16th GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.

Integrating climate change into nursing curricula and continuing education: a scoping review protocol.

INTRODUCTION: Climate change constitutes a major threat to human health. Nurses have an essential role to play in protecting populations from this threat, and to fulfil this role, they must be properly prepared. The purpose of this scoping review is to examine studies on the integration of climate change into the academic curriculum or continuing education of nurses so as to identify issues and opportunities related to this integration. METHODS AND ANALYSIS: The method being used is the methodological framework proposed by Arksey and O'Malley and Levac et al. First, a search strategy using keywords and their combinations will be developed. This strategy will be applied in four bibliographic databases: MEDLINE (PubMed), CINAHL, Embase, Web of Science. Second, an initial selection of studies based on titles and abstracts will be carried out by two members of the research team using the software Covidence. They will conduct this selection process independently, with the aim of identifying relevant studies that meet the inclusion criteria for our scoping review. Third, the second stage in the selection process will be carried out by examining the full text of each article to determine which studies to include in the review. Finally, data on year of publication, authors, geographical area, article type, study objectives, methodology and key findings will be extracted from selected articles for analysis. A search of the grey literature will also be conducted to supplement the results of the bibliographic database search. The scoping review is currently ongoing. Identification of relevant literature began in the first quarter of 2022 and is expected to be completed in the first quarter of 2023. ETHICS AND DISSEMINATION: Ethical approval is not required for this review. The results of this study will be presented in workshops and conferences and be submitted for publication to a peer-reviewed journal.

Nurses' Perceptions of Climate Change: Protocol for a Scoping Review.

BACKGROUND: Climate change is a major threat to human health. Nurses are in contact with patients suffering from the effects of climate change in their daily work. Therefore, they need to be involved in combating it at both the individual and collective levels. However, there is still very little known about nurses' perception of climate change and their role toward it. A few recent studies have embarked on the process of examining the perceptions of these health professionals relative to climate change, but no exploratory review of the literature has been conducted on nurses' perception of this phenomenon. OBJECTIVE: The purpose of this protocol is to develop a research strategy for an exploratory review of the literature focused on identifying nurses' perceptions of climate change. METHODS: Firstly, with the help of a specialized librarian, we defined keywords and their combinations, using an iterative process, to develop a documentary search strategy. This strategy was tested in the following four bibliographic databases: MEDLINE (PubMed), CINAHL, Embase, and Web of Science. A search of the grey literature will also be conducted to supplement the results of the bibliographic database search. The next step will be for 2 members of the research team to carry out a 2-stage selection process using the web-based systematic review software Covidence. They will carry out this selection process independently, with the aim of identifying relevant studies that meet the inclusion criteria for our exploratory review. Finally, data on year of publication, authors, geographic area, article type, study objectives, methodology, and key findings will be extracted from selected articles for analysis. The data will be analyzed by the research team based on an in-depth examination of the findings and will be directed toward answering the research question and fulfilling the study's objective. RESULTS: The results will help in defining nurses' perceptions of climate change more clearly as well as the role they can play and what they need to be able to bring forward solutions to this phenomenon. The findings should also serve to guide the health sector and nursing faculty's interventions aimed at preparing health professionals to act on the potential threats associated with climate change. CONCLUSIONS: The preliminary search suggests a possible gap between the importance of the nursing role in addressing the health impacts of climate change and the nurses' lack of knowledge and awareness on this matter. The results will allow for raising nurses' awareness of their role in the fight against climate change and the ways to address its health effects. This study will also open up new research perspectives on how to equip nurses to better integrate response to climate change issues into their professional practice. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/42516.

Kinetics and Dynamics of the Stiff and Flexible Tines with the Duckfoot and the Coulter after Impact with Stones Embedded in Compacted Soil. Part II.

The kinetics and dynamics of the stiff and flexible tines with the duckfoot and the coulter after impact with stones embedded in compacted soil were examined. The beak of the duckfoot was positioned in the axis of the row of stones embedded in the soil at the depth of stones thickness. The coulter covered the stone or impact the edge of the stone halfway along its length. The tools worked at a speed of 0.83-2.22 m·s-1 and a working depth of 0.05-0.10 m. The results of specific parameters were compared to the response of the tools to loads in soil without stones. For both soil conditions, the kinetics of the flexible tine was 24 times more reactive, and the dynamic loads were two times lower than for the stiff tine. The responses of both tines were suppressed along with the working depth because of the more favorable place of impact of the duckfoot beak with the stone. Along with the working speed, for a stiff tine, the specific accelerations decreased significantly, by ten times, and the specific forces increased slightly, by 1.6 times. Among the two systems of setting the coulter, the impact of the cutting edge of the coulter with the stone in the middle of its length was more unfavorable than the work of the coulter covering the stone.

Effect of Stone Impacts on Various Ground Engaging Tools (Flexible/Stiff Tines and Coulter): Part I.

Analysis of the state of knowledge showed a gap in the description of tool-stone feedback. Therefore, the aim of this study was to investigate tool-stone interactions. Spherical-like silicate stones were hit by stiff and flexible tines with a duckfoot or a coulter. The tools worked with various parameters in the depth range of 0.05-0.10 m and a speed of 0.83-2.22 m·s-1. The characteristics of stone movement were specific to the type of tool and were described by the Numerical Stone Movement Scale developed for the purpose of the research. After the impact with the stiff tine, the stones were thrown the greatest distance of 0.26-1.08 m, and these distances were strongly dependent on the working speed and slightly dependent on the working depth. Large vibrations of the flexible tine and the location of the contact point of the tine in relation to the centre of the stone thickness contributed to the random behaviour of stones that were slightly moved, rotated or displaced. The specific work required to remove the stone reflected the distance travelled by the stone as well as the specific force which largely contributed to increasing the differences in this work between both tines.

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

Immunogenicity Assessment of Lipegfilgrastim in Patients with Breast Cancer Receiving Chemotherapy.

Lipegfilgrastim is a long-acting, once-per-cycle, glycopegylated recombinant granulocyte colony-stimulating factor (G-CSF) used to prevent neutropenia in patients receiving myelosuppressive chemotherapy. This integrated analysis examined the immunogenicity of lipegfilgrastim and its potential clinical impact in two double-blind randomized studies (phases II and III) of patients with breast cancer receiving chemotherapy. Serum samples were analyzed using sequential assays for screening, confirmation, antibody titer, and characterization of antidrug antibodies (ADA). Neutropenia-related efficacy measures were reviewed for each ADA-positive patient. Among 255 patients receiving lipegfilgrastim (154 in phase II, 101 in phase III) and 155 patients receiving pegfilgrastim (54 in phase II, 101 in phase III), the incidence of treatment-emergent ADA was low and similar between the lipegfilgrastim (phase II: 1.3%; phase III: 1.0%) and pegfilgrastim (phase II: 1.9%; phase III: 1.0%) arms. None of the treatment-emergent ADA-positive samples exhibited neutralizing activity against lipegfilgrastim, pegfilgrastim, or glycosylated G-CSF in a cell-based neutralizing antibody assay. No changes were observed in neutropenia-related efficacy measures among ADA-positive patients, and no treatment-related hypersensitivity or anaphylaxis occurred. These results indicate that there is no apparent impact of ADA on lipegfilgrastim efficacy and safety.

Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans.

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.

Characterization of a CC49-based single-chain fragment-beta-lactamase fusion protein for antibody-directed enzyme prodrug therapy (ADEPT).

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

Fusion of two distinct peptide exosite inhibitors of Factor VIIa.

Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465-470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513-9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522-9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG-Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC(50) for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of (125)I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.