Impact of including two types of destoned olive cakes in pigs' diets on fecal bacterial composition and study of the relationship between fecal microbiota, feed efficiency, gut fermentation, and gaseous emissions.

The microbial population in the pig's gastrointestinal tract can be influenced by incorporating fibrous by-products into the diets. This study investigated the impact of including two types of dried olive cake (OC) in pigs' diets on fecal bacterial composition. The correlation between fecal microbiota and growth performance, nutrient digestibility, gut fermentation pattern and slurry gas emissions was also evaluated. Thirty male Pietrain x (Landrace x Large white) pigs (47.9 ± 4.21 kg) were assigned to three groups: a control group (C), a group fed a diet with 20% partially defatted OC (20PDOC), and a group fed a diet with 20% cyclone OC (20COC) for 21 days. Fecal samples collected before and after providing the experimental diets were analyzed for the V3-V4 region of the 16S rRNA gene. Pigs were weighed, and feed intake was recorded throughout the study. Potential ammonia and methane emissions from slurry were measured. No significant differences in alpha diversity indexes were found. The taxonomic analysis revealed that Firmicutes and Bacteroidota phyla were dominant at the phylum level across all groups. Differential abundance analysis using ALDEx showed significant differences among groups for various bacteria at the phylum, genus, and species levels at the end of the experiment. Pigs from 20PDOC and 20COC groups exhibited increased abundances of health-promoting bacteria, such as Plactomycetota at the phylum level and Allisonella and an unidentified genus from the Eggerthellaceae family at the genus level. These changes influenced short-chain fatty acids' (SCFA) concentration in slurries, leading to greater acetic, butyric, caproic and heptanoic acids in OC-fed groups, especially 20COC pigs. A volatility analysis revealed significant positive correlations (p < 0.05) between Uncultured_Bacteroidales and Unculured_Selenomonadaceae and energy digestibility. Monoglobus and Desulfovibrio showed a positive significant (p < 0.05) correlation with total SCFA, indicating a high impact on gut fermentation. However, growth performance parameters and potential gas emission displayed no significant correlations with a specific bacterial genus. In conclusion, our results suggest that OC inclusion into pig diets could positively modulate and contribute to the gut microbiota's favorable composition and functionality. Also, nutrient digestibility and gut fermentation patterns can be associated with specific microbial populations.

Effect of providing citrus pulp-integrated diet on fecal microbiota and serum and fecal metabolome shifts in crossbred pigs.

The study aimed to assess the impact of dehydrated citrus pulp (DCP) on growth performance, fecal characteristics, fecal bacterial composition (based on 16S rRNA analysis), and fecal and serum metabolomic profiles in crossbred pigs. 80 finishing pigs Duroc × (Landrace × Large White) were fed either a control diet (C) or a diet with 240 g/kg DCP (T) for six weeks. Including DCP in diets tended to decrease feed intake, increased (p < 0.05) the concentrations of acetic and heptanoic acids and decreased (p < 0.05) fecal butyric and branched-chain fatty acid concentrations in feces. Animals fed DCP exhibited a lower abundance of the genera Clostridium and Romboutsia, while Lachnospira significantly increased. Orthogonal partial least squares discriminant analysis plotted a clear separation of fecal and serum metabolites between groups. The main discriminant fecal metabolites were associated with bacterial protein fermentation and were downregulated in T-fed pigs. In serum, DCP supplementation upregulated metabolites related to protein and fatty acids metabolism. In conclusion, the addition of DCP as an environmentally friendly source of nutrients in pig diets, resulted in modifications of fecal bacterial composition, fermentation patterns, and overall pig metabolism, suggesting improvements in protein metabolism and gut health.

Pushing Technique Boundaries to Probe Conformational Polymorphism.

We present an extensive exploration of the solid-form landscape of chlorpropamide (CPA) using a combined experimental-computational approach at the frontiers of both fields. We have obtained new conformational polymorphs of CPA, placing them into context with known forms using flexible-molecule crystal structure prediction. We highlight the formation of a new polymorph (ζ-CPA) via spray-drying experiments despite its notable metastability (14 kJ/mol) relative to the thermodynamic α-form, and we identify and resolve the ball-milled η-form isolated in 2019. Additionally, we employ impurity- and gel-assisted crystallization to control polymorphism and the formation of novel multicomponent forms. We, thus, demonstrate the power of this collaborative screening approach to observe, rationalize, and control the formation of new metastable forms.

Self-assembly of achiral building blocks into chiral cyclophanes using non-directional interactions.

The diastereoselective assembly of achiral constituents through a single spontaneous process into complex covalent architectures bearing multiple stereogenic elements still remains a challenge for synthetic chemists. Here, we show that such an extreme level of control can be achieved by implementing stereo-electronic information on synthetic organic building blocks and templates and that non-directional interactions (i.e., electrostatic and steric interactions) can transfer this information to deliver, after self-assembly, high-molecular weight macrocyclic species carrying up to 16 stereogenic elements. Beyond the field of supramolecular chemistry, this proof of concept should stimulate the on-demand production of highly structured polyfunctional architectures.

Root-Soil Interactions for Pepper Accessions Grown under Organic and Conventional Farming.

