New insights into how freeze-drying and cryo-milling affects the fine structure and digestibility of gelatinized starch.

Freeze-drying (FD) and cryo-milling (CM) are common methods for preparing powder gelatinized starch samples. This study investigates the structural characterization of raw/gelatinized maize starches and digestibility after FD/CM processes to elucidate their effect on starch digestibility determination. Results showed that FD slightly increased digestibility, while higher initial glucose content in CM samples, especially for gelatinized samples. Only FD retained the granular morphology and relative crystallinity (RC), while gelatinized-FD decreased RC by 75%. CM decreased RC by 12%, while gelatinized-CM decreased it by 97%. Combined with short-range and chain structural results, FD tended to disrupt internal connected chains through volume stress, while CM cleaved glycosidic bonds in external chain. Stretched chains in gelatinized starch promoted the breakage of chains during shearing and their efficient binding with digestive enzymes. These findings would provide a basis for pre-treatment of powder samples and processes of starch- rich foods.

Production and characterisation of environmentally relevant microplastic test materials derived from agricultural plastics.

Soil environments across the globe, particularly in agricultural settings, have now been shown to be contaminated with microplastics. Agricultural plastics - such as mulching films - are used in close or direct contact with soils and there is growing evidence demonstrating that they represent a potential source of microplastics. There is a demand to undertake fate and effects studies to understand the behaviour and potential long-term ecological risks of this contamination. Yet, there is a lack of test materials available for this purpose. This study describes the manufacture and characterisation of five large (1-40 kg) batches of microplastic test materials derived from agricultural mulching films. Batches were produced from either polyethylene-based conventional mulching films or starch-polybutadiene adipate terephthalate blend mulching films that are certified biodegradable in soil. Challenges encountered and overcome during the micronisation process provide valuable insights into the future of microplastic test material generation from these material types. This includes difficulties in micronising virgin polyethylene film materials. All five batches were subjected to a thorough physical and chemical characterisation - both of the original virgin films and the subsequent microplastic particles generated - including a screening for the presence of chemical additives. This is a critical step to provide essential information for interpreting particle fate or effects in scientific testing. Trade-offs between obtaining preferred particle typologies and time and cost constraints are elucidated. Several recommendations emerging from the experiences gained in this study are put forward to advance the research field towards greater harmonisation and utilisation of environmentally relevant test materials.

Cryomilling-assisted high purity ß-chitin extraction from Uroteuthis edulis pens.

We report on the extraction of ß-chitin from pens (or Gladius) of Uroteuthis edulis, a squid species prevalent in the Pacific coastal regions of East Asia. In particular, we employ cryogenic mechanical grinding (or cryomilling) as a pre-treatment process for the raw squid pens. We show that the cryomilling step enables an effective pulverization of the raw materials, which facilitates the removal of protein residues allowing the extraction of high-purity ß-chitin with a high acetylation degree (∼97 %) and crystallinity (∼82 %). We also demonstrate that the Uroteuthis edulis extract ß-chitin affords a free-standing film with excellent optical transmittance and mechanical properties.

Insights into emerging organic pollutants extraction from polypropylene, polystyrene, and polyethylene microplastics.

