Parametric Assessment to Evaluate and Compare the Carbon Footprint of Diverse Manufacturing Processes for Building Complex Surfaces

At present, building design is faced with a need to properly manage complex geometries and surfaces. This fact is not only driven by the increased demand for visually stunning spaces but also stems from the rise of new design paradigms, such as “user-centred design”, that include bespoke optimization approaches. Nevertheless, the escalating adoption of customized components and one-off solutions raises valid concerns regarding the optimal use of energy and resources in this production paradigm. This study focuses on the Life Cycle Assessment of a novel Cement–Textile Composite (CTC) patented material. It combines a synthetic reinforcing textile with a customized concrete matrix, to generate rigid elements that are able to statically preserve complex spatial arrangements, particularly double-curvature surfaces. Moreover, the CTC offers a low-volume cost-effective alternative for custom-made cladding applications. The study performed a comparative carbon footprint assessment of the CTC production process in contrast to other technologies, such as CNC milling and 3D printing. To facilitate meaningful comparisons among diverse construction alternatives and to derive generalized data capable of characterizing their overall capacity, independent of specific production configurations, the present study implemented a generalized parametric shape of reference defined as a bounding box (BBOX), which encloses the volume of the target shape. Comparing different production technologies of the same shape with the same BBOX results in a significant carbon saving, up to 9/10th of the carbon footprint, when the CTC technology is adopted. The study therefore highlights the potential environmental advantages of CTC in the fields of architectural design and building engineering

A novel approach for the purification and proteomic analysis of pathogenic immunglobulin free light chains from serum

An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms

Minimal residual disease negativity by next-generation flow cytometry is associated with improved organ response in AL amyloidosis

© The Author(s) 2021.Light chain (AL) amyloidosis is caused by a small B-cell clone producing light chains that form amyloid deposits and cause organ dysfunction. Chemotherapy aims at suppressing the production of the toxic light chain (LC) and restore organ function. However, even complete hematologic response (CR), defined as negative serum and urine immunofixation and normalized free LC ratio, does not always translate into organ response. Next-generation flow (NGF) cytometry is used to detect minimal residual disease (MRD) in multiple myeloma. We evaluated MRD by NGF in 92 AL amyloidosis patients in CR. Fifty-four percent had persistent MRD (median 0.03% abnormal plasma cells). There were no differences in baseline clinical variables in patients with or without detectable MRD. Undetectable MRD was associated with higher rates of renal (90% vs 62%, p = 0.006) and cardiac response (95% vs 75%, p = 0.023). Hematologic progression was more frequent in MRD positive (0 vs 25% at 1 year, p = 0.001). Altogether, NGF can detect MRD in approximately half the AL amyloidosis patients in CR, and persistent MRD can explain persistent organ dysfunction. Thus, this study supports testing MRD in CR patients, especially if not accompanied by organ response. In case MRD persists, further treatment could be considered, carefully balancing residual organ damage, patient frailty, and possible toxicity.This study was supported by a grant from CARIPLO “Molecular mechanisms of Ig toxicity in age-related plasma cell dyscrasias no. 2015-0591”, by a grant from the Black Swan Research Initiative from the International Myeloma Foundation “Automated multidimensional flow cytometry for high-sensitive screening and to monitor response in AL amyloidosis”, by a grant from CARIPLO “Structure–function relation of amyloid: understanding the molecular bases of protein misfolding diseases to design new treatments no. 2013-0964”, by a grant from the Amyloidosis Foundation “Investigating new therapies to treat AL amyloidosis”, and by a grant from Cancer Research UK, FCAECC and AIRC under the Accelerator Award 2017 Program “Early detection and intervention: understanding the mechanisms of transformation and hidden resistance of incurable hematological malignancies”, by a grant from CARIPLO “Harnessing the plasma cell secretory capacity against systemic light chain amyloidosis” (no. 2018-0257), by a grant from the Italian Ministry of Health “Towards effective, patient-tailored anti-plasma cell therapies in AL amyloidosis: predicting drug response and overcoming drug resistance” (GR-2018-12368387). This study has also supported the Centro de Investigación Biomédica en Red—Área de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369, CB16/12/00400, and CB16/12/00489) and the Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS No. PI13/02196). G.P. is supported in part by the Bart Barlogie Young Investigator Award from the International Myeloma Society (IMS). P.M. is supported in part by a fellowship grant form Collegio Ghislieri (Pavia). We acknowledge the study coordinator and data manager Anna Carnevale Baraglia

A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders

SDS–PAGE analysis of the purified rLC.

<p>Coomassie Brilliant Blue staining (A) and western blotting (B) analysis of purified rLC compared to AL-2 Bence Jones protein (AL-2 BJ), purified from urine. Protein samples (5 µg loaded) were resolved on 12% SDS-PAGE run under non reducing and reducing conditions. Western blot analysis was performed using a rabbit anti-human λ LC antiserum as primary antibody (Dako Cytomation, Glostrup, Denmark), revealed by a goat anti-rabbit conjugated with horseradish peroxidase (Dako Cytomation), probed with diaminobenzidine (DAB) substrate. Molecular mass markers are shown in the left lane. The proteins presented the typical dimeric-monomeric species of free LC. Different degrees of protein denaturation are responsible of the minor differences in electrophoretic migration of monoclonal free LC (A), a common finding in SDS PAGE running under denaturing conditions of these proteins.</p

Schematic representation of the expression strategy.

<p>Each patient’s full-length monoclonal LC (V_L +C_L) obtained by RT-PCR was re-amplified using primers designed according to the pASK-IBA cloning strategy. Start Met codon in 5′ primer and the two stop codons in 3′ primer were introduced and shown in bold, <i>Bsm</i>BI recognition sites are indicated in lower case in both primers. After <i>Bsm</i>BI digestion, PCR product was ligated into <i>Bsa</i>I/<i>Bsa</i>I sites of pASK-IBA33plus vector.</p