An engineered enzyme embedded into PLA to make self-biodegradable plastic.

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6-8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an 'enzymated' polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20-24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

Lessons from a transplantation of zebra mussels into a small urban river: An integrated ecotoxicological assessment.

It is often difficult to evaluate the level of contamination in small urban rivers because pollution is mainly diffuse, with low levels of numerous substances. The use of a coupled approach using both chemical and biological measurements may provide an integrated evaluation of the impact of micro-pollution on the river. Zebra mussels were transplanted along a metal and organic pollution gradient in spring 2008. For two months, mussels and water samples were collected from two sites every two weeks and analyzed for metal and PAH content as well as water physicochemical parameters. Diffusive gradients in thin film (DGT) were also used to assess levels of labile metals. Exposure of mussels to contaminants and potential impact were evaluated using physiological indices and various biomarkers including condition index (CI), defense mechanisms (glutathione-S-transferase: GST), digestive enzymes (amylase and cellulase) and genotoxicity (micronucleus test: MN and comet assay: CA). For most contaminants, the water contamination was significantly higher downstream. Bioaccumulation in zebra mussels was related to water contamination in the framework of the biodynamic model, which allowed us to take into account the biological dilution that was caused by the growth of soft tissue downstream. Thus, metal influxes were on average two times higher downstream than upstream in particular for Zn, Cr, Cu and Cd. Significant differences in condition index were observed (final CI was 0.42 ± 0.03 downstream and 0.31 ± 0.03 upstream) reflecting a better food availability downstream. Moreover a significant decrease of GST activity and digestive enzymes activity in the cristalline style was observed downstream. Interpreting this decrease requires considering not only micro-pollution but also the trophic status related to the water's physicochemistry. The MN test and the CA on gill cells highlighted genotoxicity in mussels transplanted downstream compared to upstream.

Measurement of dynamic mobilization of trace metals in sediments using DGT and comparison with bioaccumulation in Chironomus riparius: first results of an experimental study.

Sediments in aquatic ecosystems are often contaminated as a result of anthropogenic activities. Sediments and benthic organisms have been used to monitor trace metals contamination. However, due to the high variability of contaminant bioavailability, the attempt to link metal concentration in sediments and contamination of the organisms or ecotoxicological effect often lead to disappointing results. The technique of diffusive gradients in thin films (DGT) has been proposed as a relevant tool to study metal bioavailability, for example for accumulation in plants. In the present study, laboratory microcosm experiments were conducted with six contaminated sediments to compare metal accumulation in DGT and bioaccumulation in a chironomid (Chironomus riparius) for Cu, Cd and Pb . Metal accumulation in DGT was measured over time then modelled to determine two parameters of the dynamic response of the metals to DGT deployment: the size of the particulate labile pool and the kinetic of the solid-dissolved phase exchange. The mobility of metals was found metal and sediment dependent. A significant relationship between metal accumulated in DGT and bioaccumulated in chironomids was found for Cu and Pb. However, total metals in sediments were the best predictors of bioaccumulation. Nevertheless, the knowledge of the metals dynamic enhanced our ability to explain the different biological uptake observed in sediments of similar total metal concentrations.

Mouse fetal trisomy 13 and hypotrophy of the spinal cord: effect on calbindin-D28k and calretinin expressed by neurons of the spinal cord and dorsal root ganglia.

Trisomy 13 was detected in 10% of mouse embryos obtained from pregnant females which were doubly heterozygous for Robertsonian chromosomes involving chromosome 13. The developing dorsal root ganglia and spinal cords were examined in trisomy 13 and littermate control mice between days 12 and 18 of gestation (E12-18). The overall size of the dorsal root ganglia and number of ganglion cells within a given ganglion were not altered, but the number of neurons immunoreactive for calbindin and calretinin was reduced. The trisomic spinal cord was reduced in size with neurons lying in a tightly compact distribution in the gray matter. In trisomic fetuses, the extent of the neuropil of the spinal cord was reduced, and may represent a diminished field of interneuronal connectivity, due to reduced arborization of dendritic processes of the neurons present, particularly of calbindin-immunostained neurons. Furthermore, the subpopulation of calretinin-immunoreactive neurons and axons was also reduced in developing trisomic gray and white matter, respectively. Thus, overexpression of genes on mouse chromosome 13 exerts a deleterious effect on the development of neuropil, affecting both dendritic and axonal arborization in the trisomy 13 mouse. The defect of calbindin or calretinin expression by subsets of dorsal root ganglion or spinal cord neurons may result from deficient cell-to-cell interactions with targets which are hypoplastic.

Antithrombotic therapy is associated with better survival in patients with severe heart failure and left ventricular systolic dysfunction (EPICAL study).

BACKGROUND: In patients with congestive heart failure (CHF), clinical trials have demonstrated the benefit of a number of drugs on morbidity and mortality. Nevertheless so far, there is no published controlled study of long-term antithrombotic therapy in patients with CHF. The aim of this work was to identify the relationship between cardiovascular drug use, especially antithrombotic therapy, and survival of CHF patients in current clinical practice, using an observational, population-based database. METHODS: The EPICAL study (Epidémiologie de l'Insuffisance Cardiaque Avancée en Lorraine) has identified prospectively all patients with severe CHF in the community of Lorraine. Inclusion criteria were age 20-80 years in 1994, at least one hospitalisation for cardiac decompensation, NYHA III/IV HF, ventricular ejection fraction < or =30% or cardiothoracic index > or =60% and arterial hypotension or peripheral and/or pulmonary oedema. A total of 417 consecutive patients surviving at hospital discharge were included in the database. The average follow-up period was 5 years. Univariate Cox models were used to test the relationship of baseline biological and clinical factors to survival. Cardiovascular drug prescriptions were tested in a multivariate Cox model adjusted by other known predictive factors. RESULTS: Duration of disease >1 year, renal failure, serum sodium > or =138 mmol/l, old age, serious comorbidity, previous decompensation, high doses of furosemide and vasodilators use were independently associated with poor prognosis at 1 and 5 years. Oral anticoagulants, aspirin, lipid lowering drugs and beta-blockers use were associated with better survival. There was no interaction between aspirin and angiotensin converting enzyme inhibitor use on survival. CONCLUSION: Antithrombotic therapy was associated with a better long-term survival in our study population of severe CHF. These results together with other previously published circumstantial evidence urge for a prospective, controlled and randomised trial specifically designed to evaluate optimal oral anticoagulants and aspirin in patients with congestive heart failure.

Pathogenesis of various forms of double outlet right ventricle in mouse fetal trisomy 13.

The pathogenesis of double outlet right ventricle with or without pulmonary infundibular atresia in mouse fetal trisomy 13 was studied at the organ level using microdissection and scanning electron microscopy. Altogether, 394 karyotyped trisomic embryos were collected between 11 days and 16 hours of gestation (presence of a vaginal plug = day 1) and 15 days of gestation at intervals of 8 hours, and at 16 days of gestation. The hearts were perfusion-fixed, microdissected, and prepared to be observed in scanning electron microscope in the following standardized orientations: frontal, right or left profile, septal and parietal halves of the right ventricle and outflow tract (conotruncus). Comparison of 276 trisomic hearts with their normal counterparts described previously has shown that: the first pathognomonic feature is the abnormal anterior position of the proximal part of the parietal outflow tract ridge or of both ridges (at 12 days and 16 hours of gestation); the abnormal anterior fusion of these ridges ("coalescence") results in a mesenchymal mass behind which is deviated the pulmonary part of the outflow tract lumen; from 14 days and 16 hours of gestation on, this lumen is either obstructed, resulting in a supravalvar stenosis of the pulmonary trunk and subsequently evolving into double outlet right ventricle with pulmonary infundibular atresia; or, in a minority of cases, this lumen is not obstructed and the heart develops into double outlet right ventricle without pulmonary infundibular atresia. The pathogenesis of these malformations differs from most of the known hypotheses based on deductions from human malformed hearts, as well as from observations of the pathogenesis of similar outflow tract malformations, such as those found in the Keeshond dog or rats treated with trimethadione.