BACKGROUND: Microplastics have the capability of retaining contaminants on their surface, increasing their persistence, preconcentrating them, and acting as transport vectors. Nevertheless, the determination of these compounds in plastic matrices poses several analytical issues and challenges, including the capability of many of these methods of only determining the extractable pollutants fractions, repeatability issues, etc. In this sense, it is primordial to evaluate the effect of the critical parameters that allow to obtain a quantitative extraction of the target analytes from microplastics, including the matrix effect of each of the studied polymers, the influence of particle size, and the effect of weathering. RESULTS: A simple and effective methodology for the extraction of 17 emerging organic pollutants from both pristine (polypropylene, polystyrene, and low- and high-density polyethylene) and weathered (polypropylene and polyethylene) microplastics has been developed, optimized, and validated, achieving recovery values of 70-120 % and low method quantification limits (9.2-35.5 ng/g). Results show the importance of cryomilling microplastics (as smaller particle sizes improve recovery and homogenization), something ignored in most publications. The differences in matrix effect for the studied pristine polymers highlights the importance of treating polymers individually, without extrapolating results. In weathered microplastics, matrix effect is overall higher than in their pristine counterparts, evidencing the necessity of always carrying out matrix effect and recovery studies in environmental microplastics. The analysis of 10 samples collected in Playa Grande (Tenerife, Canary Islands, Spain) revealed quantitative amounts of bisphenol A (10.8 ± 3.4 ng/g) in one of them. SIGNIFICANCE: For the first time, the effect of particle size, weathering and matrix effect have been simultaneously evaluated on microplastics, revealing the importance of their assessment to properly validate the methodology. Additionally, the method shows good performance in all the different polymers and has been successfully applied to the analysis of environmental samples of microplastics.

Co-Amorphous Versus Deep Eutectic Solvents Formulations for Transdermal Administration.

Transdermal administration can be considered as an interesting route to overcome the side-effects inherent to oral intake. Designing topical formulations with maximum drug efficiency requires the optimization of the permeation and the stability of the drug. The present study focuses on the physical stability of amorphous drugs within the formulation. Ibuprofen is commonly used in topical formulations and then was selected as a model drug. Additionally, its low Tg allows easy, unexpected recrystallization at room temperature with negative consequence on skin penetration. In this study, the physical stability of amorphous ibuprofen was investigated in two types of formulations: (i) in terpenes-based deep eutectic solvents (DES) and (ii) in arginine-based co-amorphous blends. The phase diagram of ibuprofen:L-menthol was mainly analyzed by low-frequency Raman spectroscopy, leading to the evidence of ibuprofen recrystallization in a wide range of ibuprofen concentration. By contrast, it was shown that amorphous ibuprofen is stabilized when dissolved in thymol:menthol DES. Forming co-amorphous arginine-ibuprofen blends by melting is another route for stabilizing amorphous ibuprofen, while recrystallization was detected in the same co-amorphous mixtures obtained by cryo-milling. The mechanism of stabilization is discussed from determining Tg and analyzing H-bonding interactions by Raman investigations in the C=O and O-H stretching regions. It was found that recrystallization of ibuprofen was inhibited by the inability to form dimers inherent to the preferential formation of heteromolecular H-bonding, regardless of the glass transition temperatures of the various mixtures. This result should be important for predicting ibuprofen stability within other types of topical formulations.

Cryomilling of Isotope-Enriched Ti Powders for HIVIPP Deposition to Manufacture Targets for Nuclear Cross Section Measurement.

The realization of isotopically enriched Ti targets for nuclear cross-section measurements requires particular attention, from the starting material preparation up to the deposition technique. In this work, a cryomilling process was developed and optimized, aimed at reducing the size of 49,50Ti metal sponge as provided by the supplier (size up to 3 mm), to the optimal size of 10 µm, to fit the High Energy Vibrational Powder Plating technique used for target manufacturing. The optimization of the cryomilling protocol and the HIVIPP deposition using natTi material was thus performed. The scarce amount of the enriched material to be treated (about 150 mg), the need to obtain a non-contaminated final powder and a uniform target thickness of about 500 µg/cm2 were taken into account. The 49,50Ti materials were then processed and 20 targets of each isotope were manufactured. Both powders and the final Ti targets produced were characterized by SEM-EDS analysis. The amount of Ti deposited was measured by weighing, indicating reproducible and homogeneous targets, with an areal density of 468 ± 110 µg/cm2 for 49Ti (n = 20) and 638 ± 200 µg/cm2 for 50Ti (n = 20). The uniformity of the deposited layer was also confirmed by the metallurgical interface analysis. The final targets were used for the cross section measurements of the 49Ti(p,x)47Sc and 50Ti(p,x)47Sc nuclear reaction routes aimed at the production of the theranostic radionuclide 47Sc.

Rapid generation of aged tire-wear particles using dry-, wet-, and cryo-milling for ecotoxicity testing.

Strict environmental laws have been enacted to regulate the emission of exhaust particulate matter (PM), which is one of the most hazardous pollutants that reduce air quality and pose a serious risk to the human health. In addition, non-exhaust PM, such as road wear, tire wear, and brake wear debris, is a significant source of airborne pollutants. Road dust less than 100 µm in size may include tire wear particles (TWPs), which are broken down into finer particles with sizes on the order of tens of micrometers because of weathering. TWPs can be transported to water bodies via runoff, potentially contaminating water systems and negatively affecting aquatic ecosystems. Therefore, ecotoxicity tests using reference TWPs are required to investigate the impact of TWPs on the human health and environment. In this study, aged TWPs were produced using dry-, wet-, and cryo-milling methods, and the dispersion stability of TWPs in dechlorinated water was evaluated. Aged TWPs prepared by dry- and wet-milling had an average particle size of 20 µm, whereas pristine TWPs had an irregular shape and average particle size of 100 µm. The capacity of the ball-milling cylinder and excessively long 28-d generation time constrain the amount of aged TWPs that can be produced through conventional milling. In contrast, cryo-milling reduces the particle size of TWPs at the rate of -275.0 µm/d, which is nine times higher than that upon dry- and wet-milling. Dispersed cryo-milled TWPs had a hydrodiameter of 2.02 µm and were more stable in the aqueous phase in relation to the other aged TWPs. The results of this study suggest that cryo-milled TWPs can be used for aquatic exposure assessments as controls for real-world TWPs.

Rapid Cryopurification of the Yeast Mitochondrial Ribosome.

Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.

In vitro and ex vivo characterization of nanonized amniotic membrane particles: An untapped modality for ocular surface reconstruction.

The pristine Human Amniotic Membrane (HAM) has portrayed outstanding potential as scaffold for ocular surface reconstruction and regeneration. However, in treatment procedures where the supporting membrane matrix of HAM is not obligatory and only the bioactive molecules are vital, the surgical practise of HAM grafting causes redundant trauma and economic burden to the patient. Hence, in our laboratory we have attempted to break down HAM to nanoscale particles and validate its potential as a competent ocular therapeutic agent; by conducting a comparative analysis between the fresh, lyophilized, micronized and Nanonized Amniotic Membrane (NAM) particles. Our results evidently showcased that the prepared NAM particles was <100 nm and the major biomolecules such as collagen and hyaluronic acid were well retained. Further, the NAM particles eluted significantly higher amounts of proteins and growth factors while maintaining its stability and isotonicity when stored at 4 °C. Its biostability was assayed in the presence of lysozyme enzyme. Its remarkable ability to promote cell proliferation in rabbit corneal cells and negative cytotoxicity is an added advantage for ocular application. The ocular biocompatibility of NAM, evaluated by the ex vivo assessment of corneal thickness, transparency, histopathology, immunohistochemistry and corneal permeability clearly indicated its suitability for ophthalmic applications.

Mechanism for Stabilizing an Amorphous Drug Using Amino Acids within Co-Amorphous Blends.

Designing co-amorphous formulations is now recognized as a relevant strategy for improving the bioavailability of low-molecular-weight drugs. In order to determine the most suitable low-molecular-weight excipients for stabilizing the drug in the amorphous state, screening methods were developed mostly using amino acids as co-formers. The present study focused on the analysis of the thermal stability of co-amorphous blends prepared by cryo-milling indomethacin with several amino acids in order to understand the stabilization mechanism of the drug in the amorphous state. Combining low- and mid-frequency Raman investigations has provided information on the relation between the physical properties of the blends and those of the H-bond network of the amorphous drug. This study revealed the surprising capabilities of L-arginine to stiffen the H-bond network in amorphous indomethacin and to drastically improve the stability of its amorphous state. As a consequence, this study suggests that amino acids can be considered as stiffeners of the H-bond network of indomethacin, thereby improving the stability of the amorphous state.

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Manufacturing Bulk Nanocrystalline Al-3Mg Components Using Cryomilling and Spark Plasma Sintering.

In the current study, pure aluminum (Al) powders were cryomilled with and without 3 wt.% pure magnesium (Mg) dopant for varying durations followed by spark plasma sintering (SPS) of powders to prepare bulk components with superior mechanical properties. The crystallite sizes were determined for powders and the bulk components by analyzing the X-ray diffraction (XRD) spectrum. The calculations indicated a reduction in crystallite size with the increase in the cryomilling duration. The results also showed a more significant decrease in the crystallite sizes for Al-3Mg samples than that of pure Al. The changes in the surface morphology of powders were characterized using scanning electron microscopy (SEM). The elemental mapping analysis at nanoscale was carried out using Energy-dispersive X-ray spectroscopy (EDX) in Scanning transmission electron microscopy (STEM). The mechanical properties of the bulk components were assessed using a Vickers Microhardness tester. The test results demonstrated an improvement in the hardness of Mg-doped components. Higher hardness values were also reported with an increase in the cryomilling duration. This article discusses the mechanisms for the reduction in crystallite size for pure Al and Al-3Mg and its subsequent impact on improving mechanical properties.

Enhanced Reactivity and Compound Mechanism of Mg/B Composite Powders Prepared by Cryomilling.

Boron is one of the highest energy density materials. The heat of boron is difficult to carry out due to its poor combustion performance. Magnesium (10 wt.%), acting as combustion adjuvant, is added into boron powder to improve the combustion performance. In this study, two kinds of boron powder were used as raw material, boron powder with an average size of 40 µm is named B1, and B2 has an average powder size of 3 µm. Mg/B composite powder was prepared though a cryomilling method. Two compound mechanisms for Mg/B composite powder were applied. For Mg/B1 composite powder, an Mg-coating structure on the surface of B was generated. For Mg/B2, a structure that small particles were agglomerated with Mg in the interior or on the surface of B was generated. Compared with either B powder or blended Mg/B powder, the reactivity of Mg/B composite powder by cryomilling is enhanced. In addition, the atomic ratio of Mg to B and activity content of Mg on the surface of Mg/B composite powder have great impacts on the improvement of reactivity.

Targeted Cross-Linking Mass Spectrometry on Single-Step Affinity Purified Molecular Complexes in the Yeast Saccharomyces cerevisiae.

Protein cross-linking mass spectrometry (XL-MS) has been developed into a powerful and robust tool that is now well implemented and routinely used by an increasing number of laboratories. While bulk cross-linking of complexes provides useful information on whole complexes, it is limiting for the probing of specific protein "neighbourhoods," or vicinity interactomes. For example, it is not unusual to find cross-linked peptide pairs that are disproportionately overrepresented compared to the surface areas of complexes, while very few or no cross-links are identified in other regions. When studying dynamic complexes along their pathways, some vicinity cross-links may be of too low abundance in the pool of heterogenous complexes of interest to be efficiently identified by standard XL-MS. In this chapter, we describe a targeted XL-MS approach from single-step affinity purified (ssAP) complexes that enables the investigation of specific protein "neighbourhoods" within molecular complexes in yeast, using a small cross-linker anchoring tag, the CH-tag. One advantage of this method over a general cross-linking strategy is the possibility to significantly enrich for localized anchored-cross-links within complexes, thus yielding a higher sensitivity to detect highly dynamic or low abundance protein interactions within a specific protein "neighbourhood" occurring along the pathway of a selected bait protein. Moreover, many variations of the method can be employed; the ssAP-tag and the CH-tag can either be fused to the same or different proteins in the complex, or the CH-tag can be fused to multiple protein components in the same cell line to explore dynamic vicinity interactions along a pathway.

Single-Step Affinity Purification (ssAP) and Mass Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae.

Cellular functions are mostly defined by the dynamic interactions of proteins within macromolecular networks. Deciphering the composition of macromolecular complexes and their dynamic rearrangements is the key to get a comprehensive picture of cellular behavior and to understand biological systems. In the past two decades, affinity purification coupled to mass spectrometry has become a powerful tool to comprehensively study interaction networks and their assemblies. To overcome initial limitations of the approach, in particular, the effect of protein and RNA degradation, loss of transient interactors, and poor overall yield of intact complexes from cell lysates, various modifications to affinity purification protocols have been devised over the years. In this chapter, we describe a rapid single-step affinity purification method for the efficient isolation of dynamic macromolecular complexes. The technique employs cell lysis by cryo-milling, which ensures nondegraded starting material in the submicron range, and magnetic beads, which allow for dense antibody-conjugation and thus rapid complex isolation, while avoiding loss of transient interactions. The method is epitope tag-independent, and overcomes many of the previous limitations to produce large interactomes with almost no contamination. The protocol as described here has been optimized for the yeast S. cerevisiae.

Influence of Cryomilling on Crystallite Size of Aluminum Powder and Spark Plasma Sintered Component.

The present investigation aims to develop nanocrystalline (NC) pure aluminum powders using cryomilling technique and manufacture bulk components using spark plasma sintering (SPS). The cryomilling was performed on pure Al powders for 2, 6, and 8 h. The cryomilled powders were then consolidated using SPS to produce bulk components. The particle morphology and crystallite size of the powders and the bulk SPS components were analyzed using scanning electron microscopy (SEM), X-ray diffraction (XRD), and transmission electron microscopy (TEM). The results showed that the crystallite size of pure Al powders decreases with increased cryomilling time. The results also showed that the SPS at elevated temperatures resulted in a slight increase in crystallite size, however, the changes were insignificant. The mechanical properties of the bulk components were determined using a Vickers microhardness tester. The hardness of the cryomilled SPS component was determined to be three times higher than that of the unmilled SPS component. The mechanism for the reduction in crystallite size with increasing cryomilling time is discussed. This fundamental study provides an insight into the development of bulk nanomaterials with superior mechanical properties for automotive, aerospace, marine, and nuclear applications.

Engineering of Injectable Antibiotic-laden Fibrous Microparticles Gelatin Methacryloyl Hydrogel for Endodontic Infection Ablation.

This study aimed at engineering cytocompatible and injectable antibiotic-laden fibrous microparticles gelatin methacryloyl (GelMA) hydrogels for endodontic infection ablation. Clindamycin (CLIN) or metronidazole (MET) was added to a polymer solution and electrospun into fibrous mats, which were processed via cryomilling to obtain CLIN- or MET-laden fibrous microparticles. Then, GelMA was modified with CLIN- or MET-laden microparticles or by using equal amounts of each set of fibrous microparticles. Morphological characterization of electrospun fibers and cryomilled particles was performed via scanning electron microscopy (SEM). The experimental hydrogels were further examined for swelling, degradation, and toxicity to dental stem cells, as well as antimicrobial action against endodontic pathogens (agar diffusion) and biofilm inhibition, evaluated both quantitatively (CFU/mL) and qualitatively via confocal laser scanning microscopy (CLSM) and SEM. Data were analyzed using ANOVA and Tukey's test (α = 0.05). The modification of GelMA with antibiotic-laden fibrous microparticles increased the hydrogel swelling ratio and degradation rate. Cell viability was slightly reduced, although without any significant toxicity (cell viability > 50%). All hydrogels containing antibiotic-laden fibrous microparticles displayed antibiofilm effects, with the dentin substrate showing nearly complete elimination of viable bacteria. Altogether, our findings suggest that the engineered injectable antibiotic-laden fibrous microparticles hydrogels hold clinical prospects for endodontic infection ablation.

