In Pursuit of Closed‐Loop Supply Chains for Critical Materials: An Exploratory Study in the Green Energy Sector

A closed‐loop supply chain (CLSC) is considered not only an important solution for ensuring sustainable exploitation of materials, but also a promising strategy for securing long‐term availability of materials. The latter is especially highlighted in the materials criticality discourse. Critical raw materials (CRMs), being exposed to supply disruptions, create an uncertain operational environment for many industries, particularly for green energy technologies that employ multiple CRMs. However, recycling rates of CRMs are very low and engagement of companies in CLSC for CRM is limited. This study examines factors influencing CLSC for CRM development in photovoltaic panels and wind turbine technologies. The aim is to analyze how the factors manifest themselves in different companies along the supply chain and to identify enabling and bottleneck conditions for implementation of CLSC for CRM. The novelty of the study is twofold: the focus on material rather than product flows, and examination of factors from a multiactor perspective. The evidence obtained suggests that the manufacturing companies and reverse supply‐chain operators engaged in the study take different perspectives (product vs. material) regarding development of CLSC for CRM and thus emphasize different factors. The findings underline the need for interactions between supply‐chain actors, a sound competitive environment for recycling processes, and investment in technologies and infrastructure development if CLSC for CRM is to be developed. The paper provides implications for practitioners and policy makers for implementation of CLSC for CRM, and suggests prospects for further research.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Drug metabolism by cultured human hepatocytes: how far are we from the in vivo reality?

The investigation of metabolism is an important milestone in the course of drug development. Drug metabolism is a determinant of drug pharmacokinetics variability in human beings. Fundamental to this are phenotypic differences, as well as genotypic differences, in the expression of the enzymes involved in drug metabolism. Genotypic variability is easy to identify by means of polymerase chain reaction-based or DNA chip-based methods, whereas phenotypic variability requires direct measurement of enzyme activities in liver, or, indirectly, measurement of the rate of metabolism of a given compound in vivo. There is a great deal of phenotypic variability in human beings, only a minor part being attributable to gene polymorphisms. Thus, enzyme activity measurements in a series of human livers, as well as in vivo studies with human volunteers, show that phenotypic variability is, by far, much greater than genotypic variability. In vitro models are currently used to investigate the hepatic metabolism of new compounds. Cultured human hepatocytes are considered to be the closest model to the human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cells in the liver raises the question of to what extent drug metabolism variability observed in vitro actually reflects that in the liver in vivo. This issue has been examined by investigating the metabolism of the model compound, aceclofenac (an approved analgesic/anti-inflammatory drug), both in vitro and in vivo. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. The patients were given the drug during the course of clinical recovery, and the metabolites, largely present in urine, were analysed. In vitro and in vivo data from the same individual were compared. There was a good correlation between the in vitro and in vivo relative abundance of oxidised metabolites (4'-OH-aceclofenac + 4'-OH-diclofenac; Spearman's rho = 0.855), and the hydrolysis of aceclofenac (diclofenac + 4'-OH-aceclofenac + 4'-OH-diclofenac; rho = 0.691), while the conjugation of the drug in vitro was somewhat lower than in vivo. Globally, the metabolism of aceclofenac in vitro correlated with the amount of metabolites excreted in urine after 16 hours (rho = 0.95). Overall, although differing among assays, the in vitro/in vivo metabolism data for each patient were surprisingly similar. Thus, the variability observed in vitro appears to reflect genuine phenotypic variability among the donors.     

Altered adhesion features and signal transduction in NRK-49F cells transformed by down-regulation of lysyl oxidase

Lysyl oxidase (LOX) down-regulation induced an oncogenic phenotype in NRK-49F. This event was accompanied by a constitutive activation of ras oncogene and down-regulation of PDGF beta receptor, among other important phenotypic and molecular modifications. In the present paper we show that ras activation is not accompanied by a constitutive activation of the MAP kinases as expected. Surprisingly, even if MAPK-independent, ras activation was accompanied by a constitutive Ser(63) and Ser(73) phosphorylation of c-jun, a further downstream target of ras. Although rare, this ras alternative pathway has been described. Since ras alone is seldom able to trigger cell transformation and the transformed phenotype showed clearly an abnormal adhesion pattern, we investigated the main molecules involved in cell-cell adhesion. In fact, we found that beta-catenin was up-regulated, escaping the glycogen synthase kinase-3 beta (GSK-3 beta) control, through unclear mechanisms. Its nuclear accumulation was accompanied by an up-regulation of cyclin D1, as classically described in the activation of the Wnt/beta-catenin signal pathway. We believe that the resulting up-regulation of cyclin D1 acted in synergy with ras to induce the cell transformation.     

Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001