Amphistomy: stomata patterning inferred from 13C content and leaf-side specific deposition of epicuticular wax.

BACKGROUND AND AIMS: The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax (EW) on the opposite leaf sides is studied. METHODS: We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from (i) wax synthesis being located exclusively in epidermal cells and (ii) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL, fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of EW, i.e. the difference in abaxial and adaxial δ(δab-δad), was used to calculate the leaf dorsi-ventral CO2 gradient. Leaf-side specific EW deposition, amphiwaxy level (AWL), was estimated and related to ASL. KEY RESULTS: In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one independently of light conditions. In high-light and low-light grown AS leaves, the isotopic polarity and CO2 gradient varied in parallel with ASL. AS leaves grown under high light increased ASL compared to low light, and δab-δad approached near-zero values. Changes in ASL occurred concomitantly with changes in AWL. CONCLUSIONS: The leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.

Macrophages play a nutritive role in post-metamorphic maturation in Drosophila.

In the body of multicellular organisms, macrophages play an indispensable role in maintaining tissue homeostasis by removing old, apoptotic and damaged cells. In addition, macrophages allow significant remodeling of body plans during embryonic morphogenesis, regeneration and metamorphosis. Although the huge amount of organic matter that must be removed during these processes represents a potential source of nutrients, their further use by the organism has not yet been addressed. Here, we document that, during metamorphosis, Drosophila larval adipose tissue is infiltrated by macrophages, which remove dying adipocytes by efferocytosis and engulf leaking RNA-protein granules and lipids. Consequently, the infiltrating macrophages transiently adopt the adipocyte-like metabolic profile to convert remnants of dying adipocytes to lipoproteins and storage peptides that nutritionally support post-metamorphic development. This process is fundamental for the full maturation of ovaries and the achievement of early fecundity of individuals. Whether macrophages play an analogous role in other situations of apoptotic cell removal remains to be elucidated.

Characterization of hFOB 1.19 Cell Line for Studying Zn-Based Degradable Metallic Biomaterials.

In vitro testing is the first important step in the development of new biomaterials. The human fetal osteoblast cell line hFOB 1.19 is a very promising cell model; however, there are vast discrepancies in cultivation protocols, especially in the cultivation temperature and the presence of the selection reagent, geneticin (G418). We intended to use hFOB 1.19 for the testing of Zn-based degradable metallic materials. However, the sensitivity of hFOB 1.19 to zinc ions has not yet been studied. Therefore, we compared the toxicity of zinc towards hFOB 1.19 under different conditions and compared it with that of the L929 mouse fibroblast cell line. We also tested the cytotoxicity of three types of Zn-based biomaterials in two types of media. The presence of G418 used as a selection reagent decreased the sensitivity of hFOB 1.19 to Zn2+. hFOB 1.19 cell line was more sensitive to Zn2+ at elevated (restrictive) temperatures. hFOB 1.19 cell line was less sensitive to Zn2+ than L929 cell line (both as ZnCl2 and extracts of alloys). Therefore, the appropriate cultivation conditions of hFOB 1.19 during biomaterial testing should be chosen with caution.

Alcohol extract of the gypsy mushroom (Cortinarius caperatus) inhibits the development of Deformed wing virus infection in western honey bee (Apis mellifera).

Deformed wing virus (DWV) transmitted by the parasitic mite Varroa destructor is one of the most significant factors contributing to massive losses of managed colonies of western honey bee (Apis mellifera) subspecies of European origin reported worldwide in recent decades. Despite this fact, no antiviral treatment against honey bee viruses is currently available for practical applications and the level of viral infection can only be controlled indirectly by reducing the number of Varroa mites in honey bee colonies. In this study, we investigated the antiviral potential of the gypsy mushroom (Cortinarius caperatus) to reduce DWV infection in honey bees. Our results indicate that the alcohol extract of C. caperatus prevented the development of DWV infection in cage experiments as well as after direct application to honey bee colonies in a field experiment. The applied doses did not shorten the lifespan of honey bees. The reduced levels of DWV in C. caperatus-treated honey bees in cage experiments were accompanied by significant changes in the gene expression of Tep7, Bap1, and Vago. The C. caperatus treatment was not effective against the trypanosomatid Lotmaria passim. No residues of C.caperatus were found in honey harvested in the spring from colonies supplemented with the mushroom extract for their winter feeding. These findings suggest that C. caperatus alcohol extract could be a potential natural remedy to treat DWV infection in honey bees.

Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection.

The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. Our data show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. We demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection.

A detailed mechanism of degradation behaviour of biodegradable as-ECAPed Zn-0.8Mg-0.2Sr with emphasis on localized corrosion attack.

In this study, advanced techniques such as atom probe tomography, atomic force microscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy were used to determine the corrosion mechanism of the as-ECAPed Zn-0.8Mg-0.2Sr alloy. The influence of microstructural and surface features on the corrosion mechanism was investigated. Despite its significance, the surface composition before exposure is often neglected by the scientific community. The analyses revealed the formation of thin ZnO, MgO, and MgCO3 layers on the surface of the material before exposure. These layers participated in the formation of corrosion products, leading to the predominant occurrence of hydrozincite. In addition, the layers possessed different resistance to the environment, resulting in localized corrosion attacks. The segregation of Mg on the Zn grain boundaries with lower potential compared with the Zn-matrix was revealed by atom probe tomography and atomic force microscopy. The degradation process was initiated by the activity of micro-galvanic cells, specifically Zn - Mg2Zn11/SrZn13. This process led to the activity of the crevice corrosion mechanism and subsequent attack to a depth of 250 µm. The corrosion rate of the alloy determined by the weight loss method was 0.36 mm·a-1. Based on this detailed study, the degradation mechanism of the Zn-0.8Mg-0.2Sr alloy is proposed.

Alcobiosis, an algal-fungal association on the threshold of lichenisation.

Alcobiosis, the symbiosis of algae and corticioid fungi, frequently occurs on bark and wood. Algae form a layer in or below fungal basidiomata reminiscent of the photobiont layer in lichens. Identities of algal and fungal partners were confirmed by DNA barcoding. Algal activity was examined using gas exchange and chlorophyll fluorescence techniques. Carbon transfer from algae to fungi was detected as 13C, assimilated by algae, transferred to the fungal polyol. Nine fungal partners scattered across Agaricomycetes are associated with three algae from Trebouxiophycae: Coccomyxa sp. with seven fungal species on damp wood, Desmococcus olivaceus and Tritostichococcus coniocybes, both with a single species on bark and rain-sheltered wood, respectively. The fungal partner does not cause any obvious harm to the algae. Algae enclosed in fungal tissue exhibited a substantial CO2 uptake, but carbon transfer to fungal tissues was only detected in the Lyomyces-Desmococcus alcobiosis where some algal cells are tightly enclosed by hyphae in goniocyst-like structures. Unlike lichen mycobionts, fungi in alcobioses are not nutritionally dependent on the algal partner as all of them can live without algae. We consider alcobioses to be symbioses in various stages of co-evolution, but still quite different from true lichens.

Novel Ultrafine-Grain Mg-Gd/Nd-Y-Ca Alloys with an Increased Ignition Temperature.

Two novel ignition-resistant magnesium alloys, Mg-2Gd-2Y-1Ca and Mg-2Nd-1Y-1Ca, were prepared in the ultrafine-grain condition by equal channel angular pressing (ECAP). In addition, four commercial alloys-AZ31, AX41, AE42 and WE43-were prepared similarly as a reference. The microstructure, mechanical properties and ignition temperature were thoroughly investigated. Both novel alloys exhibited a mean grain size of ~1 µm and dense distribution of small secondary phase particles. The mechanical strength measured by the tensile deformation test showed that the novel alloys are much stronger (~290 MPa) than all commercial alloys except WE43. However, Ca segregation into the grain boundaries caused a significant decrease in ductility (<6%). The ignition temperature of the novel alloys (~950 °C) was considerably improved by the presence of Gd/Nd, Y and Ca. This study showed that both novel alloys exhibit high strength and high ignition temperature in the ultrafine-grain condition.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Ultrafine-Grained Zn-Mg-Sr Alloy Synthesized by Mechanical Alloying and Spark Plasma Sintering.