Studies on the Vitrified and Cryomilled Bosentan.

In this paper, several experimental techniques [X-ray diffraction, differential scanning calorimetry (DSC), thermogravimetry, Fourier transform infrared spectroscopy, and broad-band dielectric spectroscopy] have been applied to characterize the structural and thermal properties, H-bonding pattern, and molecular dynamics of amorphous bosentan (BOS) obtained by vitrification and cryomilling of the monohydrate crystalline form of this drug. Samples prepared by these two methods were found to be similar with regard to their internal structure, H-bonding scheme, and structural (α) dynamics in the supercooled liquid state. However, based on the analysis of α-relaxation times (dielectric measurements) predicted for temperatures below the glass-transition temperature (Tg), as well as DSC thermograms, it was concluded that the cryoground sample is more aged (and probably more physically stable) compared to the vitrified one. Interestingly, such differences in physical properties turned out to be reflected in the lower intrinsic dissolution rate of BOS obtained by cryomilling (in the first 15 min of dissolution test) in comparison to the vitrified drug. Furthermore, we showed that cryogrinding of the crystalline BOS monohydrate leads to the formation of a nearly anhydrous amorphous sample. This finding, different from that reported by Megarry et al. [ Carbohydr. Res. 2011, 346, 1061-1064] for trehalose (TRE), was revealed on the basis of infrared and thermal measurements. Finally, two various hypotheses explaining water removal upon cryomilling have been discussed in the manuscript.

In Situ Formation of Nano Ni-Co Oxyhydroxide Enables Water Oxidation Electrocatalysts Durable at High Current Densities.

The oxygen evolution reaction (OER) limits the energy efficiency of electrocatalytic systems due to the high overpotential symptomatic of poor reaction kinetics; this problem worsens over time if the performance of the OER electrocatalyst diminishes during operation. Here, a novel synthesis of nanocrystalline Ni-Co-Se using ball milling at cryogenic temperature is reported. It is discovered that, by anodizing the Ni-Co-Se structure during OER, Se ions leach out of the original structure, allowing water molecules to hydrate Ni and Co defective sites, and the nanoparticles to evolve into an active Ni-Co oxyhydroxide. This transformation is observed using operando X-ray absorption spectroscopy, with the findings confirmed using density functional theory calculations. The resulting electrocatalyst exhibits an overpotential of 279 mV at 0.5 A cm-2 and 329 mV at 1 A cm-2 and sustained performance for 500 h. This is achieved using low mass loadings (0.36 mg cm-2 ) of cobalt. Incorporating the electrocatalyst in an anion exchange membrane water electrolyzer yields a current density of 1 A cm-2 at 1.75 V for 95 h without decay in performance. When the electrocatalyst is integrated into a CO2 -to-ethylene electrolyzer, a record-setting full cell voltage of 3 V at current density 1 A cm-2 is achieved.

Effect of cryomilling time on microstructure evolution and hardness of cryomilled AZ31 powders.

The synthesis of nanostructured AZ31 powder by cryomilling was studied in this paper. The microstructural evolution during cryomilling, including the changes of particle morphology and internal grain size, was characterized via optical microscopy, SEM, TEM and XRD. Observations during the cryomilling produced four main findings. Firstly, cryomilling can refine the grains of AZ31 particles down to 100 nm after around 1 h milling and the minimum average grain size of about 30 nm was reached when the cryomilling time was extended to 6 h or longer. Secondly, cold welding played a dominant role in the early stage of cryomilling, while fracture took place in the late stage and surpassed cold welding. The former led to a particle size increase while the latter decreased the particle size. The minimum average particle size after 6 h cryomilling was approximately 26 µm. Thirdly, a few particles were agglomerated with other particles and could not be processed by cryomilling due to cold welding. Finally, after cryomilling 6 h and longer times, the hardness reached 162 HV which was much higher than other values reported in AZ31 alloy studies.