Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling

Nucleolar integrity during interphase supports faithful Cdk1 activation and mitotic entry

The nucleolus is a dynamic nuclear body that has been demonstrated to disassemble at the onset of mitosis; the relationship between cell cycle progression and nucleolar integrity, however, remains poorly understood. We studied the role of nucleolar proteins in mitosis by performing a global analysis using small interfering RNAs specific to nucleolar proteins; we focused on nucleolar protein 11 (NOL11), with currently unknown mitotic functions. Depletion of NOL11 delayed entry into the mitotic phase owing to increased inhibitory phosphorylation of cyclin-dependent kinase 1 (Cdk1) and aberrant accumulation of Wee1, a kinase that phosphorylates and inhibits Cdk1. In addition to effects on overall mitotic phenotypes, NOL11 depletion reduced ribosomal RNA (rRNA) levels and caused nucleolar disruption during interphase. Notably, mitotic phenotypes found in NOL11-depleted cells were recapitulated when nucleolar disruption was induced by depletion of rRNA transcription factors or treatment with actinomycin D. Furthermore, delayed entry into the mitotic phase, caused by the depletion of pre-rRNA transcription factors, was attributable to nucleolar disruption rather than to G2/M checkpoint activation or reduced protein synthesis. Our findings therefore suggest that maintenance of nucleolar integrity during interphase is essential for proper cell cycle progression to mitosis via the regulation of Wee1 and Cdk1

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins

Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum–protein interactions in the design and application of antibody-based drugs with increased efficacy and safety

Irrigant Agitation Techniques versus Passive Ultrasonic Irrigation for Removing Debris from Curved Root Canals: An Environmental Scanning Electron Microscopic Study

Introduction: The aim of this study was to compare the efficacy of passive ultrasonic irrigation (PUI) versus mechanical agitation of the irrigant promoted by the EasyClean (EC) and XP-Endo Finisher (XP-F) systems in removing debris from root canal walls, using environmental scanning electron microscopy (ESEM). Methods and Materials: Twelve curved mesiobuccal canals of mandibular molars were prepared with the ProTaper Next system up to file X2 (25/0.06). The specimens were embedded in flasks containing silicone putty, sectioned lengthwise, and a 4-mm long groove was made on the canal wall of the buccal half of the specimen, extending from 2 mm up to 6 mm short of the apex. Five cross-sectional markings were made along this groove to establish standardized locations for imaging. The same specimens were used to prepare a negative control group (without debris), a positive control group (completely covered by debris), and 3 experimental groups according to the final irrigation protocol employed: PUI, EC or XP-F. ESEM images were obtained and evaluated by 3 examiners. The amount of debris observed on the images was classified according to a 4-category scoring system. The kappa test was used to assess inter-examiner agreement, and the Kruskal-Wallis and Dunn tests were used to compare the scores (P<0.05). Results: The scores attributed to the PUI, EC, and XP-F groups were statistically similar to those attributed to the negative control group (P>0.05). Conclusion: Based on this in-vitro study, the mechanical agitation of the irrigant promoted by EC and XP-F was as effective as using PUI to remove debris from the root canal walls

Removal of Obturation Material from Root Canals Using a Combination of Reciprocal Instrumentation and Different Final Irrigation Techniques

Introduction: The predictability of successful non-surgical endodontic retreatment is directly related to it’s ability to completely cleanse and remove obturation material from the canal system. The purpose of this study was to evaluate the removal of gutta-percha from curved canals using three final irrigation methods: passive ultrasonic irrigation (PUI) with a 20/01 E1 insert (Irrisonic); XP-endo Finisher (XPF); and Easy Clean (EC). Methods and Materials: Forty mesial roots of mandibular molars with an angle of curvature between 10° and 20°, two canals, and independent foramina were cut into 16-mm sections. The canals were instrumented using the Reciproc system (R25) and filled with a #25 gutta-percha cone and AH-Plus sealer by the continuous-wave condensation technique. The roots were double-sealed with Coltosol and photopolymerizable resin and stored at 37°C and 100% humidity for 30 days. They were then randomized into 4 groups (n=10): control (C), PUI, XPF, and EC. All specimens were scanned using cone-beam computed tomography (CBCT), and fill volume data (in square pixels) were calculated before retreatment, after retreatment, and after final irrigation. The images were analyzed using Tps Dig software 2.32 by two blinded, calibrated examiners (intra-class correlation coefficient=0.9967). The results were analyzed in BioEstat 4.0. The nonparametric Kruskal-Wallis test with Dunn's post-hoc and Friedman comparison were applied. Significance was accepted at 5% (P<0.05). Results: None of the final irrigation protocols completely removed remnants of obturation material from the root canal systems (P>0.05). On comparative analysis with specimens divided into thirds, all methods were found to remove material equally, with no significant differences (P>0.05). Conclusion: Based on this in vitro study, the additional cleaning methods tested were equivalent to each other and did not lead to an improvement in the removal of residual obturation material

Factors Associated with Post-Endodontic Treatment Pain Performed by Students in an Endodontic Graduate Program

Introduction: The aim of this study was to evaluate the possible associations between pre-established clinical variables and manifestation of postoperative pain after endodontic treatments performed by graduate students in endodontics, from June 2016 to December 2017. Methods and Materials: A total of 998 dental patient charts were included in the study. All the patients underwent the same clinical protocol. Possible associations between postoperative pain and clinical variables were investigated, including age, gender, type of tooth, type of treatment, pulpal diagnosis, periradicular diagnosis, instrumentation system used, number of sessions, previous symptom, procedural accident, and endodontic sealer extrusion. Patients were contacted by telephone 24 h and 7 days after treatment completion and were asked about the degree of postoperative pain they had experienced, using a four-level scoring system: 0, no pain; 1, mild pain (no medication was needed); 2, moderate pain (an analgesic or anti-inflammatory was needed); 3, severe pain. Fischer’s exact test, Pearson’s test, and logistic regression were used for the statistical analysis of the data. A significance level of 0.05 was used. Results: A total of 8.6% of the patients reported having experienced postoperative pain, 50% of which reported mild pain, 47.7%, moderate pain, and 2.3%, severe pain. The only variable significantly associated with postoperative endodontic pain was pre-endodontic treatment symptoms (Pearson’s test, P=0.0047). The logistic regression analysis indicated that the association between use of the Reciproc system and sealer extrusion posed a significant risk for postoperative endodontic pain. Conclusion: Based on this retrospective cohort study, the incidence of moderate and severe pain after endodontic treatment was low, and the only variable associated with a higher frequency of patients reporting postoperative endodontic pain was previous pain/symptoms. Therefore, in these cases, pain management methods such as the use of analgesics before treatment or immediately after treatment should be considered

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering

Inhibitory effects of local anesthetics on the proteasome and their biological actions

Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells