Phenotype and genotype characterization of Phalaenopsis amabilis (L.) Blume Orchid Transformant Harboring Construct UBI::Cas9::U3::PDS3

Phalaenopsis amabilis (L.) Blume is an Indonesian national “Flower of Charm” (“Puspa Pesona”), an ornamental plant that by using genetic engineering we could improve the flower quality. In this study, Agrobacterium tumefaciens-mediated genetic transformation and CRISPR/Cas9 genome editing system was used to edit the orchid genome more specific and precisely. Twelve months old putative transgenic plants originated from protocorm like bodies (PLBs) transformation harboring the T-DNA construct UBI::Cas9::U3::PDS3/plasmid pRGEB32. Proofing for the putative transformant plants were stably harboring the T-DNA constructs were conducted by genotype and phenotype characterization. The purpose of this study was to characterize genotypically and phenotypically the putative transformant of P. amabilis harboring T-DNA constructs UBI::Cas9::U3::PDS3 compared to non-transformant. Genotype characterization was carried out by detecting the integration of T-DNA harboring UBI::Cas9::U3::PDS3 on the P. amabilis genome using several primers for HPT, Cas9, PDS3 and trnL-F (internal control primers). Phenotype analysis was carried out by observing and analyzing chlorophyll content using spectrophotometric methods. The results showed that genome of putative P. amabilis transformant age 12 months could be amplified by the primers. Phenotypic analysis of P. amabilis transformant showed a change in plant color from green to pale (albino) with lower chlorophyll content compared to non-transformant. This indicates that the CRISPR/Cas9 technology is able to edit the genome of orchids. Keywords: Chlorophyll, CRISPR/Cas9, orchid, Phalaenopsis amabilis (L.) Blume, PHYTOENE DESATURASE 3 (PDS3), Transformant.Phalaenopsis amabilis (L.) Blume adalah tanaman hias “Puspa Pesona Indonesia” yang dapat ditingkatkan kualitasnya dengan teknik rekayasa genetika. Transformasi genetik dengan perantara Agrobacterium tumefaciens dan CRISPR/Cas9 digunakan dalam penelitian ini untuk pengeditan genom secara lebih spesifik dan presisi pada target sekuen gen PHYTOENE DESATURASE3 (PDS3) yaitu gen yang berperan penting pada biosintesis kloroplas. Dalam penelitian ini digunakan tanaman transforman umur 12 bulan yang ditumbuhkan dari protokorm yang telah diintegrasi dengan T-DNA pembawa konstruksi UBI::Cas9::U3::PDS3/plasmid pRGEB32. Pembuktian tanaman transforman tersebut masih mengandung konstruksi T-DNA tersebut perlu dilakukan, yaitu dengan karakterisasi secara genotipe dan fenotipe. Tujuan penelitian ini adalah untuk mengkarakterisasi P. amabilis transforman pembawa T-DNA dengan konstruksi UBI::Cas9::U3::PDS3 secara genotip dan fenotip dibandingkan dengan P. amabilis non-transforman. Karakterisasi genotipe dilakukan dengan mendeteksi integrasi T-DNA pembawa konstruksi UBI::Cas9::U3::PDS3 pada genom anggrek P. amabilis menggunakan beberapa primer yaitu HPT, Cas9, PDS3 dan trnL-F (primer kontrol internal). Analisis karakter fenotipe dilakukan dengan pengamatan morfologi dan analisis kadar klorofil menggunakan metode spektrofotometri. Hasil penelitian menunjukkan bahwa genom anggrek P. amabilis transforman pembawa konstruksi UBI::Cas9::U3::PDS3 umur 12 bulan dapat teramplifikasi oleh semua primer. Analisis fenotipe P. amabilis transforman menunjukkan adanya perubahan warna tanaman dari hijau menjadi albino dengan kadar klorofil lebih rendah jika dibandingkan dengan P. amabilis non-transforman. Hal ini menunjukkan bahwa teknologi CRISPR/Cas9 dapat digunakan untuk mengedit genom tanaman anggrek. Kata kunci: Anggrek, CRISPR/Cas9, klorofil, Phalaenopsis amabilis (L.) Blume, PHYTOENE DESATURASE 3 (PDS3), Transforma

ヒゾウ ゲンパツ Inflammatory pseudotumor ノ 1レイ

A case of inflammatory pseudotumor of the spleen is reported. This benign tumor in the spleen is rare. To our knowledge, 68 cases had been reported in the literature and 26 cases in the japanese literature. A splenic mass was incidentally detected by abdominal CT scan during the examination of the preoperative course of cholecystectomy for cholecystlithiasis. An abdominal CT scan revealed an slightly low density mass in the spleen, and MRI showed a low intensity mass. Cholecystectomy and extirpation of spelenic tumor was performed and the tumor measured 45×35×25mm in size. The tumor was diagnosed histologically as a inflammatory pseudotumor of the spleen

