A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Mass Mortality of Asari Clams (<i>Ruditapes philippinarum</i>) Triggered by Wind-Induced Upwelling of Hypoxic Water Masses

To investigate the mass mortality of the macrobenthos community, primarily asari clams, triggered by upwelling-driven hypoxia, we conducted continuous observations of temperature, salinity, and DO, and monthly macrobenthos monitoring on the Rokujo tidal flat in Mikawa Bay, central Japan, from 2014 to 2016. Additionally, laboratory experiments were conducted using sediments on a tidal flat containing macrobenthos to examine the possibility of hydrogen sulfide formation in tidal flats. The bottom layer at the offshore station was intermittently hypoxic, and the station of the tidal flat was occasionally hypoxic in August and September for three years. Hypoxia was mostly observed on the tidal flat when constant easterly winds were recorded offshore. The biomass of asari clams decreased considerably from September to October 2016 when hypoxia was intermittent. Hypoxia persisted for approximately one week from 20 September, which was associated with the calm weather and stagnation of tidal currents owing to the neap tide. Conversely, the hydrogen sulfide concentration in the water directly above the sediment exceeded 30 mg L−1 after 3 days of incubation in the laboratory experiment. Therefore, the possibility of oxygen consumption on tidal flats due to hydrogen sulfide formed by biological die-offs was considered in the long-term persistence of hypoxia

Adoption of inpatient family‐based treatment for anorexia nervosa: A case report

Abstract Background Family‐based treatment (FBT) is effective for the treatment of anorexia nervosa (AN) in children and adolescents. However, its availability in Japan is limited because it requires adherence to specific guidelines, commitment of sufficient time for frequent outpatient treatment, as well as the entire family's participation. We present a case of a patient with AN who was treated with modified FBT during hospitalization. Case Presentation Our patient was a 14‐year‐old girl with AN. She was hospitalized for malnutrition and dehydration, and was introduced to FBT during this period. After discharge, she continued FBT on an outpatient basis and was in remission 1 year later. Conclusion This case shows that initiation of FBT during hospitalization may be useful in patients with physically severe AN. Flexible adaptation to each of the diverse healthcare systems and cultural differences may be necessary for the widespread use of FBT

Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs

The allospecifc T cell population responding to a transplanted organ consists of both naïve and memory lymphocytes. Although it is established that naive T cells are activated by antigen within the organized structures of secondary lymphoid organs (the spleen, lymph nodes, and mucosal lympoid tissues), it is not clear whether memory T cell activation and propagation depend on homing to these organs. To answer this question, we investigated whether allospecific naïve or memory T cells can mediate acute cardiac allograft rejection in mutant mice that lack all of their secondary lymphoid tissues. The results of our experiments demonstrated that antigen-experienced memory T cells have two advantages over naïve T cells: (i) memory T cells mount a vigorous immune response that leads to allograft rejection independent of secondary lymphoid organs; and (ii) memory T cells generate more memory T cells without homing to secondary lymphoid organs. These unique properties of memory T cells were further confirmed by showing that memory-like T cells that arise from the homeostatic proliferation of naive T cells in the absence of antigenic stimulation are suboptimal at rejecting allografts and do not generate memory T cells in mice devoid of secondary lymphoid tissues