Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.

Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.

Genetic Analysis of SCN11A, SCN10A, and SCN9A in Familial Episodic Pain Syndrome (FEPS) in Japan and Proposal of Clinical Diagnostic Criteria.

Familial episodic pain syndrome (FEPS) is an early childhood onset disorder of severe episodic limb pain caused mainly by pathogenic variants of SCN11A, SCN10A, and SCN9A, which encode three voltage-gated sodium channels (VGSCs) expressed as key determinants of nociceptor excitability in primary sensory neurons. There may still be many undiagnosed patients with FEPS. A better understanding of the associated pathogenesis, epidemiology, and clinical characteristics is needed to provide appropriate diagnosis and care. For this study, nationwide recruitment of Japanese patients was conducted using provisional clinical diagnostic criteria, followed by genetic testing for SCN11A, SCN10A, and SCN9A. In the cohort of 212 recruited patients, genetic testing revealed that 64 patients (30.2%) harbored pathogenic or likely pathogenic variants of these genes, consisting of 42 (19.8%), 14 (6.60%), and 8 (3.77%) patients with variants of SCN11A, SCN10A, and SCN9A, respectively. Meanwhile, the proportions of patients meeting the tentative clinical criteria were 89.1%, 52.0%, and 54.5% among patients with pathogenic or likely pathogenic variants of each of the three genes, suggesting the validity of these clinical criteria, especially for patients with SCN11A variants. These clinical diagnostic criteria of FEPS will accelerate the recruitment of patients with underlying pathogenic variants who are unexpectedly prevalent in Japan.

Clinical and molecular features of 66 patients with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14).

BACKGROUND: Musculocontractural Ehlers-Danlos syndrome is caused by biallelic loss-of-function variants in CHST14 (mcEDS-CHST14) or DSE (mcEDS-DSE). Although 48 patients in 33 families with mcEDS-CHST14 have been reported, the spectrum of pathogenic variants, accurate prevalence of various manifestations and detailed natural history have not been systematically investigated. METHODS: We collected detailed and comprehensive clinical and molecular information regarding previously reported and newly identified patients with mcEDS-CHST14 through international collaborations. RESULTS: Sixty-six patients in 48 families (33 males/females; 0-59 years), including 18 newly reported patients, were evaluated. Japanese was the predominant ethnicity (27 families), associated with three recurrent variants. No apparent genotype-phenotype correlation was noted. Specific craniofacial (large fontanelle with delayed closure, downslanting palpebral fissures and hypertelorism), skeletal (characteristic finger morphologies, joint hypermobility, multiple congenital contractures, progressive talipes deformities and recurrent joint dislocation), cutaneous (hyperextensibility, fine/acrogeria-like/wrinkling palmar creases and bruisability) and ocular (refractive errors) features were observed in most patients (>90%). Large subcutaneous haematomas, constipation, cryptorchidism, hypotonia and motor developmental delay were also common (>80%). Median ages at the initial episode of dislocation or large subcutaneous haematoma were both 6 years. Nine patients died; their median age was 12 years. Several features, including joint and skin characteristics (hypermobility/extensibility and fragility), were significantly more frequent in patients with mcEDS-CHST14 than in eight reported patients with mcEDS-DSE. CONCLUSION: This first international collaborative study of mcEDS-CHST14 demonstrated that the subtype represents a multisystem disorder with unique set of clinical phenotypes consisting of multiple malformations and progressive fragility-related manifestations; these require lifelong, multidisciplinary healthcare approaches.

[A Case of Transcatheter Arterial Chemoembolization and Surgical Resection of Left 7th Rib Metastasis after Surgery for Hepatocellular Carcinoma].

The patient was an 81-year-old man. After a liver posterior segmentectomy for hepatocellular carcinoma, a painful bulge was observed in the left anterior thoracic region during a routine outpatient visit. Elevated tumor markers and contrast- enhanced CT scan revealed a mass with contrast effect in the left 7th rib. Ultrasound-guided biopsy revealed hepatocellular carcinoma metastatic to the left 7th rib. There were no other obvious metastases, and the diagnosis of a single bone metastasis was made. The patient did not request chemotherapy and underwent transcatheter arterial chemoembolization 4 times. The patient did not show any improvement in tumor markers or shrinkage of the tumor, and his quality of life was deteriorated due to increased pain. The patient underwent left chest wall tumor resection and chest wall reconstruction. Postoperative tumor markers were normalized and pain improved markedly. We report a case of postoperative recurrence- free survival for 2 years.

[A Case of Intussusception Due to Juvenile Colorectal Cancer with Suspected Lynch Syndrome].

A 20-year-old man presenting with abdominal pain and distension from 3 months ago was admitted to our hospital. Colonoscopy showed a type 1 tumor in the transverse colon. The biopsy was moderately differentiated adenocarcinoma. Abdominal enhanced CT revealed intussusception associated with the tumor. After non-invasive reduction of intussusception was performed, a full-body examination revealed no findings suggestive of distant metastases or other Lynch syndrome- related tumors. We performed laparoscopic right hemicolectomy. Lynch syndrome was suspected based on his family history, MMR protein immunohistochemistry and MSI-High, but genetic testing was rejected due to cost reasons. If young people have chronic abdominal symptoms and a family history of suspected hereditary colorectal cancer, a close examination of colon cancer should be performed.

[A Case of Simultaneous Double Cancer with Gastric Metastasis of Breast Cancer and Lung Cancer].

A 60s female with complaint of epigastric discomfort revealed 0-Ⅱb lesion in gastric fornix on upper gastrointestinal endoscopy and exhibited signet-ring cell carcinoma on histopathologic examination of biopsy specimens. A thoracoabdominal CT scan suggested tumors in right breast and left lung, and biopsy revealed invasive lobular carcinoma and primary lung adenocarcinoma, respectively. As results of multidisciplinary discussion of the treatment strategy for triple cancers, it was decided to perform robotic proximal gastrectomy, D1+ lymph node dissection, and esophagogastrostomy. The gastric lesion was diagnosed as gastric metastasis of breast cancer(T2N1M1, Stage Ⅳ)because immunohistochemical staining findings showed ER(+), GCDFP-15(+), and GATA-3(+). Two months later, she underwent the left upper lobectomy, and was diagnosed with lung acinar adenocarcinoma(pT2N0M0, pStage ⅠB). Six months after gastrectomy, she is currently on hormone therapy with aromatase inhibitors for breast cancer.

Complications During Continuous Renal Replacement Therapy in Critically Ill Neonates.

Background/ Aims: Owing to practical and technical developments, continuous renal replacement therapy (CRRT) has been administered even in critically ill neonates. In this study, the complications in CRRT for neonates were examined to establish a safe CRRT. METHODS: This retrospective study reviewed the clinical records of neonates who underwent CRRT at our neonatal intensive care unit between 2009 and 2017. RESULTS: Eight neonates with a body weight of 1,462-3,288 g were treated by 70 CRRT sessions with blood priming. Intradialytic hypotension (IDH) was observed in 39 sessions (55.7%), most of which occurred soon after the start of the CRRT. Body temperature decreased in 48 sessions (70.5%), and thrombocytopenia during CRRT occurred 30 times (42.9%). CONCLUSION: Complications during CRRT in neonates comprised IDH at the start of the CRRT, body temperature decline, and thrombocytopenia. These complications need to be analyzed for a safe neonatal CRRT.

Restriction glycosylases: involvement of endonuclease activities in the restriction process.

All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction­modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Validity of a disposable catheter to drain urine overnight in neurogenic bladder.

