Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.

Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

Exosome-Hijacking Drug Delivery System with Branched Arginine Linker Effectively Deliver Antisense Oligonucleotides into Lung Adenocarcinoma Cells.

Exosomes are a type of extracellular vesicles that contain diverse molecules and are present in our body fluids. They play a crucial role in transporting materials and transmitting signals between cells. Currently, there have been numerous reports on the use of exosomes in drug delivery systems (DDS). However, most existing methods for utilizing exosomes in DDS require the isolation and purification of exosomes, which raises concerns about yield and potential damage to the exosomes. Recently, we have developed a novel DDS called "ExomiR-Tracker" that harnesses exosomes without the need for isolation and purification. This system aims to deliver nucleic acid drugs effectively. ExomiR-Tracker consists of an anti-exosome antibody equipped with nona-D-arginines (9 mer) and nucleic acid drugs which have complementary sequence of target microRNA (anti-miR). In this study, we modified ExomiR-Tracker by incorporating branched nona-D-arginines (9 + 9 mer) molecules (referred to as Branch ExomiR-Tracker) and evaluated its efficacy in lung adenocarcinoma cells (A549 cells). The improved complex formation ability and enhanced cellular uptake of anti-miR, demonstrated by our findings, highlight the advantages of incorporating branched oligoarginine peptides into the ExomiR-Tracker platform. These results represent significant progress in revealing the effectiveness of Branch ExomiR-Tracker against adhesive cancer cells, which has not been shown to be effective with the conventional Linear ExomiR-Tracker.

Engineered Cytochrome P450-Catalyzed Oxidative Biaryl Coupling Reaction Provides a Scalable Entry into Arylomycin Antibiotics.

We report herein the first example of a cytochrome P450-catalyzed oxidative carbon-carbon coupling process for a scalable entry into arylomycin antibiotic cores. Starting from wild-type hydroxylating cytochrome P450 enzymes and engineered Escherichia coli, a combination of enzyme engineering, random mutagenesis, and optimization of reaction conditions generated a P450 variant that affords the desired arylomycin core 2d in 84% assay yield. Furthermore, this process was demonstrated as a viable route for the production of the arylomycin antibiotic core on the gram scale. Finally, this new entry affords a viable, scalable, and practical route for the synthesis of novel Gram-negative antibiotics.

Synthesis and Evaluation of Oligonucleotide-Containing 2'-O-{[(4,5',8-Trimethylpsoralen)-4'-ylmethoxy]ethylaminocarb-onyl}adenosine as Photo-crosslinkable Gene Targeting Tools.

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-crosslinking properties of two Ps-Oligos, 5'-Ps-Oligo and a novel Ps-Oligo containing 2'-O-{[(4,5',8-trimethylpsoralen)-4'-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5'-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the interesting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies.

Safety, speed, and effectiveness of air transportation for neonates.

BACKGROUND: In Japan, 44.3% of neonates are delivered in private clinics without an attending pediatrician. Obstetricians in the clinics must resuscitate asphyxiated neonates in unstable condition, such as respiratory failure, and they are frequently transferred to tertiary perinatal medical centers. There has been no study comparing the physiological status and prognosis of neonates transported by ambulance with those transported by helicopter. METHODS: Medical and transport records were used to compare the physiological status of neonates transported to Kagoshima City Hospital by land and those transported by air between January 1, 2013, and December 31, 2017. RESULTS: Data from 425 neonates transferred by land and 143 by air were analyzed. There were no significant differences between the two groups in mean gestational age, mean birthweight, fetal blood pH, Apgar score, or the Score for Neonatal Acute Physiology with Perinatal Extension-II (SNAPPE-II) on arrival to the tertiary center (16.3 ± 15.4 [95% confidence interval (CI): 13.2-17.7] vs 16.4 ± 15.4 [95% CI: 13.9-19.0], respectively; P = 0.999); both groups had SNAPPE-II score 10-19, indicating no difference in mortality risk. The times to starting first aid and to admission to the intensive care unit were significantly reduced in neonates transported by air than by land. In subgroup analysis of patients of a gestational age ≤28 weeks, all cases of severe intraventricular hemorrhage (IVH) were observed in the land transportation group. CONCLUSIONS: Neonatal transportation by air is as safe as land transportation, and time to first aid and intensive care are significantly reduced by transportation by air than by land. Air transport could also contribute to the prevention of IVH in neonatal transportation.

Influence of Type 2 Endoleaks on Long-Term Outcomes after Endovascular Repair for Abdominal Aortic Aneurysms: A National Hospital Organization Network Study for Abdominal Aortic Aneurysms in Japan.

BACKGROUND: Although endovascular repair (EVAR) is the first-line treatment for abdominal aortic aneurysm, type 2 endoleak (EL), which is associated with late sac enlargement or rupture, remains a concern. The present study aimed to assess the influence of type 2 EL on long-term outcomes after EVAR. METHODS: Among 550 patients who underwent EVAR between 2007 and 2013 at 14 Japanese national hospitals, 135 patients had type 2 EL diagnosed on follow-up computed tomography (CT) within 12 months after EVAR (EL2[+] group) and 415 patients did not have EL within 12 months (EL2[-] group). The cumulative incidences of sac enlargement, late intervention, and aneurysm-related death after EVAR were estimated using the cumulative incidence function method, and prognostic factors were investigated using the Fine-Gray hazard model. RESULTS: The median follow-up period was 5 years, and the 5-year cumulative incidence rates of sac enlargement, late intervention, and aneurysm-related death were 30.7% ± 4.4%, 25.3% ± 4.1%, and 2.6% ± 1.4%, respectively, in the EL2(+) group, and 8.7% ± 1.6%, 7.6% ± 1.4%, and 0.3% ± 0.3%, respectively, in the EL2(-) group. The cumulative incidence rates of sac enlargement (P = 0.002), late intervention (P < 0.001), and aneurysm-related death (P = 0.015) were significantly different between the 2 groups. As the first-line treatment for sac enlargement with type 2 EL, transcatheter coil embolization was performed in 30 patients. Information about sac behavior on CT after coil embolization was available in 20 of the 30 patients. Among these patients, no patients experienced sac shrinkage, and the aneurysmal sac dilated after coil embolization in 18 patients. CONCLUSIONS: Type 2 EL affects the long-term outcomes after EVAR. It is not recommended to observe large aneurysmal sacs conservatively as they tend to dilate in the presence of type 2 EL.

Assessment of risk factors for cystic periventricular leukomalacia.

AIM: Periventricular leukomalacia (PVL) is an important cause of cerebral palsy in premature infants, and cystic PVL is the most serious form of the disease. The risk factors for cystic PVL in singleton fetuses at a gestational age of <35 weeks are unclear. METHODS: This study included 2013 singleton birth infants delivered at a gestational age of <35 weeks in Kagoshima City Hospital between 2006 and 2017. The findings for 30 infants with cystic PVL were compared with those for 63 matched control infants by gestational age and birth weight. RESULTS: The cystic PVL was associated with increased incidence of recurrent late deceleration (L/D) (43.4% vs. 15.9%, P = 0.004) and loss of variability (LOV) (10.0% vs. 0.0%, P = 0.03) in fetal heart rate monitoring and late-onset circulatory dysfunction (LCD) (33.3% vs. 11.1%, P = 0.02). Logistic regression analysis revealed that recurrent L/D (odds ratio [OR] = 3.57, 95% confidence interval [CI]: 1.29-10.15, P = 0.01) and LCD (OR = 3.41, 95% CI: 1.09-11.04, P = 0.03) were risk factors associated with cystic PVL. LOV was not included in the multivariate analysis as there were too few cases in both the cystic PVL and control groups. CONCLUSION: Recurrent L/D, LOV and LCD are strongly associated with cystic PVL. In cases of fetal acidosis related to recurrent L/D or loss of variability, cystic PVL may occur.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

Dopamine detection on activated reaction field consisting of graphene-integrated silicon photonic cavity.

Graphene is widely recognized as an outstanding and multi-functional material in various application fields such as electronics, photonics, mechanics, and life sciences. We propose a neurotransmitter sensor with ultra-small volume for detecting the photonic light-matter response. Such detection can be achieved using surface-activated monolayer graphene sheets and CMOS-compatible silicon-photonic circuits. Patterned pieces of CVD-grown graphene are integrated on the top of a silicon micro-ring resonator, which induce the adsorption of catecholamine molecules originated from the π-stacking effect. We used dopamine to demonstrate such detection and examine the sensitivity of graphene-dopamine coupling. To avoid high optical insertion loss and degradation of resonance characteristics caused by a graphene's extremely high optical absorption coefficient in the near infrared region, a ring resonator with adjusted coupling design is used to compensate for the drawbacks. Owing to the advanced nano-sensing platform and measurement system, an activated graphene-sensing surface of only ∼30 µm2/ch enables π coupling to dopamine with enough sensitivity to detect less than 10-µM solution concentration. The detection mechanism through the surface reaction is also verified by optical simulation and atomic force microscopy measurement, revealing that the flowing dopamine molecules can only occupy the outermost surface of graphene. We expect this sensor to contribute to the development of an innovative label-free and disposable bio-sensing platform with accurate, sensitive, and fast response.

Efficient transient protein expression in tomato cultivars and wild species using agroinfiltration-mediated high expression system.

KEY MESSAGE: The new transient protein expression system using the pBYR2HS vector is applicable to several tomato cultivars and wild species with high level of protein expression. Innovation and improvement of effective tools for transient protein expression in plant cells is critical for the development of plant biotechnology. We have created the new transient protein expression system using the pBYR2HS vector that led to about 4 mg/g fresh weight of protein expression in Nicotiana benthamiana. In this study, we validated the adaptability of this transient protein expression system by agroinfiltration to leaves and fruits of several tomato cultivars and wild species. Although the GFP protein was transiently expressed in the leaves and fruits of all tomato cultivars and wild species, we observed species-specific differences in protein expression. In particular, GFP protein expression was higher in the leaves and fruits of Micro-Tom, Solanum pimpinellifolium (0043) and S. pimpinellifolium (0049-w1) than in those of cultivars and wild species. Furthermore, Agrobacterium with GABA transaminase enhanced transient expression in tomato fruits of Micro-Tom. Taken together with these results, our system is applicable to several tomato cultivars and species as well as a model tomato, even though characteristics are often different among tomato cultivars or species. Thus, the system is an effective, simple, and valuable tool to achieve rapid transgene expression to examine gene function in tomato plant cells.

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma.

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.

Small Nucleolar Noncoding RNA SNORA23, Up-Regulated in Human Pancreatic Ductal Adenocarcinoma, Regulates Expression of Spectrin Repeat-Containing Nuclear Envelope 2 to Promote Growth and Metastasis of Xenograft Tumors in Mice.

BACKGROUND & AIMS: Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes, and specific snoRNAs are dysregulated in some cancer cells. We investigated dysregulation of snoRNAs in pancreatic ductal adenocarcinoma (PDAC) cells. METHODS: We investigated snoRNA expression in PDAC cell lines by complementary DNA microarray and quantitative reverse transcription polymerase chain reaction. In PDAC (n = 133), intraductal papillary mucinous neoplasm (n = 16), mucinous cystic neoplasm-associated PDAC (n = 1), and non-tumor pancreas (n = 8) and liver (n = 3) tissues from subjects who underwent surgical resection, levels of snoRNA were measured by quantitative reverse transcription polymerase chain reaction and compared with clinicopathologic parameters and survival times determined by Kaplan-Meier analysis. To examine snoRNA function, PDAC cells were transfected with snoRNA-antisense oligonucleotides flanked with amido-bridged nucleic acids, or snoRNA-expression plasmids, and analyzed in proliferation, colony formation, spheroid formation, and invasion assays. To identify snoRNA-related factors, cells were analyzed by gene expression and proteomic profiling and immunoblot assays. Mice were given intrasplenic injections of MIA PaCa2- or Suit2-HLMC cells; tumor-bearing nude mice were then given 3 weekly injections of an antisense oligonucleotides against SNORA23, a H/ACA-box type snoRNA, and tumor growth and metastasis to liver, blood, and pancreas were analyzed. RESULTS: Levels of SNORA23 increased and accumulated at the nucleolus in highly metastatic MIA PaCa2- or Suit2-HLMC cells compared with their parental cells. We detected SNORA23 in human PDAC specimens but not in non-tumor pancreatic tissue. PDAC level of SNORA23 correlated with invasion grade and correlated inversely with disease-free survival time of patients. Expression of SNORA23 in PDAC cells increased their invasive activity and colony formation, and spheroid formation was inhibited by SNORA23 knockdown. In gene expression and proteomic profile analyses, we found SNORA23 to increase expression of spectrin repeat-containing nuclear envelope 2 (SYNE2) messenger RNA and protein. Knockdown of SYNE2 in PDAC cells reduced their invasive activities and anchor-independent survival. Administration of SNORA23 antisense oligonucleotides to mice slowed growth of xenograft tumors, tumor expression of SYNE2, tumor cell dissemination, and metastasis to liver. CONCLUSIONS: We found expression of the snoRNA SNORA23, which mediates sequence-specific pseudouridylation of ribosomal RNAs, to be increased in human PDAC tissues compared with non-tumor tissues, and levels to correlate with tumor invasion grade and patient survival time. SNORA23 increases expression of SYNE2, possibly through modulation of ribosome biogenesis, to promote PDAC cell survival and invasion, and growth and metastasis of xenograft tumors in mice.

Cyanine Photocages Enable Spatial Control of Inducible Cre-Mediated Recombination.

Optical control over protein expression could provide a means to interrogate a range of biological processes. One approach has employed caged ligands of the estrogen receptor (ER) in combination with broadly used ligand-dependent Cre recombinase proteins. Existing approaches use UV or blue wavelengths, which hinders their application in tissue settings. Additionally, issues of payload diffusion can impede fine spatial control over the recombination process. Here, we detail the chemical optimization of a near-infrared (NIR) light-activated variant of the ER antagonist cyclofen. These studies resulted in modification of both the caging group and payload with lipophilic n-butyl esters. The appendage of esters to the cyanine cage improved cellular uptake and retention. The installation of a 4-piperidyl ester enabled high spatial resolution of the light-initiated Cre-mediated recombination event. These studies described chemical modifications with potential general utility for improving spatial control of intracellular caging strategies. Additionally, these efforts will enable future applications to use these molecules in complex physiological settings.

Twin-mirror membrane distributed-reflector lasers using 20-µm-long active region on Si substrates.

We demonstrate 20-µm-long twin-mirror membrane distributed-reflector (DR) lasers for chip-to-chip optical interconnects. The lasers employ distributed Bragg reflectors (DBRs) at both ends of a 20-µm-long λ/4-phase shifted distributed feedback (DFB) section. We achieve single-mode lasing in a λ/4-phase shifted DFB mode at room temperature with a threshold current of 0.39 mA. The lasing wavelength remains stable while the injected current is varied, and it is determined by the λ/4 phase-shifted DFB. The modulation current efficiency is 11.4 GHz/mA1/2, which is measured by using relative intensity noise spectra. We also demonstrate the direct modulation of the DR lasers at a bit rate of 25.8 Gbit/s with an energy cost of 163 fJ/bit.

Identification and Characterization of a Novel NADPH Oxidase 1 (Nox1) Inhibitor That Suppresses Proliferation of Colon and Stomach Cancer Cells.

Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2-5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.

Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

Synthesis, duplex-forming ability, enzymatic stability, and in vitro antisense potency of oligonucleotides including 2'-C,4'-C-ethyleneoxy-bridged thymidine derivatives.

We synthesized thymidine derivatives of 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids with an 8'-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents. The results showed that EoDNAs and methylene-EoDNAs could be promising candidates for antisense technology.

Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.

Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory.

Extracorporeal membrane oxygenation in 61 neonates: Single-center experience.

BACKGROUND: There have been few reports on the outcome of extracorporeal membrane oxygenation (ECMO) in newborn Japanese infants. METHODS: A review was carried out of 61 neonates with ECMO between January 1995 and December 2015 at a single center. ECMO was used in neonates with oxygenation index >20 after conventional treatment. Background factors, such as etiology, vascular access mode (veno-venous [VV] or veno-arterial [VA]), number of days with ECMO, and early ECMO (within 24 h after birth), were analyzed in relation to outcome with respect to survival to hospital discharge (SHD). RESULTS: Survival to hospital discharge was achieved in 35 infants (57%), while the remaining 26 died during hospital stay. Gestational age at birth was significantly higher and number of days with ECMO was significantly lower in SHD infants compared with those with adverse outcome (median, 4.0 vs 5.5 days, respectively; P = 0.008). The SHD rate was significantly higher for those with VV than VA vascular access mode (78%, 18/23 vs 45%, 17/38, respectively; P = 0.016), and for those with than without early ECMO (72%, 28/39 vs 32%, 7/22, respectively; P = 0.003). The SHD rate was relatively high in neonates with meconium aspiration syndrome (86%, 12/14), persistent pulmonary hypertension associated with hypoxic ischemic encephalopathy (75%, 6/8), and emphysema (80%, 4/5). On stepwise logistic regression analysis two independent factors of SHD were identified: early ECMO (OR, 9.63; 95%CI: 2.47-37.6) and ECMO length <8 days (OR, 8.05; 95%CI: 1.94-33.5). CONCLUSIONS: Neonates with early ECMO and those with ECMO duration <8 days may benefit from ECMO with respect to SHD.