Carbonic anhydrase II expression pattern in mouse embryonic and fetal heart.

Carbonic anhydrase II localization was studied in mouse embryonic and fetal hearts for better understanding of the functions of this enzyme during cardiac organogenesis. Immunocytolabelling was performed on serial sections of frozen hearts after one night's fixation in 4% paraformaldehyde. In the earliest stages studied, 10, 11 and 12 ed (ed = embryonic day; vaginal plug = day 1), a sharp decrease of labelled cells was observed in the endocardium form which cushion-tissue mesenchyme is derived. During the same period, differences in the decreasing frequencies of labelled cells were also observed between three different cushion-tissue mesenchyme localizations: immunostained cells were abundant in the atrioventricular cushions, less numerous in the proximal part of the conotruncal ridges and rare in their distal part. From 13 ed their repartition was more regular along the conotruncus. From 13 to 16 ed the signal was also present in a peculiar region of the myocardium: the anterior and left walls of the left ventricle. At the 18 and 20 ed labelling was found only in some endothelial cells of coronary vessels, particularly in the interventricular septum. The pattern of expression of carbonic anhydrase II in activated endothelial cells and endothelial-derived mesenchyme cells of the cardiac cushion tissue, strongly suggests that this isoenzyme can be a useful marker for a subpopulation of endothelial cells and cells derived from this endothelium that morphologically express signs of active cell behavior (e.g., invasion, migration, proliferation).

Hydrogen peroxide (H2O2) as a source of dissolved oxygen in COD-degradation respirometric experiments.

Two different re-oxygenation techniques (aeration and hydrogen peroxide addition) were compared in respirometric experiments. As similar results were obtained in both cases, it was concluded that the addition of hydrogen peroxide does not modify the oxygen uptake rate of the biomass, under either endogenous or feeding conditions. It was hypothesized that under those experimental conditions (inhibition of nitrification with ATU), hydrogen peroxide alters neither the biomass metabolism nor the biodegradability of the tested substrates. The oxygen uptake rates obtained with the aeration system were often more scattered due to the adhesion of fine bubbles after the switch off of the aeration. Moreover, the transfer rate of oxygen to the solution is faster in the case of hydrogen peroxide addition.

Severe hypophosphatasia due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.

Hypophosphatasia is a rare autosomal recessive inborn error of metabolism characterized by a defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutation in a patient affected by infantile hypophosphatasia. This boy was the first child of non affected, non related parents. At 1 month of age he presented with palsy of the left upper limb with hypotonia. Length was - 2SD. The anterior fontanel was large. There was a markedly decreased ossification of all bones. All limbs were shortened. Ultrasonographic examination of the kidneys showed nephrocalcinosis. Level of alkaline phosphatases was decreased in the child as well as in the parents. Bone density was decreased. At 2 years of age development was delayed. Weight was - 3,5 SD and OFC - 3SD. The child had craniosynostosis. Molecular studies showed 2 missense mutations, both in exon 6 of the TNSALP gene.

A two-step alignment method for 3D computer-aided reconstruction based on fiducial markers and applied to mouse embryonic hearts.

An accurate three dimensional computer reconstruction of microscopic biological objects or distribution of molecules identified on serial sections must solve two major problems: 1) the alignment of sections using adequate extrinsic references (fiducial markers); 2) the impossibility of observing these references and the cellular or molecular structures in the microscope at the same magnification. To provide extrinsic references for objects embedded in soft media, we have modified and simplified the charcoal-paraffin method described by Langemeijer and Simons (1973). It consists of drilling three or four small holes into the paraffin block, sealing this block at the extremity of a glass holder and, from the other extremity of the holder attached to a rubber hose, aspirating a liquefied mixture of charcoal-paraffin to fill these cylindrical holes. An alignment procedure was developed using serial sections of mouse embryonic hearts with bromodeoxyuridine-labelled DNA synthesizing cells. From each fourth section, two sets of contours have been drawn and digitized: 1) at low magnification (about 40x), embryo body wall, heart, neural tube and extrinsic reference marks (black dots); 2) at higher magnification (240-300x): heart contours alone (without extrinsic references, but with individual labelled cells). Different operations of the computer-aided alignment, as well as checking of results by inverse alignment, are described in detail. This two-step alignment method offers a practical, efficient compromise between: a) purely subjective alignment based only on tissular landmarks interpreted by the operator; b) ideal perfect alignment based not only on adequate references, but on computerized correction of section deformation, as well.

