The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

DNA-functionalized colloidal crystals for macromolecular encapsulation.

Novel DNA-based structures with the ability to encapsulate nanoscale molecules, such as proteins, can be applied to a wide range of areas, including reaction fields and micro/nano drug carriers. DNA-functionalized nanoparticle (DNA-NP) colloidal crystals have emerged as a new class of programmable DNA-based structures harboring metal nanoparticles with improved mechanical properties. The encapsulation of guest molecules into empty spaces in lattice structures is theoretically possible. However, due to the lack of a strategy for versatile encapsulation of guest molecules, the feasibility of nanoscale encapsulation by DNA-NP crystals is unclear. In this study, we developed DNA-functionalized gold nanoparticle (DNA-AuNP) crystals with tunable interparticle spacing for molecular encapsulation. We demonstrated that the modification of DNA-AuNP crystals with functional moieties, that is, biotin molecules, was effective in retaining molecules in the crystals. The crystallinities before and after encapsulation of the molecules were confirmed using small-angle X-ray scattering. We also succeeded in encapsulating CRISPR/Cas9 ribonucleoproteins into DNA-AuNP crystals by harnessing their affinity for target molecules. These findings demonstrated the potential use of metal-DNA hybrid crystals as carriers for direct protein delivery via biolistic bombardment. Thus, this study provides an attractive strategy for creating a new class of DNA-based structures for macromolecular encapsulation, and an alternative research direction toward colloidal crystal engineering using DNA.

KLU/CYP78A5, a Cytochrome P450 Monooxygenase Identified via Fox Hunting, Contributes to Cuticle Biosynthesis and Improves Various Abiotic Stress Tolerances.

Acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana (Arabidopsis) accessions. Most salt-tolerant accessions exhibit acquired osmotolerance, whereas Col-0 does not. To identify genes that can confer acquired osmotolerance to Col-0 plants, we performed full-length cDNA overexpression (FOX) hunting using full-length cDNAs of halophyte Eutrema salsugineum, a close relative of Arabidopsis. We identified EsCYP78A5 as a gene that can confer acquired osmotolerance to Col-0 wild-type (WT) plants. EsCYP78A5 encodes a cytochrome P450 monooxygenase and the Arabidopsis ortholog is known as KLU. We also demonstrated that transgenic Col-0 plants overexpressing AtKLU (AtKLUox) exhibited acquired osmotolerance. Interestingly, KLU overexpression improved not only acquired osmotolerance but also osmo-shock, salt-shock, oxidative, and heat-stress tolerances. Under normal conditions, the AtKLUox plants showed growth retardation with shiny green leaves. The AtKLUox plants also accumulated higher anthocyanin levels and developed denser cuticular wax than WT plants. Compared to WT plants, the AtKLUox plants accumulated significantly higher levels of cutin monomers and very-long-chain fatty acids, which play an important role in the development of cuticular wax and membrane lipids. Endoplasmic reticulum (ER) stress induced by osmotic or heat stress was reduced in AtKLUox plants compared to WT plants. These findings suggest that KLU is involved in the cuticle biosynthesis, accumulation of cuticular wax, and reduction of ER stress induced by abiotic stresses, leading to the observed abiotic stress tolerances.

Genome-wide characterization of the TALE homeodomain family and the KNOX-BLH interaction network in tomato.

KEY MESSAGE: Comprehensive yeast and protoplast two-hybrid analyses illustrated the protein-protein interaction network of the TALE homeodomain protein family, KNOX and BLH proteins, in tomato leaf and fruit development. KNOTTED-like (KNOX, KN) proteins and BELL1-like (BLH) proteins, which belong to the same TALE homeodomain family, act together by forming KNOX-BLH heterodimer modules. These modules play crucial roles in regulating multiple developmental processes in plants, like organ differentiation. However, despite the increasing knowledge about individual KNOX and BLH functions, a comprehensive view of their functional protein-protein interaction (PPI) network remains elusive in most plants, including tomato (Solanum lycopersicum), an important model plant to study fruit and leaf development. Here, we characterized eight tomato KNOX genes (SlKN1 to SlKN8) and fourteen tomato BLH genes (SlBLH1 to SlBLH14) by expression profiling, co-expression analysis, and PPI network analysis using two-hybrid techniques in yeasts (Y2H) and protoplasts (P2H). We identified 75 pairwise KNOX-BLH interactions, including ten novel interactors of SlKN2/TKN2, a primary class I KNOX protein, and nine novel interactors of SlKN5, a primary class II KNOX protein. Based on these data, we classified KNOX-BLH modules into several categories, which made us infer the order and combination of the KNOX-BLH modules involved in differentiation processes in leaf and fruit. Notably, the co-expression and interaction of SlKN5 and fruit preferentially expressing BLH1-clade paralogs (SlBLH5/SlBEL11 and SlBLH7) suggest their important roles in regulating fruit differentiation. Furthermore, in silico modeling of the KNOX-BLH modules, sequence analysis, and P2H assay identified several residues and a linker region potentially influencing the affinity of BLHs to KNOXs within their conserved dimerization domains. Together, these findings provide insights into the regulatory mechanism of KNOX-BLH modules underlying tomato organ differentiation.

Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNAHis, producing mature tRNAHis In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G-1 Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs.

A Case of Wallenberg's Syndrome Presenting with Spontaneous Thrombosis of a Vertebral Artery Aneurysm.

Complete spontaneous thrombosis of intracranial aneurysms is uncommon. Although this type of thrombosis is largely asymptomatic, in rare cases it can be accompanied by parent artery occlusion and ischemic stroke. There are limited reports of complete thrombosis of an unruptured aneurysm of the internal carotid artery and middle cerebral artery. Furthermore, there are no reports of occlusion of the vertebral artery caused by thrombosis of an aneurysm. The mechanisms of spontaneous thrombosis are not established. However, aneurysm morphology, arteriosclerosis, and stagnation of aneurysm flow have been suggested. Herein, we present a novel case of Wallenberg's syndrome caused by a fusiform aneurysm in which complete thrombosis of the proximal vertebral artery occurred. We discuss the mechanisms of thrombosis caused by an unruptured aneurysm, which may be useful for managing such patients who present with transient ischemic attacks.

Biochemical analysis of human tRNAHis guanylyltransferase in mitochondrial tRNAHis maturation.

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at the -1 position (G-1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5'-terminal of ctRNAHis and then removes the 5'-pyrophosphate to yield the mature 5'-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHisin vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5'-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5'-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans.

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3'-5' direction onto the 5'-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the -1 position (G-1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G-1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G-1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson-Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3'-5' polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

Severe Reversible Cerebral Vasoconstriction Syndrome with Large Posterior Cerebral Infarction.

Reversible cerebral vasoconstriction syndrome is characterized by thunderclap headache and multifocal cerebral vasoconstriction. Cerebral vasoconstriction is reversible, and most cases have good prognosis. However, clinical outcome is possibly severe when it is complicated by stroke, yet detailed reports on such a case are few. We experienced a case of severe reversible cerebral vasoconstriction syndrome in a 32-year-old woman with medical history of preeclampsia 3years prior. She presented with sudden sharp headache followed by altered mental status and vasoconstriction of the bilateral posterior cerebral arteries. She was treated with intravenous and oral calcium channel blockers, edaravone, and glycerol. However, the cerebral infarction in the posterior circulation subsequently remained, and her impaired consciousness did not recover. Furthermore, although imaging findings of vasoconstriction showed improvement a day after the occurrence of symptom, the same vessels showed poor visualization 7 weeks later, which indicated the recurrence of vasoconstriction, without additional symptom due to the fixed infarction. Although most cases of reversible cerebral vasoconstriction syndrome show good prognosis, neurologists must monitor the possibility of worse clinical course and permanent neurological deficit when associated with stroke, such as cerebral infarction. Strict management and treatment are needed in these cases.

Paroxysmal Sympathetic Hyperactivity after Surgery for Cerebral Hemorrhagic Arteriovenous Malformation: A Case Report.

Paroxysmal sympathetic hyperactivity is a condition involving a sudden increase in body temperature, heart rate, blood pressure, respiratory rate, sweating, and posturing followed by severe brain injury. Most of the reported preceding disorders involve head trauma, followed by anoxic brain injury, and stroke. Here, we report an extremely rare case of 17-year-old man diagnosed with hemorrhagic arteriovenous malformation, underwent emergent surgery, was on prolonged sedation due to postoperative complications, and subsequently developed paroxysmal sympathetic hyperactivity. We recommend monitoring for paroxysmal sympathetic hyperactivity occurrence with severe brain injury patients, even when sedating.

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes.

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Structural basis for tRNA-dependent cysteine biosynthesis.

Cysteine can be synthesized by tRNA-dependent mechanism using a two-step indirect pathway, where O-phosphoseryl-tRNA synthetase (SepRS) catalyzes the ligation of a mismatching O-phosphoserine (Sep) to tRNACys followed by the conversion of tRNA-bounded Sep into cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS). In ancestral methanogens, a third protein SepCysE forms a bridge between the two enzymes to create a ternary complex named the transsulfursome. By combination of X-ray crystallography, SAXS and EM, together with biochemical evidences, here we show that the three domains of SepCysE each bind SepRS, SepCysS, and tRNACys, respectively, which mediates the dynamic architecture of the transsulfursome and thus enables a global long-range channeling of tRNACys between SepRS and SepCysS distant active sites. This channeling mechanism could facilitate the consecutive reactions of the two-step indirect pathway of Cys-tRNACys synthesis (tRNA-dependent cysteine biosynthesis) to prevent challenge of translational fidelity, and may reflect the mechanism that cysteine was originally added into genetic code.

Late-Onset Massive Epistaxis due to a Ruptured Traumatic Internal Carotid Artery Aneurysm: A Case Report.

A traumatic internal carotid artery (ICA) aneurysm is rare and difficult to treat. Trapping of ICA is commonly performed owing to the difficulty of directly approaching ICA aneurysms. Recently, coiling the aneurysm itself was recommended if possible. However, it is controversial which of methods are best to completely treat aneurysm. We present the case of a 74-year-old man, who had experienced a head injury 8 years previously, with recurrent severe epistaxis. An ICA aneurysm was detected on computed tomography. The trapping and bypass was planned. However, sudden epistaxis occurred, we performed trapping to stop the bleeding and save his life. After the operation, no right ICA or aneurysm was detected. However, severe epistaxis recurred two months after the operation. In the second operation, a ligation of the common -/- external carotid artery and a severance of an ICA portion between the ophthalmic artery and the aneurysm were insufficient to stop the bleeding. This case indicates ICA trapping, even if a trapping portion is below an ophthalmic artery, is insufficient to treat an ICA aneurysm. ICA aneurysms should be suspected when a patient present with recurrent -/- massive epistaxis, who has a head injury history, even if it is far past.

Spontaneous Intraventricular Pneumocephalus.

BACKGROUD: Pneumocephalus without a known underlying cause is defined as spontaneous pneumocephalus. Few patients of intraventricular pneumocephalus have been reported. PATIENT PRESENTATION: An 84-year-old man presented with dysarthria and incontinence. Computed tomography revealed an intraventricular pneumocephalus, thinning in the petrous bone, fluid in the air cells, and cleft in temporal lobe. A right subtemporal extradural approach was taken to detect bone-/-dural defects, and a reconstruction was performed using a musculo-pericranial flap. CONCLUSION: This is the first patient of an isolated intraventricular spontaneous pneumocephalus without any other site air involved. Surgical approaches to repair such bone and dura defects should be considered an appropriate option.

Histamine H2-Blocker and Proton Pump Inhibitor Use and the Risk of Pneumonia in Acute Stroke: A Retrospective Analysis on Susceptible Patients.

BACKGROUND: Although histamine H2-blockers (H2B) and proton pump inhibitors (PPI) are used commonly to prevent gastrointestinal bleeding in acute stroke, they are implicated in the increased risk of pneumonia in other disease populations. In acute stroke, the presence of distinctive risk factors of pneumonia, including dysphagia and impaired consciousness, makes inclusive analysis vulnerable to confounding. Our aim was to assess whether acid-suppressive drugs increase pneumonia in acute stroke in a population controlled for confounding. METHODS: We analyzed acute stroke patients admitted to a tertiary care hospital. To minimize confounding, we only included subjects who could not feed orally during 14 days of hospitalization. Exposure was defined as H2B or PPI, given in days; the outcome was development of pneumonia within this period. The incidence was calculated from the total number of pneumonias divided by the sum of person-days at risk. We additionally performed multivariate Poisson regression and propensity score analyses, although the restriction largely eliminated the need for multivariate adjustment. RESULTS: A total of 132 pneumonias occurred in 3582 person-days. The incidence was 3.69%/person-day (95% confidence interval (CI); 3.03-4.37%/day). All subjects had dysphagia. Stroke severity and consciousness disturbances were well-balanced between the groups exposed to H2B, PPI, or none. The relative risk (RR) compared with the unexposed was 1.22 in H2B (95%CI; 0.83-1.81) and 2.07 in PPI (95% CI; 1.13-3.62). The RR of PPI compared with H2B was 1.69 (95%CI; 0.95-2.89). In multivariate regression analysis, the RRs of H2B and PPI were 1.24 (95% CI; 0.85-1.81) and 2.00 (95% CI; 1.12-3.57), respectively; in propensity score analyses they were 1.17 (95% CI; 0.89-1.54) and 2.13 (95% CI; 1.60-2.84). CONCLUSIONS: The results of this study suggested that prophylactic acid-suppressive therapy with PPI may have to be avoided in acute stroke patients susceptible to pneumonia.

Delayed Acute Subdural Hematoma Associated With Percutaneous Coronary Intervention.

BACKGROUND: Delayed acute subdural hematoma (DASH) is a subdural hematoma which is detected later. An initial computed tomography (CT) does not reveal any intracranial hemorrhage at all. Few patients of DASH after mild traumatic brain injury associated with percutaneous coronary intervention (PCI) have been published. PATIENT PRESENTATION: A 63-year-old woman presented with cardiac pulmonary arrest due to acute myocardial infarction and lethal arrhythmia. She had hit her head on the road. The initial CT did not reveal any hemorrhage in the intra-cranium. She fully recovered after PCI. However, 1 hour after PCI, she lost consciousness and immediate CT showed acute subdural hematoma and subarachnoid hemorrhage. The period from losing consciousness to brain herniation presenting as anisocoria was very short-only 30 minutes in our patient. Although emergent evacuation of hematoma and external decompression were performed, the patient died 1 day after the operation. CONCLUSION: The authors encountered a patient of DASH after PCI that resulted in death. Clinicians should be aware that subdural hemorrhage can occur after PCI if no hemorrhage is noted in the initial head CT, and the operation should be performed as soon as possible when the consciousness level decreases.

Malignant isolated cortical vein thrombosis with type II protein S deficiency: a case report.

BACKGROUND: The incidence of cerebral venous thrombosis (CVT) is low, and in particular, isolated cortical vein thrombosis (ICVT) is very rare. The diagnosis of ICVT is difficult by using conventional computed tomography (CT) and magnetic resonance imaging (MRI). However, with appropriate treatment, ICVT has a good prognosis. CASE PRESENTATION: Herein, we present a rare case of a 40-year-old woman with ICVT and type II protein S (PS) deficiency, who experienced a stroke. She initially presented with generalized convulsions. A CT scan showed intracerebral hemorrhage (ICH) in the left temporoparietal region. However, her condition rapidly deteriorated and she went into a coma approximately 20 h after admission. A second CT scan revealed significant ICH expansion and transfalcine herniation. Decompressive hemicraniectomy with duraplasty was performed, and ICVT was confirmed owing to abnormal vascular tone and black appearance of the cortical vein. She underwent anticoagulation therapy and rehabilitation, and gradually recovered. CONCLUSION: We experienced an extremely rare case of isolated cortical vein thrombosis related with type II PS deficiency. CT-digital subtraction angiography is a useful supportive technique in the diagnosis of ICVT. Decompressive hemicraniectomy is effective for hemorrhage extension cases, and ICVT with hemorrhage might require early anticoagulation therapy.

Template-dependent nucleotide addition in the reverse (3'-5') direction by Thg1-like protein.

Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5'-end of truncated transfer RNA (tRNA) species in a Watson-Crick template-dependent manner. The reaction proceeds in two steps: the activation of the 5'-end by adenosine 5'-triphosphate (ATP)/guanosine 5'-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3'-5' direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg(2+) ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3'-OH of the incoming nucleotide and the 5'-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3'-5' elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNA(His)-specific G-1 addition enzyme. Each tRNA(His) binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible ß-hairpin. Furthermore, mutational analyses show that tRNA(His) is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode.