Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.This research was supported by Cancer Research UK (T.T.)

BdWRKY38 is required for the incompatible interaction of Brachypodium distachyon with the necrotrophic fungus Rhizoctonia solani

Rhizoctonia solani is a soil‐borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3‐1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession‐specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3‐1 and Tek‐3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY‐dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling

Semaphorin3f in the maturation of the outer retina

Cells of multicellular organisms have the remarkable ability to coordinate and control dynamic cellular activities in response to changes in their environment. From development into tissue homeostasis, cells communicate with each other through a myriad of intercellular signalling mechanisms. The large family of Semaphorins is a group of well-known extracellular signalling molecules implicated in a wide range of diverse physiological functions. In particular, the expression of secreted Class III Semaphorins (Sema3s) in the retina, not only during development but also in adult tissue, raises interesting questions about their tissue-specific spatiotemporal roles. The vertebrate retina is a highly complex, light-sensitive tissue that lines the back of our eyes. Within the retina, there are two key players that enables our ability to see: the retinal pigment epithelium (RPE) and the photoreceptors. Located in the outermost layer of the retina, the RPE and photoreceptors develop and mature together throughout the lifetime of the organism, forming an interdependent relationship that is highly critical for visual function. This thesis explores, using CRISPR/Cas9-generated loss-of-function mutants (sema3faca304), cell autonomous and non-cell autonomous roles of an RPE-secreted protein, Semaphorin3f (Sema3f), in the zebrafish retina. First, I demonstrate that both cell types, the RPE and photoreceptors, express multiple members of well-known Sema3 receptors, Nrp and PlxnA families. Second, I define a cell autonomous role of Sema3fa in maturing RPE. The loss of Sema3fa does not affect the maturation of the RPE at the transcriptional and morphological level, but does result in the perturbation of appropriate physiological responses to light conditions. Last, I demonstrate the non-cell autonomous role of Sema3fa in the development and specification of maturing photoreceptors. My work elucidates one of the many endogenous roles of Sema3fa as a regulator of the maturing retina to add to the growing literature of Sema signalling in events other than development

Extended phase-correlated rescanning irradiation to improve dose homogeneity in carbon-ion beam liver treatment

We previously reported that an adequate number of simple layered phase-correlated rescanning (PCR) irradiations improve dose conformation to a moving target. However, if PCR is not completed within a single gating window due to the particular irradiation specifications selected in advance and/or layer size, the iso-energy layer is completed using next gating window, and the effect of rescanning is effectively nullified. To solve this problem, we developed extended PCR irradiation. Extended PCR is conceptually based on simple PCR. It differs in that the dose rate is adjusted to irradiate the number of rescans within multiple gating windows when simple PCR is not finished within a single gating window. This process is repeated until the total prescribed dose is given. Here, 4DCT imaging was performed under free-breathing conditions in 10 patients with hepatocellular carcinoma. 4D dose distributions for a single field approach were performed with simple PCR and extended PCR using 4DCT. A total dose of 11.25 Gy(RBE) was administered to the field-specific target volume. The number of rescans was changed from 1 to 10. Respiratory gating of a 30% duty cycle around exhalation was applied. Dose assessment metrics (CTV-D95, CTV-Dmin, CTV-Dmax) with PCR improved with an increasing number of rescans, and largely plateaued at five or more rescans. Minimum D95 and Dmin values, and maximum Dmax and HI values fluctuated at odd and even numbers of rescans. Extended PCR improved all dose assessment metrics whereas PCR did not. Values with extended PCR were mostly constant with three or more rescans, or in other words independent of the number of rescans. Extended PCR improved dose conformation to a moving target with greater efficacy than simple PCR when more than two gating windows were necessary. Extended PCR can be applied to irradiation machines having a low maximum dose rate

Reproduction of marble-mouth frogfish Lophiocharon lithinostomus (Lophiiformes, Antennariidae) and the evolution of parental care among frogfishes

Here, we observed the reproductive behavior of marble-mouth frogfish (Lophiocharon lithinostomus) and the morphology of newly hatched juveniles under captive conditions. Adult males showed pursuit behavior towards females approximately seven days before spawning; spawning and ejaculation took place almost simultaneously. An adult female cared for a fertilized egg mass adhered to their right of the left side of the flank. The position of the adhered eggs on the flank was left-biased (3 on the right and 25 on the left). The females exhibited a proactive fanning behavior towards the egg masses using their dorsal fins; the fanning frequency increased over time after spawning. Meanwhile, the males did not display any form of parental care behavior. The eggs had hook-like structures that might enable the eggs to attach to the flank of the parent. Hatching occurred between 22 and 28 days after spawning. Newly hatched juveniles already had a full set of fin rays; their morphology was similar to that of adult fish. Moreover, we reviewed the evolution of parental care behaviors and egg types among frogfish family

Lower DNA methylation levels in CpG island shores of CR1, CLU, and PICALM in the blood of Japanese Alzheimer's disease patients.

The aim of the present study was to (1) investigate the relationship between late-onset Alzheimer's disease (AD) and DNA methylation levels in six of the top seven AD-associated genes identified through a meta-analysis of recent genome wide association studies, APOE, BIN1, PICALM, CR1, CLU, and ABCA7, in blood, and (2) examine its applicability to the diagnosis of AD. We examined methylation differences at CpG island shores in the six genes using Sanger sequencing, and one of two groups of 48 AD patients and 48 elderly controls was used for a test or replication analysis. We found that methylation levels in three out of the six genes, CR1, CLU, and PICALM, were significantly lower in AD subjects. The combination of CLU methylation levels and the APOE genotype classified AD patients with AUC = 0.84 and 0.80 in the test and replication analyses, respectively. Our study implicates methylation differences at the CpG island shores of AD-associated genes in the onset of AD and suggests their diagnostic value

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex), respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots