Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein Dynamin related protein 5B (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Life cycle and functional genomics of the unicellular red alga Galdieria for elucidating algal and plant evolution and industrial use.

Sexual reproduction is widespread in eukaryotes; however, only asexual reproduction has been observed in unicellular red algae, including Galdieria, which branched early in Archaeplastida. Galdieria possesses a small genome; it is polyextremophile, grows either photoautotrophically, mixotrophically, or heterotrophically, and is being developed as an industrial source of vitamins and pigments because of its high biomass productivity. Here, we show that Galdieria exhibits a sexual life cycle, alternating between cell-walled diploid and cell wall-less haploid, and that both phases can proliferate asexually. The haploid can move over surfaces and undergo self-diploidization or generate heterozygous diploids through mating. Further, we prepared the whole genome and a comparative transcriptome dataset between the diploid and haploid and developed genetic tools for the stable gene expression, gene disruption, and selectable marker recycling system using the cell wall-less haploid. The BELL/KNOX and MADS-box transcription factors, which function in haploid-to-diploid transition and development in plants, are specifically expressed in the haploid and diploid, respectively, and are involved in the haploid-to-diploid transition in Galdieria, providing information on the missing link of the sexual life cycle evolution in Archaeplastida. Four actin genes are differently involved in motility of the haploid and cytokinesis in the diploid, both of which are myosin independent and likely reflect ancestral roles of actin. We have also generated photosynthesis-deficient mutants, such as blue-colored cells, which were depleted in chlorophyll and carotenoids, for industrial pigment production. These features of Galdieria facilitate the understanding of the evolution of algae and plants and the industrial use of microalgae.

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene.

BACKGROUND: Astrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections. RESULTS: To establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36-33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12-12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES. CONCLUSIONS: The optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Formation of internal carotid artery aneurysms following gamma knife radiosurgery for pituitary adenomas: a case series and literature review.

Only two aneurysm formations in the internal carotid artery after gamma knife radiosurgery (GKRS) for pituitary adenomas are reported so far. Here, out of the 482 patients who underwent GKRS for pituitary adenomas at our institute, at least five developed aneurysms within the area of high single-dose irradiation. Three patients presented with epistaxis due to aneurysmal rupture and one presented with abducens paralysis due to nerve compression, while one was asymptomatic. The interval between irradiation and aneurysmal detection ranged from 14 to 21 years. Aneurysm formation in those conditions may be higher than previously thought.

Reconversion to ventriculoperitoneal shunt following ventriculoatrial shunt malfunction in children.

PURPOSE: To analyze the long-term efficacy of the ventriculoatrial shunt (VAS) in pediatric patients with hydrocephalus, focusing on the atrial catheter and suitable revision procedures of the distal catheter following VAS malformation performed at our institution. METHODS: The authors retrospectively analyzed data of 28 pediatric patients under the age of 10 years who were treated with VAS for hydrocephalus and who had a follow-up period of at least 5 years. RESULTS: A total of 42 atrial tube revision procedures were performed in 28 patients during the study period. The median atrial tube survival time due to atrial tube obstruction was 2.32 years (n = 31, range: 0.4-8.08 years). Atrial tube survival time was shorter in younger children (p < 0.0001) and in children who were shorter in height (p = 0.0001). As a revision procedure following atrial tube malfunction, 22 (78.6%) out of the 28 patients who had an inserted VAS had the VAS reconversion into a VPS at the last follow-up. CONCLUSIONS: VAS can be a useful alternative to VPS, but it requires frequent atrial tube revisions, especially in younger children. Reconversion to VPS after VAS malfunction is a reasonable option and is associated with longer shunt survival time despite its previously observed difficulties.

Embryogenesis of flattened colonies implies the innovation required for the evolution of spheroidal colonies in volvocine green algae.

BACKGROUND: Volvocine algae provide a suitable model for investigation of the evolution of multicellular organisms. Within this group, evolution of the body plan from flattened to spheroidal colonies is thought to have occurred independently in two different lineages, Volvocaceae and Astrephomene. Volvocacean species undergo inversion to form a spheroidal cell layer following successive cell divisions during embryogenesis. During inversion, the daughter protoplasts change their shape and develop acute chloroplast ends (opposite to basal bodies). By contrast, Astrephomene does not undergo inversion; rather, its daughter protoplasts rotate during successive cell divisions to form a spheroidal colony. However, the evolutionary pathways of these cellular events involved in the two tactics for formation of spheroidal colony are unclear, since the embryogenesis of extant volvocine genera with ancestral flattened colonies, such as Gonium and Tetrabaena, has not previously been investigated in detail. RESULTS: We conducted time-lapse imaging by light microscopy and indirect immunofluorescence microscopy with staining of basal bodies, nuclei, and microtubules to observe embryogenesis in G. pectorale and T. socialis, which form 16-celled or 4-celled flattened colonies, respectively. In G. pectorale, a cup-shaped cell layer of the 16-celled embryo underwent gradual expansion after successive cell divisions, with the apical ends (position of basal bodies) of the square embryo's peripheral protoplasts separated from each other. In T. socialis, on the other hand, there was no apparent expansion of the daughter protoplasts in 4-celled embryos after successive cell divisions, however the two pairs of diagonally opposed daughter protoplasts shifted slightly and flattened after hatching. Neither of these two species exhibited rotation of daughter protoplasts during successive cell divisions as in Astrephomene or the formation of acute chloroplast ends of daughter protoplasts as in volvocacean inversion. CONCLUSIONS: The present results indicate that the ancestor of Astrephomene might have newly acquired the rotation of daughter protoplasts after it diverged from the ancestor of Gonium, while the ancestor of Volvocaceae might have newly acquired the formation of acute chloroplast ends to complete inversion after divergence from the ancestor of Goniaceae (Gonium and Astrephomene).

Colorectal endoscopic mucosal resection with submucosal injection of epinephrine versus hypertonic saline in patients taking antithrombotic agents: propensity-score-matching analysis.

BACKGROUND: Endoscopic mucosal resection (EMR) to remove colon polyps is increasingly common in patients taking antithrombotic agents. The safety of EMR with submucosal saline injection has not been clearly demonstrated in this population. AIMS: The present study aimed to evaluate the efficacy and safety of submucosal injection of saline-epinephrine versus hypertonic saline in colorectal EMR of patients taking antithrombotic agents. METHODS: This study enrolled 204 patients taking antithrombotic agents among 995 consecutive patients who underwent colonic EMR from April 2012 to March 2018 at Ureshino Medical Center. Patients were divided into two groups according to the injected solution: saline-epinephrine or hypertonic (10%) saline (n = 102 in each group). Treatment outcomes and adverse events were evaluated in each group and risk factors for immediate and post-EMR bleeding were investigated. RESULTS: There were no differences between groups in patient or polyp characteristics. The main antithrombotic agents were low-dose aspirin, warfarin, and clopidogrel. Propensity-score matching created 80 matched pairs. Adjusted comparisons between groups showed similar en bloc resection rates (95.1% with saline-epinephrine vs. 98.0% with hypertonic saline). There were no significant differences in adverse events (immediate EMR bleeding, post-EMR bleeding, perforation, or mortality) between groups. Multivariate analyses revealed that polyp size over 10 mm was associated with an increased risk of immediate EMR bleeding (odds ratio 12.1, 95% confidence interval 2.0-74.0; P = 0.001). CONCLUSIONS: Two tested solutions in colorectal EMR were considered to be both safe and effective in patients taking antithrombotic agents.

Alternative evolution of a spheroidal colony in volvocine algae: developmental analysis of embryogenesis in Astrephomene (Volvocales, Chlorophyta).

BACKGROUND: Volvocine algae, which range from the unicellular Chlamydomonas to the multicellular Volvox with a germ-soma division of labor, are a model for the evolution of multicellularity. Within this group, the spheroidal colony might have evolved in two independent lineages: Volvocaceae and the goniacean Astrephomene. Astrephomene produces spheroidal colonies with posterior somatic cells. The feature that distinguishes Astrephomene from the volvocacean algae is lack of inversion during embryogenesis; the volvocacean embryo undergoes inversion after successive divisions to orient flagella toward the outside. The mechanisms of inversion at the molecular and cellular levels in volvocacean algae have been assessed in detail, particularly in Volvox carteri. However, embryogenesis in Astrephomene has not been subjected to such investigations. RESULTS: This study relied on light microscopy time-lapse imaging using an actively growing culture of a newly established strain to conduct a developmental analysis of Astrephomene as well as to perform a comparison with the similar spheroidal volvocacean Eudorina. During the successive divisions involved in Astrephomene embryogenesis, gradual rotation of daughter protoplasts resulted in movement of their apical portions toward the embryonic posterior, forming a convex-to-spheroidal cell sheet with the apical ends of protoplasts on the outside. Differentiation of the posterior somatic cells from the embryo periphery was traced based on cell lineages during embryogenesis. In contrast, in Eudorina, the rotation of daughter protoplasts did not occur during successive cell divisions; however, inversion occurred after such divisions, and a spheroidal embryo was formed. Indirect immunofluorescence microscopy of basal bodies and nuclei verified this difference between Astrephomene and Eudorina in the movement of embryonic protoplasts. CONCLUSIONS: These results suggest different tactics for spheroidal colony formation between the two lineages: rotation of daughter protoplasts during successive cell divisions in Astrephomene, and inversion after cell divisions in Eudorina. This study will facilitate further research into the molecular and genetic mechanisms of the parallel evolution of the spheroidal colony in volvocine algae.

Post-discharge sedentary behavior and light-intensity physical activity-associated stroke recurrence in patients with minor ischemic stroke: A preliminary retrospective observational study.

BACKGROUND AND PURPOSE: Evidence regarding whether reducing sedentary behavior and increasing physical activity levels to prevent stroke recurrence is insufficient. Therefore, this study preliminarily investigated whether post-discharge sedentary behavior and physical activity levels in patients with minor ischemic stroke were associated with stroke recurrence. METHODS: This retrospective observational study included 73 patients (aged 72.0 years) with minor ischemic stroke from a previous study. The outcome was recurrent stroke 2 years after stroke onset, assessed using medical records. Exposure factors including sedentary behavior and physical activity levels 6 months post-discharge were measured using accelerometers; patients were classified into the recurrence or non-recurrence groups. Logistic regression analyses were then conducted to determine whether sedentary behavior and physical activity 6 months after discharge were associated with stroke recurrence. RESULTS: Six patients experienced stroke recurrence (recurrence rate, 8.2%). The recurrence group showed greater sedentary behavior (recurrence group 68.0%, non-recurrence group 52.0%, p = 0.007) and less light-intensity physical activity (LPA) (recurrence group 21.0%, non-recurrence group 37.0%, p = 0.002) than in the non-recurrence group. Logistic regression analysis showed that sedentary behavior (odds ratio = 1.083, 95% confidence interval = 1.007-1.165, p = 0.032) and LPA (odds ratio = 0.874, 95% confidence interval = 0.785-0.975, p = 0.015) were independent factors for recurrence of stroke. DISCUSSION: Post-discharge sedentary behavior and LPA in patients with minor ischemic stroke were associated with stroke recurrence. Results suggest that reducing post-discharge sedentary behavior and increasing LPA may be crucial for reducing the risk of stroke recurrence in patients with minor ischemic stroke.

Clinical features of 26 cases of COVID-19-associated conjunctivitis.

PURPOSE: To explore the clinical features of COVID-19-associated conjunctivitis with the objective of preventing the spread of infection. STUDY DESIGN: Retrospective cohort study. METHODS: From March 2020 to March 2021, we retrospectively reviewed 26 (9.8%) consecutive COVID-19 patients with conjunctivitis among 282 COVID-19 cases admitted to our hospital. Clinical symptoms, onset date of conjunctivitis, time to patient recovery, and eye drop intervention were investigated. In addition, risk factors for developing conjunctivitis were statistically examined among 206 inpatients available for within 5 days of the onset. A multivariate analysis of conjunctivitis risk factors was performed. RESULTS: Among the 282 COVID-19 patients, 4 (1.4%) had conjunctival hyperemia as the primary symptom. The median time of onset was 4 days after the COVID-19 onset. Hyperemia was observed in all cases, but other ocular symptoms were rare. The median duration of hyperemia was 3 days. A multiple logistic regression analysis revealed that a young age (p=0.005) and current smoking habit (p=0.027) were independent risk factors for conjunctivitis after COVID-19. CONCLUSIONS: COVID-19-associated conjunctivitis is rare in the elderly and strongly associated with a history of smoking. It often occurs in the early stages of infection, and while hyperemia is recognized as a clinical symptom, other ocular symptoms are rare or non-existent. Many cases recover within a short time.

Selenoprotein P expression in glioblastoma as a regulator of ferroptosis sensitivity: preservation of GPX4 via the cycling-selenium storage.

Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.

Identifying Cerebral Large Vessel Occlusion in Acute Ischemic Stroke by MRI Positioning Scanning.

Various approaches have been tried for acute ischemic stroke (AIS) treatment to shorten the time from onset to recanalization. MRI positioning scanning (PS), which must be taken before any MRI sequences, was examined whether it can detect cerebral large vessel occlusion. A total of 68 consecutive patients with AIS who underwent MRI and were treated with intravenous recombinant tissue plasminogen activator or mechanical thrombectomy at our hospital were retrospectively included in this study. Occluded vessels were identified on the axial or coronal views of PS images, and these images were compared with 3D time-of-flight MRA and digital subtraction angiogram. The sensitivities, positive predictive values (PPVs), and negative predictive values (NPVs) for internal carotid artery (ICA), the proximal M1, distal M1, and M2 segment of the middle cerebral artery occlusion were assessed, and the number of PS slices was assessed. The sensitivities of the axial slices for ICA, proximal M1, distal M1, and M2 occlusion were 62%, 21%, 35%, and 86%, respectively. The PPVs of the axial slices for ICA, proximal M1, distal M1, and M2 occlusion were 81%, 88%, 100%, and 97%, respectively, and the NPVs of the axial slices for ICA, proximal M1, distal M1, and M2 occlusion were 94%, 90%, 86%, and 100%, respectively. The detection rate for the ICA was significantly higher with three axial slices (91%) than with two slices (47%) (p <0.01). MRI PS is warranted to be referred to detect large cerebral vessel occlusion.

A Super-selective Wada Test Successfully Detected an Artery That Supplied Broca's Area in a Case of Left Frontal Lobe Glioblastoma: Technical Case Report.

In cases of malignant gliomas located at language eloquent area, it is often difficult to preoperatively detect those area with functional MRI. Awake surgery is often used to spare the language eloquent area during surgery for such tumors; it is not available for a patient whose intracranial pressure is elevated due to the malignant tumor. The Wada test involves infusing anesthetic agents into the internal carotid artery to determine language dominancy before surgery for epilepsy or brain tumor. The super-selective Wada test is a technique to detect more detailed functional localization by infusing anesthetics into far distal middle cerebral artery branches. We present a 37-year-old man suffering from a left frontal lobe glioblastoma, in whom detection of an artery supplying Broca's area was attempted by a super-selective Wada test. The super-selective Wada test successfully detected the branch of middle cerebral artery supplying Broca's area. Total resection of the contrast-enhancing area was achieved without damaging the artery supplying Broca's area without any neurological sequelae. This is the first report describing the usefulness of the super-selective Wada test in glioblastoma treatment. Our findings suggest that the super-selective Wada test is a powerful and useful means to distinguish the artery that supplies the language area from the tumor feeding artery in cases of tumors in the language eloquent area.

Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

Morphology, taxonomy and mating-type loci in natural populations of Volvox carteri in Taiwan.

BACKGROUND: Volvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the production of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a > 1 Mbp linear chromosome. Fifteen sex-specific genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identified in natural populations of this species. RESULTS: During a recent fieldwork in Taiwan, we encountered natural populations of V. carteri that had not previously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identified as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to > 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specific genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specific gene HMG1f was not amplified by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain. CONCLUSIONS: Male spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon.