Transcription factor old astrocyte specifically induced substance is a novel regulator of kidney fibrosis.

Prevention of kidney fibrosis is an essential requisite for effective therapy in preventing chronic kidney disease (CKD). Here, we identify Old astrocyte specifically induced substance (OASIS)/cAMP responsive element-binding protein 3-like 1 (CREB3l1), a CREB/ATF family transcription factor, as a candidate profibrotic gene that drives the final common pathological step along the fibrotic pathway in CKD. Although microarray data from diseased patient kidneys and fibrotic mouse model kidneys both exhibit OASIS/Creb3l1 upregulation, the pathophysiological roles of OASIS in CKD remains unknown. Immunohistochemistry revealed that OASIS protein was overexpressed in human fibrotic kidney compared with normal kidney. Moreover, OASIS was upregulated in murine fibrotic kidneys, following unilateral ureteral obstruction (UUO), resulting in an increase in the number of OASIS-expressing pathological myofibroblasts. In vitro assays revealed exogenous TGF-ß1 increased OASIS expression coincident with fibroblast-to-myofibroblast transition and OASIS contributed to TGF-ß1-mediated myofibroblast migration and increased proliferation. Significantly, in vivo kidney fibrosis induced via UUO or ischemia/reperfusion injury was ameliorated by systemic genetic knockout of OASIS, accompanied by reduced myofibroblast proliferation. Microarrays revealed that the transmembrane glycoprotein Bone marrow stromal antigen 2 (Bst2) expression was reduced in OASIS knockout myofibroblasts. Interestingly, a systemic anti-Bst2 blocking antibody approach attenuated kidney fibrosis in normal mice but not in OASIS knockout mice after UUO, signifying Bst2 functions downstream of OASIS. Finally, myofibroblast-restricted OASIS conditional knockouts resulted in resistance to kidney fibrosis. Taken together, OASIS in myofibroblasts promotes kidney fibrosis, at least in part, via increased Bst2 expression. Thus, we have identified and demonstrated that OASIS signaling is a novel regulator of kidney fibrosis.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

Transgene-independent heredity of RdDM-mediated transcriptional gene silencing of endogenous genes in rice.

To induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double-strand RNA of each promoter region and thus induced RNA-directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulphide isomerase (PDIL1-1) and luminal binding protein (BiP1); an endoplasmic reticulum stress-inducible gene (OsbZIP50); and genes with seed-specific expression encoding α-globulin (Glb-1) and glutelin-B4 (GluB4). TGS of four genes was obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50, 63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1-1 (33.3%) and Glb-1 TGS lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T-DNA) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the -1 to -250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post-transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of RdDM to new plant breeding techniques in rice are discussed.

Biotechnology and apple breeding in Japan.

Apple is a fruit crop of significant economic importance, and breeders world wide continue to develop novel cultivars with improved characteristics. The lengthy juvenile period and the large field space required to grow apple populations have imposed major limitations on breeding. Various molecular biological techniques have been employed to make apple breeding easier. Transgenic technology has facilitated the development of apples with resistance to fungal or bacterial diseases, improved fruit quality, or root stocks with better rooting or dwarfing ability. DNA markers for disease resistance (scab, powdery mildew, fire-blight, Alternaria blotch) and fruit skin color have also been developed, and marker-assisted selection (MAS) has been employed in breeding programs. In the last decade, genomic sequences and chromosome maps of various cultivars have become available, allowing the development of large SNP arrays, enabling efficient QTL mapping and genomic selection (GS). In recent years, new technologies for genetic improvement, such as trans-grafting, virus vectors, and genome-editing, have emerged. Using these techniques, no foreign genes are present in the final product, and some of them show considerable promise for application to apple breeding.

Juxtaglomerular apparatus-mediated homeostatic mechanisms: therapeutic implication for chronic kidney disease.

INTRODUCTION: Juxtaglomerular apparatus (JGA)-mediated homeostatic mechanism links to how sodium-glucose cotransporter 2 inhibitors (SGLT2is) slow progression of chronic kidney disease (CKD) and may link to how tolvaptan slows renal function decline in autosomal dominant polycystic kidney disease (ADPKD). AREA COVERED: JGA-mediated homeostatic mechanism has been hypothesized based on investigations of tubuloglomerular feedback and renin-angiotensin system. We reviewed clinical trials of SGLT2is and tolvaptan to assess the relationship between this mechanism and these drugs. EXPERT OPINION: When sodium load to macula densa (MD) increases, MD increases adenosine production, constricting afferent arteriole (Af-art) and protecting glomeruli. Concurrently, MD signaling suppresses renin secretion, increases urinary sodium excretion, and counterbalances reduced sodium filtration. However, when there is marked increase in sodium load per-nephron, as in advanced CKD, MD adenosine production increases, relaxing Af-art and maintaining sodium homeostasis at the expense of glomeruli. The beneficial effects of tolvaptan on renal function in ADPKD may also depend on the JGA-mediated homeostatic mechanisms since tolvaptan inhibits sodium reabsorption in the thick ascending limb.The JGA-mediated homeostatic mechanism regulates Af-arts, constricting to relaxing according to homeostatic needs. Understanding this mechanism may contribute to the development of pharmacotherapeutic compounds and better care for patients with CKD.

Long-distance transport of Gibberellic Acid Insensitive mRNA in Nicotiana benthamiana.

BACKGROUND: The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock. RESULTS: Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion. CONCLUSIONS: Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.

Progressive spinal destruction secondary to minimal vertebral fracture in a patient with diffuse idiopathic skeletal hyperostosis: a case report.

Early operative fixation is widely recognized as essential for managing spinal fractures in patients with diffuse idiopathic skeletal hyperostosis (DISH). However, no report to date has addressed the occurrence of minimal vertebral fractures diagnosable only through magnetic resonance imaging (MRI) in these patients and the associated temporal changes in the fracture site. In this report, we describe a rare clinical case involving an 81-year-old man who developed progressive spinal destruction secondary to a minimal vertebral fracture. MRI showed minimum-intensity changes in the T12 vertebral body, whereas X-ray and computed tomography examinations showed DISH and no spinal fracture. Despite experiencing severe low back pain, the patient did not undergo operative therapy for 2 months, resulting in progressive spinal destruction. Spinal fusion with posterior instrumentation was performed, and the patient was followed for 1 year with no symptoms and good functional status. This case emphasizes the importance of clinicians being cautious to avoid overlooking and undervaluing minimal vertebral fractures diagnosable only through MRI in patients with DISH.

Distribution of MdACS3 null alleles in apple (Malus × domestica Borkh.) and its relevance to the fruit ripening characters.

Expression of MdACS3a, one of the ripening-related ACC synthase genes, plays a pivotal role in initiating the burst of ethylene production by MdACS1 in apple fruit. Although previous studies have demonstrated the presence of MdACS3a-null alleles through deficiency of transcription activity or loss of enzyme activity due to amino acid substitution, which may affect the storage properties of certain fruit cultivars, an overall picture of these null alleles in cultivars is still lacking. The present study investigated the distribution of null allelic genes in 103 cultivars and 172 breeding selections by using a simple sequence repeat (SSR) marker linked to them. The results indicated that both allelic genes were widely distributed throughout the examined cultivars and selections, some occurring as the null genotype, either homozygously or heterozygously, with each null allele. The implications of MdACS3a distribution results and the influence of its null allelotypes in fruit characters are discussed.

A mobile signal transported over a long distance induces systemic transcriptional gene silencing in a grafted partner.

Transcriptional gene silencing (TGS) can be induced by promoter-targeted small interfering RNA (siRNA). Long-distance transmission of TGS by viral infection in plants has been reported. However, systemic TGS has not been observed in the case of using an inverted repeat transgene as the silencing trigger. Here it is reported that a mobile signal, presumably the siRNA, produced from a hairpin structure transgene controlled by a companion cell-specific promoter can also induce transmissible TGS in both a modified agroinfiltration and a grafting system. Although the transmissible TGS occurred only in cells located in the vicinity of a leaf vein in the scion, very strong silencing was observed in the root system, especially the lateral roots, including the root apical meristem. The transmissible TGS was maintained through tissue culture and subsequently inherited by the progeny. The results suggest the potential application of mobile promoter-targeting siRNA in horticulture for improvement of plant cultivars by grafting.

The Arabidopsis CORI3 promoter contains two cis-acting regulatory regions required for transcriptional activity in companion cells.

Companion cells are metabolically active and functionally specialized cells that behave as terminals for the transport of materials between phloem and the surrounding tissue. Although previous research has clarified the distinct function of companion cells, it is still largely unknown how plants establish and maintain the special identity of these cells. To shed further light on this issue, we carried out expressed sequence tag (EST) analysis. To minimize the difficulty of dissociating and gathering intact companion cells, vascular strings with an abundant content of companion cells were excised from the petioles of Brassica napus. By random sequencing with a string-specific cDNA library derived by suppression subtractive hybridization between the string itself and the petiole from which it had been removed, we identified 377 ESTs and assembled them into 247 EST groups. The most frequent EST was ExBdl-102 (15 of 377 ESTs), which showed the highest sequence similarity to the Arabidopsis CORI3 (CORONATINE INDUCED 3) gene. The CORI3 promoter:GUS showed predominant expression in the vascular tissue of Arabidopsis. Through transient expression assay using Brassica vasculature and gene-gun-mediated transient assay, we found two integrated cis-regulatory regions of the CORI3 promoter. This work has provided not only string-specific EST information and shown that two novel cis-regulatory regions sustain transcriptional activity in companion cells, but also a series of procedures for efficiently examining the transcriptional framework of companion cells by exploiting the histochemical advantage of B. napus as an experimental material.

Relief of Low Back Pain After Posterior Decompression for Lumbar Spinal Stenosis.

STUDY DESIGN: A retrospective study. OBJECTIVE: The aim of this study was to confirm that decompression for lumbar spinal stenosis (LSS) relieves low back pain (LBP) as adequately as it relieves leg pain and to identify predictors for inadequate LBP relief. SUMMARY OF BACKGROUND DATA: Although decompression for LSS is generally thought to yield worse results for LBP than for leg pain, some studies have reported similar improvements in pain scores between LBP and leg pain. To treat LBP or take measures to prevent inadequate LBP relief, reliable predictors for LBP relief should be identified. METHODS: We retrospectively reviewed 175 patients who underwent posterior element-preserving decompression and evaluated the relief of LBP and leg pain using numeric rating scales (NRSs). Associations between demographic, clinical, or imaging parameters and LBP relief at 1 and 4 years were analyzed by stepwise linear regression analyses. The imaging parameters included Modic change type 1, disc degeneration, foraminal stenosis, vertebral slipping (within Grade 1), scoliosis (<15°) and lordosis. RESULTS: The mean improvements in LBP and leg pain NRS scores from baseline were 5.22 and 4.70 points (P = 0.064, paired t test) at 1 year and 5.12 and 4.62 points (P = 0.068) at 4 years, respectively. Poor LBP scores at 4 years were significantly associated with long-lasting LBP (beta = 0.31, P < 0.0001) and moderate or severe arm symptoms with cervical spinal cord compression or intramedullary hyperintense signal on T2-weighted MRI (beta = 0.22, P = 0.0014). The imaging parameters of the lumbar spine failed to show clear associations with poor LBP scores at 4 years, although Modic change type 1 showed a significant association with poor LBP scores at 1 year (beta = 0.28, P < 0.0001). CONCLUSION: Posterior decompression relieves LBP as well as leg pain. Long-lasting LBP and concurrent symptomatic cervical myelopathy are important predictors for inadequate LBP relief. There were no reliable imaging parameters predictive of inadequate LBP relief.Level of Evidence: 4.

Identification of a cis-regulatory element that acts in companion cell-specific expression of AtMT2B promoter through the use of Brassica vasculature and gene-gun-mediated transient assay.

The molecular basis underlying the development, maintenance and function of companion cells in plants is largely unknown. The identification of several genes expressed specifically in companion cells implies the contribution of specific transcriptional elements to the identity of companion cells. However, less is known about the companion cell-specific transcriptional regulation of promoters. We established a novel assay method using gene-gun delivery of partially deleted promoters to string-containing vascular bundles excised from the petiole of Brassica napus for the rapid identification of cis-elements. To test this system, we analyzed the Arabidopsis METALLOTHIONEIN 2B (MT2B) gene, which is expressed in companion cells. The assay revealed a 49-bp region possessing two predicted cis-regulatory elements: a G-box and an evening element-related sequence (EEr), and EEr showed higher activity. We confirmed the reliability of the result with stable transformants harboring a deleted MT2B promoter:GUS transgene. The lack of EEr completely eliminated the MT2B-like expression, but the lack of G-box did not eliminate it. We conclude that EEr is a major cis-regulatory element of the MT2B promoter. Our method will help to explain the transcriptional background of companion cells through the rapid identification of cis-regulatory regions.

Expansive Suspension Laminoplasty Using a Spinous Process-Splitting Approach for Lumbar Spinal Stenosis: Surgical Technique and Outcomes Over 8 Years of Follow-up.

INTRODUCTION: To maximize the benefits of posterior decompression for severe multilevel lumbar spinal stenosis, we refined the expansive laminoplasty technique using a spinous process-splitting approach. This study tests the hypothesis that the surgical benefit of adequate decompression with posterior element preservation is maintained in the long term, over 8 years of follow-up. METHODS: Fifty-eight patients were followed up yearly for 8 years. Eight patients having nonlumbar spine surgery or Parkinson disease were excluded. The noninferiority of the 8-year versus peak-year outcomes was tested, with margins of 5 points for the Oswestry disability index and 1 point for the numeric rating scales (NRSs). RESULTS: In the 50 patients available for follow-up, the peak values of the mean improvements from baseline within the first 7 years were 35.8, 5.7, 5.9, and 2.8 points for the Oswestry disability index, low back pain NRS, leg pain NRS, and leg numbness NRS, respectively. The 95% lower confidence limits for the differences between the mean improvements from baseline at 8 years and the peak year were within the noninferiority margins for each scale. CONCLUSION: Our technique was associated with substantial improvement from baseline for each scale. The initial improvements in function and symptoms were maintained for 8 years.

MdERFs, two ethylene-response factors involved in apple fruit ripening.

Two MdERFs (ethylene-response factors) were isolated from ripening apple (Malusxdomestica Borkh. cv. Golden Delicious) fruit. The features of their conserved motifs indicated that MdERF1 and MdERF2 belong to group VII and group IX categories in Arabidopsis, respectively. MdERF1 was expressed predominantly in ripening fruit, although a small degree of expression was also observed in non-fruit tissues, whereas MdERF2 was expressed exclusively in ripening fruit. The increased expression in ripening fruit was repressed by treatment with 1-methylcyclopropene (1-MCP: a potent antagonist of ethylene receptors), indicating that transcription is regulated positively by the ethylene signalling system. Indeed, it was a tendency for cultivars with low ethylene production to show lower MdERFs expression than those with high ethylene production. On the basis of concomitant analyses of the expression of some genes related to ripening, the functions of MdERFs and the role of ethylene in the ripening process are discussed.

Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor.

In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops.

RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains.

Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.

Identification of structurally diverse growth hormone secretagogue agonists by virtual screening and structure-activity relationship analysis of 2-formylaminoacetamide derivatives.

Two molecules with known growth hormone secretagogue (GHS) agonist activity were used as templates to computationally screen approximately 80000 compounds. A total of 108 candidate compounds were selected, and five of them were found to be active in the low-micromolar range in both cell-based and direct binding assays. These compounds were structurally diverse and significantly differed from known GHS agonists. The most active compound was subjected to SAR evaluation, which slightly increased its potency and identified molecular regions important for specific GHS agonist activity.

Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus.

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively.

Scion on a stock producing siRNAs of potato spindle tuber viroid (PSTVd) attenuates accumulation of the viroid.

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

Design of libraries targeting protein-protein interfaces.

TARGETING PPIS: A novel strategy for designing libraries targeting protein-protein interfaces enabled us to identify diverse chemical entry points to interact with therapeutic targets for which conventional screening libraries delivered no or only few hit structures. The concept was experimentally validated by early hit evaluation in biochemical screens and early ADMET profiling.