can kill both tumor vessel cells and tumor cells expressing VEGFRs. Here we show an exploratory clinical study of VEGFRs peptide vaccine in seven patients with progressive NF2-derived schwannomas. Hearing improves in 2/5 assessable patients (40%) as determined by international guidelines, with increases in word recognition scores. Tumor volume reductions of ≥20% are observed in two patients, including one in which bevacizumab had not been effective. There are no severe adverse events related to the vaccine. Both VEGFR1-specific and VEGFR2-specific CTLs are induced in six patients. Surgery is performed after vaccination in two patients, and significant reductions in the expression of VEGFRs in schwannomas are observed. Therefore, this clinical immunotherapy study demonstrates the safety and preliminary efficacy of VEGFRs peptide vaccination in patients with NF2.
Subject(s)
Brain Neoplasms/therapy , Cancer Vaccines/administration & dosage , Neurofibromatosis 2/therapy , Vascular Endothelial Growth Factor Receptor-1/immunology , Vascular Endothelial Growth Factor Receptor-2/immunology , Adolescent , Adult , Bevacizumab/therapeutic use , Brain/diagnostic imaging , Brain/pathology , Brain/surgery , Brain Neoplasms/immunology , Brain Neoplasms/pathology , Cancer Vaccines/adverse effects , Cancer Vaccines/immunology , Combined Modality Therapy/adverse effects , Combined Modality Therapy/methods , Female , Humans , Magnetic Resonance Imaging , Male , Neurofibromatosis 2/immunology , Neurofibromatosis 2/pathology , Radiosurgery , Tomography, X-Ray Computed , Treatment Outcome , Tumor Burden/immunology , Vaccination/adverse effects , Vaccination/methods , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/adverse effects , Vaccines, Subunit/immunology , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Young AdultABSTRACT
Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.
Subject(s)
Guanosine/analogs & derivatives , Mutagenesis/genetics , RNA, Transfer/genetics , Thermococcus/genetics , Base Sequence , Chromatography, Liquid , Gene Expression Regulation, Archaeal , Guanosine/chemistry , Guanosine/genetics , Nucleosides/chemistry , Nucleosides/genetics , Tandem Mass Spectrometry , TemperatureABSTRACT
OBJECTIVE: The purpose of this study was to clarify the clinical and molecular characteristics associated with long-term survival in patients with glioblastoma. METHODS: We analyzed the characteristics of 96 glioblastoma patients. Long-term survivors (LTSs) were classified into moderate LTSs (mLTSs), who survived >3 years, and LTSs, who survived >5 years, and compared with short-term survivors (STSs). Clinical and molecular factors were investigated. RESULTS: Younger age, better recursive partitioning analysis class, lack of subventricular zone (SVZ) involvement, promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene, and loss of 19q were associated with mLTSs as compared with STSs. After adjustment for these factors, younger age and MGMT methylation remained independently associated with mLTSs. Younger age, better recursive partitioning analysis class, lack of SVZ involvement, and loss of 19q were associated with LTSs as compared with STSs. After adjustment, younger age and better preoperative Karnofsky performance scale (KPS) score remained independently associated with LTSs. Kaplan-Meier analyses revealed that younger age (<50 years), better preoperative KPS score (≥70), lack of SVZ involvement, and loss of 19q were associated with longer overall survival. In the multivariate analysis, only age was significantly associated with overall survival. CONCLUSIONS: Younger age and better preoperative KPS score were the characteristics associated with LTSs as compared with STSs. MGMT promoter methylation was associated with mLTSs, but not with LTSs. In addition, lack of SVZ involvement and loss of 19q might be prognostic for longer survival.
Subject(s)
Brain Neoplasms/mortality , Glioblastoma/mortality , Adult , Age Factors , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Combined Modality Therapy , DNA Methylation/genetics , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Female , Glioblastoma/genetics , Glioblastoma/therapy , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Prognosis , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Pure aqueductal tumor (PAT) typically originates from pure aqueductal region and is extremely rare. It is radiographically similar to tectal glioma. We examined two patients with PATs who were diagnosed with pilocytic astrocytoma and rosette-forming glioneuronal tumor. Both cases showed a progressive clinical course. It is important to distinguish between PAT and tectal glioma by radiographic imaging because the treatment strategy is different. While observation is common for tectal gliomas, a biopsy is recommended at the same time of endoscopic third ventriculostomy for PAT with hydrocephalus. Low-grade PATs show an aggressive clinical course in some cases. Our two cases also showed aggressive course in spite of no genetic aggressive mutations. Sagittal view by constructive interference in steady state (CISS) imaging was helpful to make an accurate diagnosis of PAT. Close observation is needed if PAT is diagnosed as low-grade tumor.
Subject(s)
Astrocytoma/pathology , Brain Neoplasms/pathology , Cerebral Aqueduct/pathology , Adult , Astrocytoma/diagnostic imaging , Astrocytoma/genetics , Astrocytoma/surgery , Biomarkers, Tumor/genetics , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/genetics , Brain Neoplasms/surgery , Cerebral Aqueduct/diagnostic imaging , Diagnosis, Differential , Humans , Isocitrate Dehydrogenase/genetics , Male , Proto-Oncogene Proteins B-raf/genetics , VentriculostomyABSTRACT
ALKBH1 is a 2-oxoglutarate- and Fe2+-dependent dioxygenase responsible for multiple cellular functions. Here, we show that ALKBH1 is involved in biogenesis of 5-hydroxymethyl-2Î-O-methylcytidine (hm5Cm) and 5-formyl-2Î-O-methylcytidine (f5Cm) at the first position (position 34) of anticodon in cytoplasmic tRNALeu, as well as f5C at the same position in mitochondrial tRNAMet. Because f5C34 of mitochondrial tRNAMet is essential for translation of AUA, a non-universal codon in mammalian mitochondria, ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f5C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. We reconstituted formation of f5C34 on mitochondrial tRNAMetin vitro, and found that ALKBH1 first hydroxylated m5C34 to form hm5C34, and then oxidized hm5C34 to form f5C34. Moreover, we found that the frequency of 1-methyladenosine (m1A) in two mitochondrial tRNAs increased in ALKBH1-knockout cells, indicating that ALKBH1 also has demethylation activity toward m1A in mt-tRNAs. Based on these results, we conclude that nuclear and mitochondrial ALKBH1 play distinct roles in tRNA modification.
Subject(s)
AlkB Homolog 1, Histone H2a Dioxygenase/genetics , Cytidine/analogs & derivatives , Protein Biosynthesis , RNA, Transfer, Met/genetics , AlkB Homolog 1, Histone H2a Dioxygenase/deficiency , Anticodon/chemistry , Anticodon/metabolism , Base Sequence , CRISPR-Cas Systems , Cytidine/metabolism , Cytosol/metabolism , Gene Knockout Techniques , Genetic Complementation Test , HEK293 Cells , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , Mitochondria/metabolism , Nucleic Acid Conformation , Oxidation-Reduction , Oxidative Phosphorylation , RNA, Transfer, Met/metabolismABSTRACT
BACKGROUND: Leukoencephalopathy, cerebral calcifications, and cysts (LCC) is a rare disease that was first reported by Labrune in 1996. A case of adult-onset LCC was successfully followed up for a long period. CASE PRESENTATION: A 30-year-old female presented with visual field disturbance and seizure on several occasions. Radiographic images revealed multiple supratentorial cysts and calcifications in the bilateral nucleus basalis and cerebella. Aspiration, Ommaya reservoir placement, and nodule removal were performed for the responsible cysts, and the patient had a good postoperative course. DISCUSSION: A tiny, strongly enhanced nodule was identified before cyst formation on her radiographic images. Thus, cyst growth may be related to nodule microbleeding. According to our review, if the responsible cyst is located on the noneloquent area, surgical removal of the cyst should be considered. However, if the responsible cyst is located on the eloquent area, the nodule should be first removed because nodules can bleed and enlarge cysts. CONCLUSION: Careful follow-up is needed, especially for cysts with a strongly enhanced nodule.
Subject(s)
Calcinosis/diagnosis , Calcinosis/therapy , Central Nervous System Cysts/diagnosis , Central Nervous System Cysts/therapy , Leukoencephalopathies/diagnosis , Leukoencephalopathies/therapy , Adult , Brain/diagnostic imaging , Brain/pathology , Brain/surgery , Calcinosis/diagnostic imaging , Calcinosis/pathology , Central Nervous System Cysts/diagnostic imaging , Central Nervous System Cysts/pathology , Diagnosis, Differential , Female , Follow-Up Studies , Humans , Leukoencephalopathies/diagnostic imaging , Leukoencephalopathies/pathologyABSTRACT
OBJECTIVE Transcranial motor evoked potential (tMEP) monitoring is popular in neurosurgery; however, the accuracy of tMEP can be impaired by craniotomy. Each craniotomy procedure and changes in the CSF levels affects the current spread. The aim of this study was to investigate the influence of several craniotomies on tMEP monitoring by using C3-4 transcranial electrical stimulation (TES). METHODS The authors used the finite element method to visualize the electric field in the brain, which was generated by TES, using realistic 3D head models developed from T1-weighted MR images. Surfaces of 5 layers of the head (brain, CSF, skull, subcutaneous fat, and skin layer) were separated as accurately as possible. The authors created 5 models of the head, as follows: normal head; frontotemporal craniotomy; parietal craniotomy; temporal craniotomy; and occipital craniotomy. The computer simulation was investigated by finite element methods, and clinical recordings of the stimulation threshold level of upper-extremity tMEP (UE-tMEP) during neurosurgery were also studied in 30 patients to validate the simulation study. RESULTS Bone removal during the craniotomy positively affected the generation of the electric field in the motor cortex if the motor cortex was just under the bone at the margin of the craniotomy window. This finding from the authors' simulation study was consistent with clinical reports of frontotemporal craniotomy cases. A major decrease in CSF levels during an operation had a significantly negative impact on the electric field when the motor cortex was exposed to air. The CSF surface level during neurosurgery depends on the body position and location of the craniotomy. The parietal craniotomy and temporal craniotomy were susceptible to the effect of the changing CSF level, based on the simulation study. A marked increase in the threshold following a decrease in CSF was actually recorded in clinical reports of the UE-tMEP threshold from a temporal craniotomy. However, most frontotemporal craniotomy cases were minimally affected by a small decrease in CSF. CONCLUSIONS Bone removal during a craniotomy positively affects the generation of the electric field in the motor cortex if the motor cortex is just under the bone at the margin of the craniotomy window. The CSF decrease and the shifting brain can negatively affect tMEP ignition. These changes should be minimized to maintain the original conductivity between the motor cortex and the skull, and the operation team must remember the fluctuation of the tMEP threshold.
Subject(s)
Craniotomy/methods , Evoked Potentials, Motor , Monitoring, Intraoperative/methods , Neurosurgical Procedures , Transcranial Direct Current Stimulation , Adult , Aged , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
OBJECTIVE The aim of this study was to determine the most effective electrode montage to elicit lower-extremity transcranial motor evoked potentials (LE-tMEPs) using a minimum stimulation current. METHODS A realistic 3D head model was created from T1-weighted images. Finite element methods were used to visualize the electric field in the brain, which was generated by transcranial electrical stimulation via 4 electrode montage models. The stimulation threshold level of LE-tMEPs in 52 patients was also studied in a practical clinical setting to determine the effects of each electrode montage. RESULTS The electric field in the brain radially diffused from the brain surface at a maximum just below the electrodes in the finite element models. The Cz-inion electrode montage generated a centrally distributed high electric field with a current direction longitudinal and parallel to most of the pyramidal tract fibers of the lower extremity. These features seemed to be effective in igniting LE-tMEPs. Threshold level recordings of LE-tMEPs revealed that the Cz-inion electrode montage had a lower threshold on average than the C3-C4 montage, 76.5 ± 20.6 mA and 86.2 ± 20.6 mA, respectively (31 patients, t = 4.045, p < 0.001, paired t-test). In 23 (74.2%) of 31 cases, the Cz-inion montage could elicit LE-tMEPs at a lower threshold than C3-C4. CONCLUSIONS The C3-C4 and C1-C2 electrode montages are the standard for tMEP monitoring in neurosurgery, but the Cz-inion montage showed lower thresholds for the generation of LE-tMEPs. The Cz-inion electrode montage should be a good alternative for LE-tMEP monitoring when the C3-C4 has trouble igniting LE-tMEPs.