Detection and Typing of Genital High-Risk HPV DNAs in Cervical Scrapes Using the E6E7-Specific Consensus PCR

Specific types of human papillomaviruses (HPVs) are closely associated with the development of genital carcinomas. We previousy reported a PCR method which amplifies the E6E7 sequence from 6 different high-risk genital HPVs (HPV16, 18, 31, 33, 52 and 58) (J Gen Virol 1991, 72 : 1039-1044.). To amplify broader types of genital high-risk HPVs, we have modified our consensus primers by extending 9 nucleotides at the 3 end of the sense primer and changing 5 nucleotides at the 5 end of the anitisense primer. Genotype diag-nosis was carried out by AvaII plus Rsal digestion. This modified PCR method enabled the detection of trace levels of at least 11 types of genital high-risk HPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58), at subpicogram to sub-nanogram amounts of cloned DNA, amplified after the consensus PCR. We applied this method to analyze 155 cervical scrapes from patients who had been diagnosed with premalignant or malignant cervical lesions. HPVs were detected in 63.0% of mild dysplasia (17/27), 100% of moderate dysplasia (all 12 cases), 91.7% of severe dysplasia (11/12), 95.8% of carcinoma in situ (23/24), and 80.0% of invasive cervical cancer (52/65). HPV16 was present predominant-ly (60.9%), followed by HPV58 (153%), HPV52 (13.9%), HPV31 (13.0%), HPV18 (6.1%), HPV35 (2.6%), HPV51 (2.6%), HPV56 (2.6%), HPV33 (1.7%) and HPV39 (1.7%). Five cases contained unclassified types (4.3%). The results indicate that this modified E6E7 consensus PCR method provides a quick and easy way to detect and diagnose genotypes of the high-risk genital HPVs from scraped cells

A novel salt- and organic solvent-tolerant phosphite dehydrogenase from Cyanothece sp. ATCC 51142

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0–10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na+, K+, and NH4+. It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD+. Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD+-binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD+ under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L-tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent

Efficient genome editing and its application to conditional genetic analysis in M. polymorpha

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/ Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors

Negative Confidence-Aware Weakly Supervised Binary Classification for Effective Review Helpfulness Classification

The incompleteness of positive labels and the presence of many unlabelled instances are common problems in binary classification applications such as in review helpfulness classification. Various studies from the classification literature consider all unlabelled instances as negative examples. However, a classification model that learns to classify binary instances with incomplete positive labels while assuming all unlabelled data to be negative examples will often generate a biased classifier. In this work, we propose a novel Negative Confidence-aware Weakly Supervised approach (NCWS), which customises a binary classification loss function by discriminating the unlabelled examples with different negative confidences during the classifier's training. NCWS allows to effectively, unbiasedly identify and separate positive and negative instances after its integration into various binary classifiers from the literature, including SVM, CNN and BERT-based classifiers. We use the review helpfulness classification as a test case for examining the effectiveness of our NCWS approach. We thoroughly evaluate NCWS by using three different datasets, namely one from Yelp (venue reviews), and two from Amazon (Kindle and Electronics reviews). Our results show that NCWS outperforms strong baselines from the literature including an existing SVM-based approach (i.e. SVM-P), the positive and unlabelled learning-based approach (i.e. C-PU) and the positive confidence-based approach (i.e. P-conf) in addressing the classifier's bias problem. Moreover, we further examine the effectiveness of NCWS by using its classified helpful reviews in a state-of-the-art review-based venue recommendation model (i.e. DeepCoNN) and demonstrate the benefits of using NCWS in enhancing venue recommendation effectiveness in comparison to the baselines

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle

Suzaku Observations of the Local and Distant Hot ISM

Suzaku observed the molecular cloud MBM12 and a blank field less than 3 degrees away to separate the local and distant components of the diffuse soft X-ray background. Towards MBM12, a local (D< 275 pc) O VII emission line was clearly detected with an intensity of 3.5 ph cm^{-2} s^{-1} sr^{-1}$ (or line units, LU), and the O VIII flux was <0.34 LU. The origin of this O VII emission could be hot gas in the Local Hot Bubble (LHB), charge exchange between oxygen ions in the solar wind (SWCX) and geocoronal or interplanetary material, or a combination of the two. If entirely from the LHB, the implied temperature and emission measure would predict 1/4 keV emission in excess of observations. There is no evidence in the X-ray light curve or solar wind data for a significant contribution from geocoronal SWCX. In any case, the observed O VII flux represents an upper limit to both the LHB emission and interplanetary SWCX in this direction. The off-cloud observation was performed immediately following the on-cloud. The net off-cloud O VII and O VIII intensities were (respectively) 2.34\pm0.33 and 0.77\pm0.16 LU, after subtracting the on-cloud foreground emission. Assuming the LHB and SWCX components did not change, these increases can be attributed to more distant Galactic disk, halo, or extragalactic emission. If the distant O VII and O VIII emission is from a thermal plasma in collisional equilibrium beyond the Galactic disk, a temperature of (2.1\pm0.1)\times10^6 K with an emission measure of (4\pm0.6)\times10^-3} cm^{-6}pc is inferred.Comment: 10 pages, 8 figures, accepted by PAS