BACKGROUND: Overnight catheter drainage (OCD) is introduced to avoid overdistention of the bladder at night-time when clean intermittent catheterization proves ineffective for daytime management of neurogenic bladder. We adopted OCD using disposable silicone no-balloon (DSnB) catheters, with the distal end outside the body opening into diapers. OCD using DSnB catheter, however, had risks of retrograde bacterial contamination. Therefore, in this study, the validity of equipping DSnB catheters with check valves to prevent retrograde bacterial contamination was examined. METHODS: For the in vitro study, excised saline-filled swine bladders were drained using DSnB catheters with or without check valves, and the time required for intravesical pressure to reach 5 cmH2 O was measured. For the in vivo study, in cross-over experiments comparing DSnB catheters with and without check valves, OCD using DSnB catheters for 10 h was performed in rabbits under analgesia. Bacterial growth from urine samples before and after OCD and residual urine volume were examined. RESULTS: For the in vitro experiment, the median drainage time was 368.2 s (range, 88-1,085 s) and 344.7 s (range, 28-840 s) with and without check valves, respectively (n = 6), which was not significantly different. For the in vivo experiment, in cross-over experiments (n = 8) new bacterial growth rates after OCD did not differ, and median residual urine volume was 17.1 mL (range, 0-75 mL) and 1.2 mL (range, 0-5 mL) with and without check valves, respectively (P = 0.055). CONCLUSIONS: Installing a check valve in the DSnB catheter did not decrease new bacterial growth, while tending to increase residual urine volume. DSnB catheters without check valves appear to be better for continuous drainage of urine from bladder.

High-performance switchable grating based on pre-transitional effect of antiferroelectric liquid crystals.

An optical phase grating prototype based on the homeotropic aligned antiferroelectric liquid crystal (AFLC) is demonstrated. By applying an in-plane electric field using comb-like electrodes, the helical structure of AFLC is deformed with the molecules rotating parallel to the electric field because of dielectric anisotropy. This deformation is called the pre-transitional effect of AFLC and induces biaxiality. By using this effect, a switchable phase grating is constructed using a 40µm thick cell filled with (S)-MHPOBC at 85°C. For 532nm TM polarized incident light, the maximum diffraction efficiency of 37.0% is achieved at the electric field of 1.8V/µm for the ± 1st order diffraction. The rise and decay times for the 1st order diffraction pattern are 510µs and 210µs, respectively. The high diffraction efficiency achieved under low field makes it promising for future electro-optical applications.

Phase modulator mode based on the pre-transitional effect of antiferroelectric liquid crystals.

We demonstrate a novel phase-only modulation mode based on the pre-transitional effect of the antiferroelectric liquid crystal. 2π phase modulation without changing the polarization state of the incident light is achieved with a low field (1.8 V/µm). This phase modulation mode also shows an ultrafast response time (less than 300 µs) and a uniform optical texture with an easy fabrication process. This phase modulator can be applied to laser beam steering, virtual reality, and holograms, etc., in the future.

Successful Management of Fulminant Guillain-Barré Syndrome and Its Complications.

A 3-year-old girl presented with muscle weakness of her limbs and trunk 6 days after developing symptoms of common cold. Two days later, she experienced respiratory arrest with a Glasgow Coma Scale score of 3, necessitating endotracheal intubation. Therefore, she was transferred to our hospital with suspected acute encephalopathy. Although no abnormalities were observed on brain and spinal magnetic resonance imaging and electroencephalography, peripheral nerve conduction velocity tests failed to evoke motor and sensory nerve action potentials. Thus, we gave a diagnosis of fulminant Guillain-Barré syndrome and initiated immunoglobulin therapy. On day 3 of admission, she developed sinus tachycardia that induced circulatory failure and oliguria, which was successfully treated with landiolol. Subsequently, we performed plasmapheresis followed by immunoglobulin and steroid pulse therapies. She was weaned off the mechanical ventilator by day 20 of admission, was ambulatory by day 44, and had completely recovered without any adverse sequelae by day 55. In conclusion, landiolol was effective for treating acute sinus tachycardia-induced circulatory failure and played a key role in saving the life of this patient.

Photomanipulation of the anchoring strength using a spontaneously adsorbed layer of azo dendrimers.

We systematically studied the photoinduced anchoring transition in a nematic liquid crystal containing azo dendrimers. Because the azo dendrimers in the trans-isomer state were spontaneously adsorbed at substrate surfaces, which was confirmed by optical second-harmonic generation (SHG), a homeotropic orientation was established at the first stage. Ultraviolet (UV) light irradiation triggered a transition into a planar state which was accompanied by a suppression of the SH generation. The monotonic decrease of the effective scalar order parameter with increasing UV light intensity was determined by polarized attenuated total reflection infrared (ATR-IR) spectroscopy. The variation of anchoring strength and extrapolation length was evaluated by observing the Fréedericksz transition as a function of UV light intensity at a certain visible (VIS) light intensity. Such a photoinduced variation can be interpreted as a variation of the anchoring strength depending on the trans/cis ratio at the surfaces based on a modified Rapini-Papoular model. Thus, this system provides the opportunity for a controlled change in the anchoring strength.

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Thermodynamically Anchoring-Frustrated Surface to Trigger Bulk Discontinuous Orientational Transition.

Surface-specific liquid crystal (LC) nanostructures provide a unique platform for studying surface-wetting phenomena and also for technological applications. The most important studies on LC properties are related to bulk alignment, surface anchoring, and so on. Here, we study an LC system with a nematic liquid crystal (NLC) on a perfluoropolymer-coated substrate, in which a discontinuous bulk orientational transition has recently been found. Using free-energy analysis based on experimental results of the newly-conducted grazing-incidence X-ray diffraction (GI-XRD) measurements, we have confirmed a thermodynamic growth process of smectic liquid crystalline wetting nanosheets on the surface and successfully explained that a frustrated surface of planar and vertical anchoring states accompanied by an elastic energy cost kinetically triggers the bulk reorientation in the first-order manner. This interfacial bottom-up process may offer a general insight into how interfacial hierarchical molecular architectures alter the bulk properties of matter thermodynamically.

Ability of a novel system for neonatal extracorporeal renal replacement therapy with an ultra-small volume circuit to remove solutes in vitro.

BACKGROUND: We automated our manual, syringe-driven extracorporeal renal replacement therapy (eRRT) system with an ultra-small volume circuit (3.2 ml) that is suitable for neonates without blood priming. Our objective was to determine the solute clearance and water balance of the automated and manual systems in vitro. METHODS: Stored whole blood samples containing exogenous urea, creatinine (Cr), potassium (K), and ammonia (NH3) to imitate acute kidney injury (AKI) and hyperammonemia were dialyzed for 3 h (blood flow, 4.0 ml/min; dialysate flow, 600 ml/h) with a continuous infusion of heparin. Solute clearance and sample weight were then compared with values before dialysis. RESULTS: The median clearance of blood urea nitrogen, Cr, K, and NH3 ranged from 1.7 to 2.3 and from 2.4 to 2.6 ml/min, and the median weight of the samples was decreased by 3.8 g and increased by 8.3 g after 3 h of dialysis using the manual and automated systems, respectively. CONCLUSIONS: The automated system effectively cleared solutes, but safety concerns were associated with platelet consumption and fluid balance. Additional studies are needed to establish the safety and accuracy of this novel system for clinical use in neonates and preterm infants.

Totally intracorporeal delta-shaped B-I anastomosis following laparoscopic distal gastrectomy using the Tri-Staple™ reloads on the manual Ultra handle: a prospective cohort study with historical controls.

BACKGROUND: A delta-shaped anastomosis in totally laparoscopic Billroth I gastrectomy could be performed easily and sufficiently using only laparoscopic linear staplers. However, the restricted maneuverability and severe blurring of these staplers along with their limited hemostability induced strain. In this study, we determined the feasibility and safety of performing delta-shaped anastomosis using the Endo GIA™ Reloads with Tri-Staple™ Technology combined with Endo GIA™ Ultra Universal stapler (Tri-Staple) with a particular focus on short-term surgical outcomes. METHODS: We performed a single-institutional prospective interventional study (UMIN 000008014). The Tri-Staple was prospectively used on 23 consecutive patients who underwent a curative totally laparoscopic Billroth I gastrectomy with delta-shaped anastomosis. These patients were matched with the 19 patients previously treated using the ENDOPATH(®) ETS Articulating Linear Cutters (ETS) on clinical and demographic characteristics. RESULTS: There were no differences between the groups in anastomosis-related local complications, morbidity, non-anastomosis-related local complications, total systemic complications, and short-term outcomes with the exception of significantly reduced blood loss in the Tri-Staple group (ETS vs. Tri-Staple: 37 [10-306] vs. 15 [5-210] mL, p = 0.02). Intraoperative bleeding from the staple line was significantly reduced in the Tri-Staple group. The postoperative drain indwelling period (ETS vs. Tri-Staple, 6 [4-10] vs. 4 [2-43] days, p = 0.032), fasting period (5 [3-7] vs. 3 [3-24] days, p = 0.022), and hospital stay (14 [10-47] vs. 11 [6-58] days, p = 0.025) were significantly shorter in the Tri-Staple group. There was no mortality in this series. Acceleration assessed as indices of blurring of stapler tip might have a significant adverse influence on staple-line bleeding at stapling sites. CONCLUSION: Totally laparoscopic Billroth I distal gastrectomy using Tri-Staple was feasible and safe with favorable short-term surgical outcomes. Reduced blurring while stapling may be a novel endpoint which newly developed stapling devices should target.