ジュウニシチョウ gastrointestinal stromal tumor ノ 1レイ

77 year-old male presented with prolonged melena and anemia. There was a well-defined enhancing mass in the descending portion of the duodenum that was approximately 6 cm in diameter on contrast-enhanced computed tomography (CT). Large ulcerated mass was seen in the duodenum on endoscopy. Preoperative biopsy was diagnostic for gastrointestinal stromal tumor (GIST) and partial resection of the duodenum was performed. Postoperative diagnosis was malignant GIST. Immunohistochemistry demonstrated negative S-100, negative actin, positive C-kit and partially positive CD34

Spontaneous rupture of a dissection of the left ovarian artery

A 53-year-old female was suddenly hospitalized with acute left lateral abdominal pain. There was no history of trauma to the abdomen. She had received no abdominal operation. X-ray showed a soft tissue shadow in the left flank which displaced the bowel shadows medially. Plain abdominal CT showed a left retroperitoneal hematoma. Dynamic abdominal CT showed an outflow of medium from a blood vessel in the hematoma. At laparotomy, the source of bleeding was found to be the left ovarian artery. The ovarian artery was dilated and meandered remarkably. The ovarian artery and vein were ligated proximally and left adenectomy was performed. The patient made an uneventful recovery. Histological examination suggested a spontaneous rupture of a dissection of the left ovarian artery

タンノウ ネンテンショウ ノ 2レイ

Torsion of the gallbladder is a rare entity and pre-operative diagnosis of the disease is uncommon. Since it was first described by A.V. Wendel in 1898, about 400 cases have been reported in the literature. Two cases of torsion of the gallbladder were presented. First case was 82 year old male who was admitted with 48 hour history of right upper quadrant abdominal pain, fever, and vomiting. On physical examination there was right upper quadrant tenderness with muscle guarding and rebound tenderness. An abdominal ultrasonogram and CT showed a distended gallbladder with thickness of the wall and a stone. A diagnosis of cholecystitis due to the stone was made and conservative therapy was tried. But the condition didn’t become better and a cholecystectomy was performed 2days later. The gallbladder was found to be necrotic with a 360 degree in an anticlock wise direction. Post operative recovery was unremarkable and the patient discharged at 12 days. Second case was 80 year old female who was admitted with 11 hour history of right upper quadrant abdominal pain and vomiting. A physical examination was similar to the first case. An abdominal ultrasonogram showed a distended and floating gallbladder. A diagnosis of torsion of the gallbladder was made without CT and a cholecystectomy was performed immediately. The gallbladder was found to be necrotic with a 360 degree in an anticlock wise direction. Post operative recovery was unremarkable and the patient discharged at 16 days

Impairment of cerebellar long‑term depression and GABAergic transmission in prion protein deficient mice ectopically expressing PrPLP/Dpl

Prion protein (PrPC) knockout mice, named as the “Ngsk” strain (Ngsk Prnp0/0 mice), show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Our previous study indicated that the mutant mice also exhibited alterations in cerebellum-dependent delay eyeblink conditioning, even at a young age (16 weeks of age) when neurological changes had not occurred. Thus, this electrophysiological study was designed to examine the synaptic function of the cerebellar cortex in juvenile Ngsk Prnp0/0 mice. We showed that Ngsk Prnp0/0 mice exhibited normal paired-pulse facilitation but impaired long-term depression of excitatory synaptic transmission at synapses between parallel fibres and PCs. GABAA-mediated inhibitory postsynaptic currents recorded from PCs were also weakened in Ngsk Prnp0/0 mice. Furthermore, we confirmed that Ngsk Prnp0/0 mice (7–8-week-old) exhibited abnormalities in delay eyeblink conditioning. Our findings suggest that these alterations in both excitatory and inhibitory synaptic transmission to PCs caused deficits in delay eyeblink conditioning of Ngsk Prnp0/0 mice. Therefore, the Ngsk Prnp0/0 mouse model can contribute to study underlying mechanisms for impairments of synaptic transmission and neural plasticity, and cognitive deficits in the central nervous system

Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids

Orchid is an important ornamental plant in Indonesia due to their natural beauty of flowers. In the tropical forest, orchids are being acquired for trading and commercial market. Thus, the effort is required to proliferate orchid in large quantities for conservation and improve the floral variation for plant breeding. The purpose of this study is to develop a firmed methodology of molecular breeding of orchids using CRISPR/Cas9 KO system. The plant material used was Phalaenopsis amabilis protocorms growth on NP medium+pepton (2 g/L). Protocorm were submerged in the culture of Agrobacterium tumefaciens that Ti‐plasmid had been filled with a T‐DNA construct of a pRGEB32 vector harboring sgRNA with PDS3 sequence. Detection for transformants was confirmed by PCR using HPT primers (545 bp), Cas9 primers (402 bp), PDS primers (280 bp) and trnL‐F (1200 bp) as an internal control. The results showed that 0.96% PDS transformants were obtained from PDS3T2 lines. Several transformant showed pale leaf color compared to non‐transformant plants. This study suggests that the target gene has successfully edited by CRISPR/Cas9 system and could be applied for that functional gene editing in orchids

Spatiotemporal T790M Heterogeneity in Individual Patients with EGFR-Mutant Non–Small-Cell Lung Cancer after Acquired Resistance to EGFR-TKI

IntroductionEpidermal growth factor receptor (EGFR) mutation T790M accounts for approximately half of acquired resistances to EGFR-tyrosine kinase inhibitor (TKI). Because T790M is mediated by TKI exposure, its penetration and “on–off” may affect T790M status.MethodsWe retrospectively reviewed T790M status and clinical course of patients who had undergone multiple rebiopsies after acquired resistance to EGFR-TKI.ResultsOf 145 patients with EGFR-mutant NSCLC receiving rebiopsy after acquired resistance, 30 underwent multiple site rebiopsies, and 24 received repeated rebiopsies at the same lesion. In 22 patients who underwent rebiopsies from both central nervous system (CNS; 20 cerebrospinal fluids [CSF] and 2 brain tumoral tissues) and thoracic lesions (7 lung tissues, 14 pleural effusions, and 1 lymph node), 12 were thoracic-T790M-positive. Of these 12 patients, 10 were CNS-T790M-negative, despite exhibiting thoracic-T790M-positive. All 10 thoracic-T790M-negatives were CNS-T790M-negative. Three patients revealed a spatial heterogeneous T790M status among their thoracic lesions. In 24 patients receiving repeated rebiopsies at the same lesion (12 lung tissues, 6 CSFs, and 6 pleural effusions), T790M status of lung lesions varied in five patients after TKI-free interval. In all five patients whose T790M status changed from positive to negative, EGFR-TKI rechallenge was effective. In three of these five patients, after further TKI exposure, T790M status changed from negative to positive again. There was also a patient whose CSF T790M status changed from negative to positive after high-dose erlotinib therapy.ConclusionsT790M status in an individual patient can be spatiotemporally heterogeneous because of selective pressure from EGFR-TKI

RUPTURED CEREBRAL ANEURYSMS AND SEVERE PD

BACKGROUND: The pathophysiology of subarachnoid hemorrhages (SAHs) due to ruptured intracranial aneurysms (IAs) remains unclear.Although a relationship between SAHs and periodontal disease (PD) has been suggested, the mechanism requires clarification. OBJECTIVE: To evaluate the relationship between PD and SAHs and to identify periodontal pathogens associated with SAHs. METHODS: This prospective study included consecutive patients with ruptured (n = 11) and unruptured (n = 14) IAs and healthy controls (n = 8). The plasma and plaque subgingival bacterial deoxyribonucleic acid (DNA) levels in PD were evaluated by a dentist using the Community Periodontal Index of Treatment Needs (CPITN). Plasma levels of matrix metalloproteinase (MMP-9), tissue inhibitors of matrix metalloproteinase (TIMP2), and procollagen I were analyzed. RESULTS: Patients with ruptured IAs, had significantly higher CPITN scores than the controls, suggesting that ruptured IAs were associated with severe PD. Although no rupture-specific bacteria were identified, the positive rate of plaque subgingival bacterial DNA was significantly higher in patients with severe PD than in those without severe PD. Multivariate logistic regression analysis indicated that bleeding on probing (BOP)was associated with ruptured IAs (odds ratio, 1.10; 95% confidence interval 1.04–1.20; P = .0001). BOP was positively associated with plasma MMP-9 levels and a disequilibrium in the MMP-9/TIMP2 ratio. BOP was negatively correlated with plasma procollagen I levels (P < .05, for each). This suggested that local inflammation with severe PD might have systemic effects and lead to ruptured IAs. CONCLUSION: Disequilibrium of plasma protease/anti-protease associated with a high BOP rate in severe PD may be attributable to IA rupture

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells.

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI