The majority of animal genes are required for wild-type fitness.

Almost all eukaryotic genes are conserved, suggesting that they have essential functions. However, only a minority of genes have detectable loss-of-function phenotypes in experimental assays, and multiple theories have been proposed to explain this discrepancy. Here, we use RNA-mediated interference in C. elegans to examine how knockdown of any gene affects the overall fitness of worm populations. Whereas previous studies typically assess phenotypes that are detectable by eye after a single generation, we monitored growth quantitatively over several generations. In contrast to previous estimates, we find that, in these multigeneration population assays, the majority of genes affect fitness, and this suggests that genetic networks are not robust to mutation. Our results demonstrate that, in a single environmental condition, most animal genes play essential roles. This is a higher proportion than for yeast genes, and we suggest that the source of negative selection is different in animals and in unicellular eukaryotes.

A compendium of RNA-binding motifs for decoding gene regulation.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

Implant fracture failure rate and potential associated risk indicators: An up to 12-year retrospective study of implants in 5,124 patients.

OBJECTIVES: This study investigated fracture rates and risk indicators for fractures in internal connection dental implants. MATERIAL AND METHODS: We performed a retrospective analysis of 19,006 internal connection implants used in fixed restoration in 5,124 patients (4,570 males, 554 females) at the Dental Hospital of Veterans Health Service Medical Center between 2006 and 2015. Patients were followed through June 2018 (0.03-12.39 years post-installation). Clinical factors (age, sex, implant diameter, implant length, placement site, bone graft, fixture material, cervical feature, abutment connection, microthread, and platform switching) were recorded. Kaplan-Meier survival analysis identified risk indicators associated with an implant fracture. Cox regression models elucidated potential fracture risks. RESULTS: One hundred and seventy-four implants fractured in 135 patients, for an incidence rate of 0.92% after an average of 4.95 ± 2.14 years of use. Kaplan-Meier estimates showed that the 3-, 5-, and 10-year survival rates of implants were 99.8%, 99.2%, and 97.7%, respectively. In the multivariable Cox regression model, the diameter, location, history of bone graft, and microthread presence were significantly correlated with implant fractures. Wide-diameter implants had a reduced fracture risk within 90 months, after which the diameter did not correlate with fractures. Implants placed in the anterior mandible had a lower fracture risk within 90 months; mandibular premolar implants corresponded with a lower risk after 90 months. Implants without a history of bone graft or microthreads were more likely to fracture throughout the follow-up time. CONCLUSIONS: These results elucidate risk indicators for implant fractures and facilitate their reduction in clinical practice.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

Tuning Semiconductor Performance of Nickel Complexes through Crystal Transformation.

Crystal transformation between two polymorphs (green, 1-G, and red, 1-R) of the square-planar nickel complex NiL2 (L = 2-ethoxy-6-( N-methyliminomethyl)phenolate) and their tuning effect to semiconductor properties were studied both experimentally and theoretically. When 1-G is heated to 413 K, it converts to 1-R, whereas soaking 1-R in several kinds of solvents causes it to revert to 1-G. Crystallographic and PXRD studies reveal the dramatic changes in crystal dimensions due to the changes of packing models. Heating device made from 1-G (D-1-G(298)) at 413 K significantly increases the electrical conductivity from 6.55 × 10-4 S cm-1 for D-1-G(298) to 1.11 × 10-3 S cm-1 for D-1-G(413), showing significant crystal form dependence. Heat-treating D-1-G and D-1-R devices at different temperatures clearly reveals the reason for the conductivity tuning. Thus, the conductivity of NiL2-based devices could be well tuned through crystal transformation by heating or by soaking in solvent. Theoretical calculations clearly revealed the reason for such conductivity changes and also predicted that both polymorphs are good p-type semiconductors with hole mobilities of 1.63 × 10-2 (1-G) and 2.11 × 10-1 cm2 V-1 s-1 (1-R).

Feasibility of electromagnetically guided transjugular intrahepatic portosystemic shunt procedure.

OBJECTIVES: To develop an electromagnetic navigation technology for transjugular intrahepatic portosystemic shunt (TIPS) creation and translate it from phantom to an in-vivo large animal setting. MATERIAL AND METHODS: A custom-designed device for TIPS creation consisting of a stylet within a 5 French catheter as well as a software prototype were developed that allow real-time tip tracking of both stylet and catheter using an electromagnetic tracking system. Feasibility of navigated TIPSS creation was tested in a phantom by two interventional radiologists (A/B) followed by in-vivo testing evaluation in eight domestic pigs. Procedure duration and number of attempts needed for puncture of the portal vein were recorded. RESULTS: In the phantom setting, intervention time to gain access to the portal vein (PV) was 144 ± 67 s (A) and 122 ± 51 s (B), respectively. In the in-vivo trials, TIPS could be successfully completed in five out of eight animals. Mean time for the complete TIPS was 245 ± 205 minutes with a notable learning curve towards the last animal. CONCLUSIONS: TIPS creation with the use of electromagnetic tracking technology proved to be feasible in-vitro as well as in-vivo. The system may be useful to facilitate challenging TIPSS procedures.

Stepwise Assembly of MII7 Clusters Revealed by Mass Spectrometry, EXAFS, and Crystallography.

The square-planar monomer NiL2 (Ni1 ), L=2-ethoxy-6-(N-methyl-iminomethyl)phenolate, reacts with M(H2 O)6 (ClO4 )2 , M=Ni or Co, to form heptanuclear disks [Cox Ni7-x (OH)6 (L)6 ](ClO4 )2 ⋅2 CH3 CN (Cox Ni7-x , x=0-7) and the co-crystal [Cox Ni7-x (OH)6 L6 ][NiL2 ](ClO4 )2 ⋅2 CH3 CN (Cox Ni7-x -Ni1 ) under ambient conditions. It has proved possible to explore the bottom-up assembly process of Cox Ni7-x and Cox Ni7-x -Ni1 in real time. The final products have been characterized by thermogravimetric analysis, IR, elemental analysis, ICP-MS, and single-crystal X-ray diffraction. Time-dependent mass spectrometry (MS) revealed the following reaction steps: Ni1 →[M2 L3 ]+ →[M4 (OH)2 L4 ]2+ →[M7 (OH)6 L6 ]2+ . In contrast, the reaction of Ni1 with Zn2+ only reaches halfway, and crystallographic evidence indicates a butterfly structure for [Zn2 Ni2 (OH)2 Cl2 ] (Zn2 Ni2 ), an intermediate that is difficult to isolate in the above Ni-Co series. A summation method has been used to analyze the MS of bimetallic clusters with very similar atomic masses, as is the case for Co and Ni. The results provide ample information on the distribution of Co and Ni within each cluster and their statistical distribution within selected crystals.

Shedding light on minipig drug metabolism - elevated amide hydrolysis in vitro.

1. In recent years, the minipig is increasingly used as a test species in non-clinical assessment of drug candidates. While there is good scientific evidence available concerning cytochrome P450-mediated metabolism in minipig, the knowledge of other metabolic pathways is more limited. 2. The aim of this study was to provide an understanding of when, why, and how drug metabolism in minipig differs from other species commonly used in non-clinical studies. In-house cross-species metabolite profile comparisons in hepatocytes and microsomes of 38 Roche development compounds were retrospectively analyzed to compare the metabolism among minipig, human, rat, dog, monkey, rabbit and mouse. 3. A significant contributor to the elevated metabolism observed for certain compounds in minipig was identified as amide hydrolysis. The hepatic amide hydrolysis activity in minipig was further investigated in subcellular liver fractions and a structure-activity relationship was established. When structural motifs according to the established SAR are excluded, coverage of major human metabolic pathways was shown to be higher in minipig than in dog, and only slightly lower than in cynomolgus monkey. 4. A strategy is presented for early identification of drug compounds which might not be suited to further investigation in minipig due to excessive hydrolytic metabolism.

Endovascular placement of an extraluminal arterial bypass graft - in vitro feasibility study.

PURPOSE: A novel approach for arterial bypass grafting using exclusively endovascular techniques was established in-vitro in a phantom model. MATERIAL AND METHODS: The experimental setting consisted of a gel-wax phantom with two embedded parallel fluid-filled silicon tubes simulating the superficial femoral vessels. Through an 8-French sheath, a re-entry catheter (OUTBACK®, Cordis) was placed in the simulated artery and used to puncture the vascular wall. Then a 0.014-inch guide wire was advanced into the extravascular space. With the curved needle of the re-entry catheter, the guide wire was steered on a course parallel to the vessel wall in the extravascular space for 5-10 cm. At the desired reentry site, the re-entry catheter was used to puncture the vascular wall again in order to regain access to the endovascular space. Once the tip of the guide wire had safely been placed in the vascular lumen, a self-expandable stent graft (VIABAHN® GORE®) was deployed to complete the extraluminal bypass. RESULTS: Endovascular placement of an extraluminal bypass was successfully achieved in 20 attempts. The mean duration of the procedure amounted to 14:58 (minutes: seconds) (SD ± 3:56). CONCLUSIONS: This in-vitro study suggests that endovascular placement of an extraluminal arterial bypass graft is technically feasible.

Current status of the novel food ingredient safety evaluation system.

Increasing demand for new foods, technological development, and vegan market growth have led to an increase in new food ingredients, so the need for safety assessment of these ingredients is important. Representative safety assessment systems are the Generally Recognized as Safe (GRAS) notification of the Food and Drug Administration in the USA and the novel food system of the European Food Safety Authority in the European Union. GRAS is a notification system for information on food ingredients, food additives and functional foods under the responsibility of the applicant, while the novel food system assesses the safety of food ingredients excluding food additives. In Korea, a safety evaluation system is established for temporary food ingredients, which includes food ingredients without a domestic intake history. However, safety assessment systems for novel foods from other countries and food ingredients produced by the application of new technology need to be improved.

Enterococcus diestrammenae sp. nov., isolated from the gut of Diestrammena coreana.

A novel Gram-stain-positive, facultatively anaerobic, non-motile and lactic-acid-producing bacterium, designated strain ORL-24(T), was isolated from the gut of the camel cricket, Diestrammena coreana. Optimal growth occurred at 37 °C, pH 8 and with 0 % (w/v) NaCl. The ratio of l-lactate to d-lactate in strain ORL-24(T) was 96 : 4. Lancefield antigen D was not detected. The strain was negative for oxidase activity and catalase activity. According to a phylogenetic analysis based on 16S rRNA gene sequences, strain ORL-24(T) was most closely related to the type strain of Enterococcus asini (96.9 % similarity). Comparative pheS and rpoA sequence analyses of strain ORL-24(T) indicated that the strain belonged to the genus Enterococcus. The major fatty acids were C16 : 0 and C18 : 1ω9c. The DNA G+C content was 41.3 mol%. Based on phenotypic, genotypic and phylogenetic analyses, strain ORL-24(T) represents a novel species of the genus Enterococcus, for which the name Enterococcus diestrammenae is proposed. The type strain is ORL-24(T) ( = KACC 16708(T) = JCM 18359(T)).

Comparison of four multilocus sequence typing schemes and amino acid biosynthesis based on genomic analysis of Bacillus subtilis.

Bacillus subtilis, a valuable industrial microorganism used in starter cultures in soybean fermentation, is a species of bacteria with interspecies diversity. Here, four multilocus sequence typing (MLST) schemes developed to assess the diversity of B. subtilis or Bacillus spp. were applied and compared to confirm the interspecies diversity of B. subtilis. In addition, we analyzed correlations between amino acid biosynthesis genes and sequence types (STs); this is important because amino acids are key taste components in fermented foods. On applying the four MLST methods to 38 strains and the type strain of B. subtilis, 30 to 32 STs were identified. The discriminatory power was 0.362-0.964 for the genes used in the MLST methods; the larger the gene, the greater the number of alleles and polymorphic sites. All four MLST methods showed a correlation between STs and strains that do not possess the hutHUIG operon (which contains genes required for the production of glutamate from histidine). This correlation was verified using 168 further genome-sequence strains.

ComQXPA quorum-sensing systems contribute to enhancing the protease activity of Bacillus velezensis DMB05 from fermented soybeans.

Bacillus velezensis DMB05, isolated from traditionally fermented soybean, meju, exhibited no protease activity on a TSA plate containing skim milk. To shed light on the genetic background behind this phenotypic non-protease activity, we analyzed the complete genome sequence of strain DMB05 and compared it with those of two B. velezensis strains which did exhibit protease activity. Comparative genome analyses showed no significant difference in the kind or number of proteases between the genomes of the three strains and that all strains possessed the degSU two-component system involved in the gene regulation of protease. However, strain DMB05 possessed a truncated comP which is part of the comQXPA operon that regulates the expression of degQ involved in the activation of DegSU. When the entire comQXPA operon derived from DMB06 was introduced into DMB05, the recombinant expressed proteolytic activity. The results of this experimental study provide evidence for the presence of regulatory genes involved in protease activity, one of several important factors involved in fermentation.

Increased Production of γ-Aminobutyric Acid from Brewer's Spent Grain Through Bacillus Fermentation.

Brewer's spent grain (BSG) is a waste product of the beer industry, and γ-aminobutyric acid (GABA) is a physiologically active substance important for brain and neuron physiology. In this study, we used the bacterial strains Bacillus velezensis DMB06 and B. licheniformis 0DA23-1, respectively, to ferment BSG and produce GABA. The GABA biosynthesis pathways were identified through genomic analysis of the genomes of both strains. We then inoculated the strains into BSG to determine changes in pH, acidity, reducing sugar content, amino-type nitrogen content, and GABA production, which was approximately doubled in BSG inoculated with Bacillus compared to that in uninoculated BSG; however, no significant difference was observed in GABA production between the two bacterial strains. These results provide the experimental basis for expanding the use of BSG by demonstrating the potential gain in increasing GABA production from a waste resource.