Molecular characterization of cancer reveals interactions between ionizing radiation and chemicals on rat mammary carcinogenesis.

Although various mechanisms have been inferred for combinatorial actions of multiple carcinogens, these mechanisms have not been well demonstrated in experimental carcinogenesis models. We evaluated mammary carcinogenesis initiated by combined exposure to various doses of radiation and chemical carcinogens. Female rats at 7 weeks of age were γ-irradiated (0.2-2 Gy) and/or exposed to 1-methyl-1-nitrosourea (MNU) (20 or 40 mg/kg, single intraperitoneal injection) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (40 mg/kg/day by gavage for 10 days) and were observed until 50 weeks of age. The incidence of mammary carcinoma increased steadily as a function of radiation dose in the absence of chemicals; mathematical analysis supported an additive increase when radiation was combined with a chemical carcinogen, irrespective of the chemical species and its dose. Hras mutations were characteristic of carcinomas that developed after chemical carcinogen treatments and were overrepresented in carcinomas induced by the combination of radiation and MNU (but not PhIP), indicating an interaction of radiation and MNU at the level of initiation. The expression profiles of seven classifier genes, previously shown to distinguish two classes of rat mammary carcinomas, categorized almost all examined carcinomas that developed after individual or combined treatments with radiation (1 Gy) and chemicals as belonging to a single class; more comprehensive screening using microarrays and a separate test sample set failed to identify differences in gene expression profiles among these carcinomas. These results suggest that a complex, multilevel interaction underlies the combinatorial action of radiation and chemical carcinogens in the experimental model.

Discontinuous thermal diffusivity change due to the anchoring transition of a liquid crystal on a perfluoropolymer surface.

Thermal diffusivity of a liquid crystal, 4'-butyl-4-heptyl-bicyclohexyl-4-carbonitrile, was measured using a temperature wave method. The liquid crystal was sandwiched by two glass substrates, which were treated with three different surface agents for providing distinct molecular orientations. Here, we demonstrate that: 1) a large thermal diffusivity anisotropy arising from different orientations, that is, planar and homeotropic states, was found in the nematic and smectic A phases; 2) when substrates were coated with a perfluoropolymer, abrupt changes of the thermal diffusivity were observed in the nematic phase both on cooling and heating due to the discontinuous anchoring transition between planar and homeotropic states. The temperature dependence of the thermal diffusivity anisotropy was well described by a power law, with an exponent of 0.27 according to the mean-field theory.

A genotoxic stress-responsive miRNA, miR-574-3p, delays cell growth by suppressing the enhancer of rudimentary homolog gene in vitro.

MicroRNA (miRNA) is a type of non-coding RNA that regulates the expression of its target genes by interacting with the complementary sequence of the target mRNA molecules. Recent evidence has shown that genotoxic stress induces miRNA expression, but the target genes involved and role in cellular responses remain unclear. We examined the role of miRNA in the cellular response to X-ray irradiation by studying the expression profiles of radio-responsive miRNAs and their target genes in cultured human cell lines. We found that expression of miR-574-3p was induced in the lung cancer cell line A549 by X-ray irradiation. Overexpression of miR-574-3p caused delayed growth in A549 cells. A predicted target site was detected in the 3'-untranslated region of the enhancer of the rudimentary homolog (ERH) gene, and transfected cells showed an interaction between the luciferase reporter containing the target sequences and miR-574-3p. Overexpression of miR-574-3p suppressed ERH protein production and delayed cell growth. This delay was confirmed by knockdown of ERH expression. Our study suggests that miR-574-3p may contribute to the regulation of the cell cycle in response to X-ray irradiation via suppression of ERH protein production.