Bioaccumulation of waterborne Ni in Dreissena polymorpha: a stable isotope experiment to assess the effect of zinc, calcium, and dissolved organic matter.

The effect of Ca, Zn, and dissolved natural organic matter (NOM) on waterborne Ni accumulation was investigated in a freshwater mussel. An enriched stable metal isotope tracer was required to measure the Ni uptake rate accurately. Zebra mussels were exposed to environmentally relevant concentrations of (62) Ni (from 0.5 to 8 µg/L) for 48 h in media spiked with Ca, Zn, or dissolved NOM. The (62)Ni uptake was inhibited by Ca (from 0.138 ± 0.021 to 0.061 ± 0.010 L/g/d for Ca concentrations ranging from 43 to 133 mg/L) and enhanced by Zn (from 0.051 ± 0.006 to 0.109 ± 0.007 L/g/d for Zn concentrations ranging from 6.6 to 38.3 µg/L). The mechanisms behind the synergistic effect of Zn remain unclear, yet it can be hypothesized that Ni uptake is facilitated by Zn-dependent transport sites. To formalize the effects of Ca and Zn, a model was proposed to express the Ni uptake rate as a function of the mussels' filtration rate and of Ca and Zn concentrations. The (62)Ni uptake increased at low NOM concentrations and decreased at higher concentrations. This could be explained by the influence of NOM on both the speciation of Ni and the filtration activity of mussels. At high NOM concentrations, a modification of the membrane's permeability might also have favored Ni uptake, although this was not clearly established in this study. Therefore, the effect of water composition on Ni bioavailability to zebra mussels cannot be predicted by competition and complexation models alone, because it also influences the animal's physiology.

Normal stages of cardiac organogenesis in the mouse: I. Development of the external shape of the heart.

Normal development of the mouse embryonic heart was studied at the organ level using microdissection and scanning electron microscopy (SEM). Altogether 225 embryos, sampled at 8-hour intervals between 11ed (ed = embryonic day; day of vaginal plug = 1ed) and 15ed were collected. Their hearts were fixed by high flow-low pressure perfusion, microdissected, and observed in SEM. Standardized frontal, right profile, and left profile SEM micrographs were obtained and analyzed. The main purpose of this study was to create a series of normal stages of mouse cardiac development as a reference for ongoing studies in experimental cardiac teratology (e.g., in fetal mouse trisomies). Comparisons with chick, human, and dog embryonic hearts, prepared using the same technique, show that the mouse embryonic heart is characterized by a relatively deep interventricular sulcus. The absence of a conoventricular sulcus in the mouse results in poor definition of the boundary between the conus and the right ventricle. The external separation of the aorta and the pulmonary artery is evident from 13ed onward. The respective positions of the great arteries (aorta dextroposterior, pulmonary artery sinistroanterior) does not change until the end of cardiac organogenesis (15ed in the mouse).

Normal stages of cardiac organogenesis in the mouse: II. Development of the internal relief of the heart.

Normal stages of intracardiac development of the mouse heart are illustrated by a collection and analysis of two complementary SEM views, the septal and parietal halves of the right ventricle and conotruncus. These views are particularly suitable for understanding the septation of the outflow tract of the heart. They represent references for the studies of the pathogenesis of conotruncal malformations. The analysis is based on 90 hearts between 11ed and 15ed at intervals of 8 hours with an additional stage at 16ed. They were prepared by perfusion fixation, microdissection, and critical point drying and were examined in SEM. The following main features of the intracardiac morphogenesis were observed: 1) the presence of two spirally positioned conotruncal ridges, their disto-proximal fusion, and the formation of the semilunar valves at their distal part; 2) the trabecular transformation of the conotruncal wall; and 3) the presence of two different interventricular foramina (FIV II and FIV III), with the final closure of FIV III at 14ed16h-15ed. As a result of these observations, our description of the outflow tract septation and its interpretation are different from other conceptions based on mechanisms such as bulbar shift, bulbar absorption, torsions, or transfer of the aorta into the left ventricle.

Rabbit oviduct mucosa healing after accidental transmucosal suture passage.

The purpose of this study is to show by SEM the characteristics of mucosal healing related to the presence of intraluminal sutures of nylon (nonabsorbable) or dexon (absorbable) in the rabbit oviduct. Altogether, 30 animals with anastomosis in the isthmus were killed 2, 4, 8, or 12 weeks after the operation. Tubular structures or pieces of thread partially or completely covered by the epithelium were found n 36.8% of these cases. Independently of the suture material used, already 2 weeks after surgery, the thread was covered by an epithelium composed mostly of squamous cells and some rare ciliated cells. At 4 weeks, the proportion of ciliated cells was increased. Regions with the usual cellular morphology and repartition of the different cellular types were also observed. However, at 8 and 12 weeks, islets of atypical squamous cells persisted in areas of transmucosal passage of the suture material. At 12 weeks, the dexon suture was not yet completely absorbed.

Do food processing industries contribute to the eutrophication of aquatic systems?

Eutrophication is the enrichment of water bodies with plant nutrients and precursors, typically nitrogen, phosphorus, and organic matter. There exists a "natural" and slow eutrophication, which, over geological times, turns a lake into a marsh and then dries it entirely. Today, however, eutrophication is mostly referred to the human process that "results in the stimulation of an array of symptomatic changes, among which increased production of algae and macrophytes, deterioration of water quality and other symptomatic changes are found to be undesirable and interfere with water uses" as defined by the OECD in 1982. This undesirable process is observed mostly in enclosed water bodies, such as lakes, but also in some rivers, some estuaries, and some coastal zones. In most freshwater systems, phosphorus has been identified as the "limiting nutrient" to phytoplankton development. This nutrient is brought to aquatic environments from rock weathering, soil leaching, and rain (natural sources), but also and mostly from agricultural runoff and domestic sewage. Some food processing industries (meat, vegetables, cheese processing) also contribute significantly to the phosphorus budget, even though the pollution may be due to floor and utensil cleaning rather than to direct food wastes.

Developmental patterning of the myocardium.

The heart in higher vertebrates develops from a simple tube into a complex organ with four chambers specialized for efficient pumping at pressure. During this period, there is a concomitant change in the level of myocardial organization. One important event is the emergence of trabeculations in the luminal layers of the ventricles, a feature which enables the myocardium to increase its mass in the absence of any discrete coronary circulation. In subsequent development, this trabecular layer becomes solidified in its deeper part, thus increasing the compact component of the ventricular myocardium. The remaining layer adjacent to the ventricular lumen retains its trabeculations, with patterns which are both ventricle- and species-specific. During ontogenesis, the compact layer is initially only a few cells thick, but gradually develops a multilayered spiral architecture. A similar process can be charted in the atrial myocardium, where the luminal trabeculations become the pectinate muscles. Their extent then provides the best guide for distinguishing intrinsically the morphologically right from the left atrium. We review the variations of these processes during the development of the human heart and hearts from commonly used laboratory species (chick, mouse, and rat). Comparison with hearts from lower vertebrates is also provided. Despite some variations, such as the final pattern of papillary or pectinate muscles, the hearts observe the same biomechanical rules, and thus share many common points. The functional importance of myocardial organization is demonstrated by lethality of mouse mutants with perturbed myocardial architecture. We conclude that experimental studies uncovering the rules of myocardial assembly are relevant for the full understanding of development of the human heart.