Smaller sulfur molecules promise better lithium-sulfur batteries.

The lithium-sulfur battery holds a high theoretical energy density, 4-5 times that of today's lithium-ion batteries, yet its applications have been hindered by poor electronic conductivity of the sulfur cathode and, most importantly, the rapid fading of its capacity due to the formation of soluble polysulfide intermediates (Li(2)S(n), n = 4-8). Despite numerous efforts concerning this issue, combatting sulfur loss remains one of the greatest challenges. Here we show that this problem can be effectively diminished by controlling the sulfur as smaller allotropes. Metastable small sulfur molecules of S(2-4) were synthesized in the confined space of a conductive microporous carbon matrix. The confined S(2-4) as a new cathode material can totally avoid the unfavorable transition between the commonly used large S(8) and S(4)(2-). Li-S batteries based on this concept exhibit unprecedented electrochemical behavior with high specific capacity, good cycling stability, and superior rate capability, which promise a practicable battery with high energy density for applications in portable electronics, electric vehicles, and large-scale energy storage systems.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Transcriptomic analysis of Staphylococcus equorum KM1031 from the high-salt fermented seafood jeotgal under chloramphenicol, erythromycin and lincomycin stresses.

Staphylococcus equorum strain KM1031 is resistant to chloramphenicol, erythromycin and lincomycin. To shed light on the genetic factors underlying these antibiotic resistances, we determined the global gene expression profile of S. equorum KM1031 using RNA sequencing. During chloramphenicol, erythromycin and lincomycin treatment, 8.3% (183/2,336), 16.0% (354/2,336), and 2.9% (63/2,336) of S. equorum KM1031 genes exhibited significant differences in expression, respectively. These three antibiotics upregulated genes related to efflux and downregulated genes related to transporters. Antibiotic treatment also upregulated osmoprotectant-related genes involved in salt tolerance. To identify specific genes functionally related to antibiotic resistance, we compared the genome of strain KM1031 with those of three S. equorum strains that are sensitive to these three antibiotics. We identified three genes of particular interest: an antibiotic biosynthesis monooxygenase gene (abm, AWC34_RS01805) related to chloramphenicol resistance, an antibiotic ABC transporter ATP-binding protein gene (msr, AWC34_RS11115) related to erythromycin resistance, and a lincosamide nucleotydyltransferase gene (lnuA, AWC34_RS13300) related to lincomycin resistance. These genes were upregulated in response to the corresponding antibiotic; in particular, msr was upregulated more than fourfold by erythromycin treatment. Finally, the results of RNA sequencing were validated by quantitative real-time PCR. This transcriptomic analysis provides genetic evidence regarding antibiotic stress responses of S. equorum strain KM1031.

Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Bacterial Community of Galchi-Baechu Kimchi Based on Culture-Dependent and - Independent Investigation and Selection of Starter Candidates.

In this study, the bacterial community of galchi-baechu kimchi was determined using culture-based and culture-independent techniques (next generation sequencing:NGS), and showed discrepancies between results. Weissella koreensis and Pediococcus inopinatus were the dominant species according to the NGS results, while Bacillus species and P. inopinatus were dominant in the culture-dependent analysis. To identify safe starter candidates, sixty-five Bacillus strains isolated from galchi-baechu kimchi using culture-dependent methods were evaluated for their antibiotic resistance, presence of toxin genes, and hemolytic activity. Strains were then assessed for salt tolerance and protease and lipase activity. As a result, four strains-B. safensis GN5_10, B. subtilis GN5_19, B. velezensis GN5_25, and B. velezensis GT8-were selected as safe starter candidates for use in fermented foods.

Safety Assessment Systems for Microbial Starters Derived from Fermented Foods.

Microorganisms involved in food fermentation not only improve the aroma and taste of the food, but also enhance its preservation. Thus, they are added as starter cultures to boost the final product quality of commercial fermented foods. Although these microorganisms originate from fermented foods and have a long history of consumption, the European Union recently applied the concept of Qualified presumption of Safety (QPS), which is a safety evaluation system for microorganisms used in food or feed in Europe. The QPS system is a species-level safety system and shares results with the European Novel Food System, a strain-level safety evaluation system. In the United States, microorganisms added to fermented foods are considered as food additives or Generally Recognized as Safe substance. In Korea, food microbe lists are presented at the species level. Moreover, the nation has established a strain-oriented evaluation system that applies temporary safety evaluation methods for food raw materials as well as new raw materials. However, when it comes to microorganisms isolated from traditional fermented foods and other fermented food products, there is no definition of the term "species," and there is a lack of an evaluation system at the species level. Therefore, such an evaluation system for microbial species used in Korean fermented foods is necessary.