Zinc materials are considered promising candidates for bioabsorbable medical devices used for the fixation of broken bones or stents. Materials for these applications must meet high mechanical property requirements. One of the ways to fulfil these demands is related to microstructure refinement, particularly the decrease in grain size. In the present work, we combine two powder metallurgy techniques (mechanical alloying-MA, and spark plasma sintering-SPS) to prepare Zn-1Mg-0.5Sr nanograin material. The microstructure of compacted material consisted of Zn grains and particles of Mg2Zn11 intermetallic phases from 100 to 500 nm in size, which resulted in high values of hardness and a compressive strength equal to 86 HV1 and 327 MPa, respectively. In this relation, the combination of the suggested techniques provides an innovative way to form extremely fine microstructures without significant coarsening during powder compaction at increased temperatures.

Microstructural and Mechanical Characterization of Newly Developed Zn-Mg-CaO Composite.

In this study, the Zn-0.8Mg-0.28CaO wt.% composite was successfully prepared using different conditions of ball milling (rotations and time) followed by a direct extrusion process. These materials were characterized from the point of view of microstructure and compressive properties, and the correlation between those characteristics was found. Microstructures of individual materials possessed differences in grain size, where the grain size decreased with the intensified conditions (milling speed and time). However, the mutual relation between grain size and compressive strength was not linear. This was caused by the effect of other factors, such as texture, intermetallic phases, and pores. Material texture affects the mechanical properties by a different activity ratio between basal and pyramidal slips. The properties of intermetallic particles and pores were determined in material volume using micro-computed tomography (µCT), enhancing the precision of our assumptions compared with commonly applied methods. Based on that, and the analysis after the compressive tests, we were able to determine the influence of aspect ratio, feret diameters, and volume content of intermetallic phases and pores on mechanical behavior. The influence of the aspects on mechanical behavior is described and discussed.

Honeybees control the gas permeability of brood and honey cappings.

Some bee species use wax to build their nests. They store honey and raise their brood in cells made entirely from wax. How can the bee brood breathe and develop properly when sealed in wax cells? We compared the chemical composition and structural properties of the honey cappings and worker brood cappings of the honeybee Apis mellifera carnica, measured the worker brood respiration, and calculated the CO2 gradients across the two types of cappings. We identified microscopic pores present in the brood cappings that allow efficient gas exchange of the developing brood. In contrary, honey cappings are nearly gas impermeable to protect honey from fermenting. Similar principles apply in bumble bees. Our data suggest the control of gas exchange of cappings as a selective pressure in the evolution of wax-building bees that drives their adaptation for using wax in two highly contrasting biological contexts.

Concurrence of High Corrosion Resistance and Strength with Excellent Ductility in Ultrafine-Grained Mg-3Y Alloy.

In the field of magnesium-based degradable implantable devices, the Mg-Y-RE-Zr alloying system (WE-type) has gained popularity due to its satisfying degradation rate together with mechanical strength. However, utilization of RE and Zr in the WE-type alloys was originally driven to improve Mg-based alloys for high-temperature applications in the industry, while for medical purposes, there is a question of whether the amount of alloying elements may be further optimized. For this reason, our paper presents the Mg-3Y (W3) magnesium alloy as an alternative to the WE43 alloy. This study shows that the omission of RE and Zr elements did not compromise the corrosion resistance and the degradation rate of the W3 alloy when compared with the WE43 alloy; appropriate biocompatibility was preserved as well. It was shown that the decrease in the mechanical strength caused by the omission of RE and Zr from the WE43 alloy could be compensated for by severe plastic deformation, as achieved in this study, by equal channel angular pressing. Ultrafine-grained W3 alloy exhibited compression yield strength of 362 ± 6 MPa and plastic deformation at maximum stress of 18 ± 1%. Overall, the early results of this study put forward the motion of avoiding RE elements and Zr in magnesium alloy as a suitable material for biodegradable applications and showed that solo alloying of yttrium is sufficient for maintaining desirable properties of the material at once.

Corrosion Properties of Boron- and Manganese-Alloyed Stainless Steels as a Material for the Bipolar Plates of PEM Fuel Cells.

Stainless steels are materials that could be used for constructing not only the bearing parts of fuel cells but also the functional ones, particularly the bipolar plates. The advantage of stainless steel is its valuable electrical and thermal conductivity, reasonably low cost, excellent mechanical properties, and good formability. Paradoxically, the self-protection effect resulting from passivation turns into the main disadvantage, which is unacceptable interfacial contact resistance. The aim of this study was to test a number of possible stainless steels in a simulated fuel cell environment, especially those alloyed with boron and manganese, which were found to improve the contact resistance properties of stainless steels. The primary focus of the study is to determine the corrosion resistance of the individual materials tested. Electrochemical tests and contact resistance measurements were performed following the DOE requirements. Manganese-alloyed LDX stainless steel achieved the best results in the electrochemical tests; the worst were achieved by boron-containing steels. Boron-containing stainless steels suffered from localized corrosion resulting from chromium-rich boride formation. All steels tested exceeded the DOE limit in the contact resistance measurement, with 316L reaching the lowest values.

Heat Treatment of Aluminum Alloys with the Natural Combination of Dopants.

Aluminothermic reduction without the separation of individual metals is currently considered as a possible method for processing ferromanganese sea nodules and creating new alloys. In this study, the product of their reduction-a manganese-based polymetallic mixture-was added to pure aluminum, as a mixture of alloying elements in their natural ratios. After extrusion, two new aluminum alloys with a total percentage of metallic additives ranging from 1 to 6 percent were prepared. The possibilities of the precipitation strengthening of these aluminum alloys, especially those containing Mn, Fe, Si, Ni, and Cu, were investigated under a wide range of heat treatment conditions. After each tested combination of annealing and artificial aging temperatures, the phase composition and the microstructure changes were recorded by X-ray diffraction, optical, and scanning electron microscopy with EDS analysis. Under none of the tested heat treatment conditions is a significant hardening effect observed, even though the precipitate phases are observed by TEM. However, the changes in the morphology of the present intermetallic phases caused by the heat treatment are revealed, which highlights the further possible development of these multicomponent alloys.

Advanced Zinc-Magnesium Alloys Prepared by Mechanical Alloying and Spark Plasma Sintering.

Zinc and its alloys are considered as promising materials for the preparation of biodegradable medical devices (stents and bone fixation screws) due to their enhanced biocompatibility. These materials must achieve an ideal combination of mechanical and corrosion properties that can be influenced by alloying or thermomechanical processes. This paper presents the effects of different mechanical alloying (MA) parameters on the composition of Zn-1Mg powder. At the same time, this study describes the influence of preparation by MA on Zn-6Mg and Zn-16Mg alloys. The selected powders were compacted by the spark plasma sintering (SPS) method. Subsequently, their microstructures were studied and their mechanical properties were tested. The overall process led to a significant grain refinement (629 ± 274 nm for Zn-1Mg) and the formation of new intermetallic phases (Mg2Zn11, MgZn2). The compressive properties of the sintered samples were mainly related to the concentration of the alloying elements, where an increase in concentration led to an improvement in strength but a deterioration in ductility. According to the obtained results, the best properties were obtained for the Zn-1Mg alloy.

Microstructure and Mechanical Properties of Ti-25Nb-4Ta-8Sn Alloy Prepared by Spark Plasma Sintering.

As the commercially most-used Ti-6Al-4V alloy has a different modulus of elasticity compared to the modulus of elasticity of bone and contains allergenic elements, ß-Ti alloy could be a suitable substitution in orthopedics. The spark plasma sintering (SPS) method is feasible for the preparation of materials, with very low porosity and fine-grained structure, leading to higher mechanical properties. In this study, we prepared quaternary Ti-25Nb-4Ta-8Sn alloy using the spark plasma sintering method. The material was also heat-treated in order to homogenize the structure and compare the microstructure and properties in as-sintered and annealed states. The SPS sample had a modulus of elasticity of about 63 ± 1 GPa, which, after annealing, increased to the value of 73 ± 1 GPa. The tensile yield strength (TYS) of the SPS sample was 730 ± 52 MPa, ultimate tensile strength (UTS) 764 ± 10 MPa, and ductility 22 ± 9%. Annealed samples reached higher values of TYS and UTS (831 ± 60 MPa and 954 ± 48 MPa), but the ductility decreased to the value of 3 ± 1%. The obtained results are discussed considering the observed microstructure of the alloy.

A Complex Evaluation of the In-Vivo Biocompatibility and Degradation of an Extruded ZnMgSr Absorbable Alloy Implanted into Rabbit Bones for 360 Days.

The increasing incidence of trauma in medicine brings with it new demands on the materials used for the surgical treatment of bone fractures. Titanium, its alloys, and steel are used worldwide in the treatment of skeletal injuries. These metallic materials, although inert, are often removed after the injured bone has healed. The second-stage procedure-the removal of the plates and screws-can overwhelm patients and overload healthcare systems. The development of suitable absorbable metallic materials would help us to overcome these issues. In this experimental study, we analyzed an extruded Zn-0.8Mg-0.2Sr (wt.%) alloy on a rabbit model. From this alloy we developed screws which were implanted into the rabbit tibia. After 120, 240, and 360 days, we tested the toxicity at the site of implantation and also within the vital organs: the liver, kidneys, and brain. The results were compared with a control group, implanted with a Ti-based screw and sacrificed after 360 days. The samples were analyzed using X-ray, micro-CT, and a scanning electron microscope. Chemical analysis revealed only small concentrations of zinc, strontium, and magnesium in the liver, kidneys, and brain. Histologically, the alloy was verified to possess very good biocompatibility after 360 days, without any signs of toxicity at the site of implantation. We did not observe raised levels of Sr, Zn, or Mg in any of the vital organs when compared with the Ti group at 360 days. The material was found to slowly degrade in vivo, forming solid corrosion products on its surface.

Zn-0.8Mg-0.2Sr (wt.%) Absorbable Screws-An In-Vivo Biocompatibility and Degradation Pilot Study on a Rabbit Model.

In this pilot study, we investigated the biocompatibility and degradation rate of an extruded Zn-0.8Mg-0.2Sr (wt.%) alloy on a rabbit model. An alloy screw was implanted into one of the tibiae of New Zealand White rabbits. After 120 days, the animals were euthanized. Evaluation included clinical assessment, microCT, histological examination of implants, analyses of the adjacent bone, and assessment of zinc, magnesium, and strontium in vital organs (liver, kidneys, brain). The bone sections with the implanted screw were examined via scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS). This method showed that the implant was covered by a thin layer of phosphate-based solid corrosion products with a thickness ranging between 4 and 5 µm. Only negligible changes of the implant volume and area were observed. The degradation was not connected with gas evolution. The screws were fibrointegrated, partially osseointegrated histologically. We observed no inflammatory reaction or bone resorption. Periosteal apposition and formation of new bone with a regular structure were frequently observed near the implant surface. The histological evaluation of the liver, kidneys, and brain showed no toxic changes. The levels of Zn, Mg, and Sr after 120 days in the liver, kidneys, and brain did not exceed the reference values for these elements. The alloy was safe, biocompatible, and well-tolerated.