[STOPPFrail version 2: a tool for deprescription in medicine, validated in Italian language.] / Strumenti a supporto della deprescrizione in medicina: la versione italiana dello STOPPFrail2.

OBJECTIVE: Deprescribing, i.e. the suspension of drugs whose existing or potential harms outweigh the benefits in the context of care for the individual patient, is an increasingly frequently encountered topic in various congresses today. This issue becomes predominant especially in patients with chronic pathologies with a life expectancy of less than a year, in whom the goal of the treatments passes from healing to caring. Currently there are few validated deprescribing tools, one of these is certainly the STOPPFrail, currently available in its second version. Therefore, we decided to provide for the translation into Italian, to make the description for the elderly patient with limited life expectation more applicable. METHODS: For the translation, we used the method expressed by the European organisation for research and treatment of cancer (Eortc), using forward-backward translation and a Pilot Testing to verify the clarity and comprehensibility of the translation itself. RESULTS: We interviewed 15 experts, of whom 13 responded and completed the evaluation, without bringing to light any unclear sections or sources of misunderstanding. CONCLUSIONS: STOPPFrail2 can be a valid deprescribing tool in the elderly with limited life expectancy; the Italian version can help the physicians in the therapeutic appropriateness in this time of life where it is necessary to focus on the quality of life and on the ethical aspect of the choices, as well as being of help in a "cost-opportunity" logic.

Preparation and Use of Cellular Reagents: A Low-resource Molecular Biology Reagent Platform.

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Constructing Cell-Free Expression Systems for Low-Cost Access.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

Producing molecular biology reagents without purification.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

Homebrew reagents for low cost RT-LAMP.

RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis.

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.

Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum.

The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, particularly in galactolipids such as mono- and di-galactosyldiacylglycerol. LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analyzed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of triacylglycerol. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.

Homebrew reagents for low-cost RT-LAMP.

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Multifunctionalizing the marine diatom Phaeodactylum tricornutum for sustainable co-production of omega-3 long chain polyunsaturated fatty acids and recombinant phytase.

There is an urgent requirement for sustainable sources of food and feed due to world population growth. Aquaculture relies heavily on the fish meal and fish oils derived from capture fisheries, challenging sustainability of the production system. Furthermore, substitution of fish oil with vegetable oil and fish meal with plant seed meals in aquaculture feeds reduces the levels of valuable omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, and lowers the nutritional value due to the presence of phytate. Addition of exogenous phytase to fish feed is beneficial for enhancing animal health and reducing phosphorus pollution. We have engineered the marine diatom Phaeodactylum tricornutum, accumulating high levels of EPA and DHA together with recombinant proteins: the fungal Aspergillus niger PhyA or the bacterial Escherichia coli AppA phytases. The removal of the N-terminal signal peptide further increased phytase activity. Strains engineered with fcpA and CIP1 promoters showed the highest level of phytase activity. The best engineered strain achieved up to 40,000 phytase activity units (FTU) per gram of soluble protein, thus demonstrating the feasibility of development of multifunctionalized microalgae to simultaneously produce industrially useful proteins and fatty acids to meet the demand of intensive fish farming activity.

Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803.

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L-lactic acid.

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.

The transporter SynPAM71 is located in the plasma membrane and thylakoids, and mediates manganese tolerance in Synechocystis PCC6803.

Manganese (Mn) is an essential constituent of photosystem II (PSII) and therefore indispensable for oxygenic photosynthesis. Very little is known about how Mn is transported, delivered and retained in photosynthetic cells. Recently, the thylakoid-localized transporter PAM71 has been linked to chloroplast Mn homeostasis in Arabidopsis thaliana. Here, we characterize the function of its homolog in Synechocystis (SynPAM71). We used a loss-of-function line (ΔSynPAM71), wild-type (WT) cells exposed to Mn stress and strains expressing a tagged variant of SynPAM71 to characterize the role of SynPAM71 in cyanobacterial Mn homeostasis. The ΔSynPAM71 strain displays an Mn-sensitive phenotype with reduced levels of chlorophyll and PSI accumulation, defects in PSII photochemistry and intracellular Mn enrichment, particularly in the thylakoid membranes. These effects are attributable to Mn toxicity, as very similar symptoms were observed in WT cells exposed to excess Mn. Moreover, CyanoP, which is involved in the early steps of PSII assembly, is massively upregulated in ΔSynPAM71. SynPAM71 was detected in both the plasma membrane and, to a lesser extent, the thylakoid membranes. Our results suggest that SynPAM71 is involved in the maintenance of Mn homeostasis through the export of Mn from the cytoplasm into the periplasmic and luminal compartments, where it can be stored without interfering with cytoplasmic metabolic processes.

Awareness and consideration of malnutrition among oncologists: Insights from an exploratory survey.

OBJECTIVES: The attitude toward malnutrition varies considerably among oncologists and many malnourished cancer patients receive inadequate nutritional support. The aim of this brief report was to report the results of the exploratory national survey conducted by the Italian Society of Medical Oncology (AIOM) and the Italian Society of Artificial Nutrition and Metabolism (SINPE) before publication of a consensus document aimed at evaluating current attitudes toward malnutrition and management of nutrition, among Italian medical oncologists. METHODS: Between January and July 2015, the AIOM and the SINPE conducted a national web-based exploratory survey to investigate the attitude of oncologists toward malnutrition, and the management of nutritional support, before publication of an intersociety consensus document. RESULTS: Of the 2375 AIOM members, 135 (5.7%) participated in the survey, with a satisfactory distribution across all Italian regions. Nutritional assessment and support were routinely integrated into patient care for 38 (28%) responders. According to 66 (49%) participants, nutritional assessment was carried out only at the patients' request (n = 62), or not at all (n = 4). Availability of clinical nutritionists was reported by 88 (65%) participants. For 131 responders (97%), nutritional status was decisive (n = 63) or often crucial (n = 68) in assessing whether anticancer treatment was practicable or would be tolerated. CONCLUSIONS: The low response rate may reflect the lack of awareness and consideration of nutritional issues among Italian oncologists. Although malnutrition and nutritional support seemed to be perceived by the responders as relevant factors for the efficacy of oncologic treatments, it seems that nutritional care practices may well be inappropriate. The lack of collaboration between oncologists and clinical nutritionists may be the first obstacle to overcome. Educational intersociety initiatives aimed at improving nutritional support management for cancer patients in Italy appear urgently needed.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

The Arabidopsis Protein CGLD11 Is Required for Chloroplast ATP Synthase Accumulation.

ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the ß subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.

The Arabidopsis protein CONSERVED ONLY IN THE GREEN LINEAGE160 promotes the assembly of the membranous part of the chloroplast ATP synthase.

The chloroplast F1Fo-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b', and c) and a peripheral domain (subunits α, ß, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism.

Weekly non-pegylated liposomal doxorubicin chemotherapy in heavily pre-treated patients with metastatic breast cancer.

BACKGROUND: Due to its low cardiac toxicity, non-pegylated liposomal doxorubicin (NPLD) may represent an attractive therapeutic option as salvage therapy for patients with metastatic breast cancer who have already received anthracycline-based chemotherapy. PATIENTS AND METHODS: We retrospectively reviewed 47 consecutive patients with metastatic breast cancer treated with NPLD at our Institution between 2008 and 2012. Patients received weekly NPLD at a dose of 20 mg/m(2) i.v. until disease progression or unacceptable toxicity. RESULTS: Nine patients (19.1%) achieved a partial response and 11 (23.4%) had stable disease, with a disease control rate of 42.6%; 27 patients (57.4%) had progressive disease. The median progression-free survival and overall survival were 2.7 and 11.5 months, respectively. Grade 3 and 4 adverse events did not occur. No cardiac events were observed. CONCLUSION: Weekly NPLD represents a safe and effective therapy and may be considered a new therapeutic option for heavily pre-treated patients with metastatic breast cancer.