Modern agriculture has boosted the production of food based on the use of pesticides and fertilizers and improved plant varieties. However, the impact of some such technologies is high and not sustainable in the long term. Although the importance of rhizospheres in final plant performance, nutrient cycling, and ecosystems is well recognized, there is still a lack of information on the interactions of their main players. In this paper, four accessions of pepper are studied at the rhizosphere and root level under two farming systems: organic and conventional. Variations in soil traits, such as induced respiration, enzymatic activities, microbial counts, and metabolism of nitrogen at the rhizosphere and bulk soil, as well as measures of root morphology and plant production, are presented. The results showed differences for the evaluated traits between organic and conventional management, both at the rhizosphere and bulk soil levels. Organic farming showed higher microbial counts, enzymatic activities, and nitrogen mobilization. Our results also showed how some genotypes, such as Serrano or Piquillo, modified the properties of the rhizospheres in a very genotype-dependent way. This specificity of the soil-plant interaction should be considered for future breeding programs for soil-tailored agriculture.

Prevalence and Characterization of Beta-Lactam and Carbapenem-Resistant Bacteria Isolated from Organic Fresh Produce Retailed in Eastern Spain.

Fresh fruits and vegetables are potential reservoirs for antimicrobial resistance determinants, but few studies have focused specifically on organic vegetables. The present study aimed to determine the presence of third-generation cephalosporin (3GC)- and carbapenem-resistant Gram-negative bacteria on fresh organic vegetables produced in the city of Valencia (Spain). Main expanded spectrum beta-lactamase (ESBL)- and carbapenemase-encoding genes were also detected in the isolates. One hundred and fifteen samples were analyzed using selective media supplemented with cefotaxime and meropenem. Resistance assays for twelve relevant antibiotics in medical use were performed using a disc diffusion test. A total of 161 isolates were tested. Overall, 33.5% presented multidrug resistance and 16.8% were resistant to all ß-lactam antibiotics tested. Imipenem resistance was observed in 18% of isolates, and low resistance levels were found to ceftazidime and meropenem. Opportunistic pathogens such as Acinetobacter baumannii, Enterobacter spp., Raoultella sp., and Stenotrophomonas maltophilia were detected, all presenting high rates of resistance. PCR assays revealed blaVIM to be the most frequently isolated ESBL-encoding gene, followed by blaTEM and blaOXA-48. These results confirm the potential of fresh vegetables to act as reservoirs for 3GC- and carbapenem-producing ARB. Further studies must be carried out to determine the impact of raw organic food on the spread of AMRs into the community.

Accurate extrinsic and intrinsic peak broadening modelling for time-resolved in situ ball milling reactions via synchrotron powder X-ray diffraction.

The debate on the mechanisms which underpin mechanochemical reactions via ball mill grinding is still open. Our ability to accurately measure the microstructural (crystal size and microstrain) evolution of materials under milling conditions as well as their phase composition as a function of time is key to the in-depth understanding of the kinetics and driving forces of mechanochemical transformations. Furthermore, all ball milling reactions end with a steady state or milling equilibrium - represented by a specific phase composition and relative microstructure - that does not change as long as the milling conditions are maintained. The use of a standard sample is essential to determine the instrumental contribution to the X-ray powder diffraction (XRPD) peak broadening for time-resolved in situ (TRIS) monitoring of mechanochemical reactions under in operando conditions. Using TRIS-XRPD on a ball milling setup, coupled with low-energy synchrotron radiation, we investigated different data acquisition and analysis strategies on a silicon standard powder. The diffraction geometry and the microstructural evolution of the standard itself have been studied to model the instrumental contribution to XRPD peak broadening throughout the grinding activity. Previously proposed functions are here challenged and further developed. Importantly, we show that minor drifts of the jar position do not affect the instrumental resolution function significantly. We here report and discuss the results of such investigations and their application to TRIS-XRPD datasets of inorganic and organic ball mill grinding reactions.

Using Solid Catalysts in Disulfide-Based Dynamic Combinatorial Solution- and Mechanochemistry.

It was shown for the first time that solid amines can act as catalysts for disulfide-based dynamic combinatorial chemistry (DCC) by ball mill grinding. The mechanochemical equilibrium for the two disulfide reactions studied was reached within 1-3 h using ten different amine catalysts. This contrasts with the weeks to months to achieve solution equilibrium for most solid amine catalysts at 2 %mol mol-1 concentration in a 2 mMolar disulfide dynamic combinatorial library in a suitable solvent. The final mechanochemical equilibrium was independent of the catalyst used but varied with other ball mill grinding factors such as the presence of traces of solvent. The different efficiencies of the amines tested were discussed.

Changing the game of time resolved X-ray diffraction on the mechanochemistry playground by downsizing.

Time resolved in situ (TRIS) monitoring has revolutionised the study of mechanochemical transformations but has been limited by available data quality. Here we report how a combination of miniaturised grinding jars together with innovations in X-ray powder diffraction data collection and state-of-the-art analysis strategies transform the power of TRIS synchrotron mechanochemical experiments. Accurate phase compositions, comparable to those obtained by ex situ measurements, can be obtained with small sample loadings. Moreover, microstructural parameters (crystal size and microstrain) can be also determined with high confidence. This strategy applies to all chemistries, is readily implemented, and yields high-quality diffraction data even using a low energy synchrotron source. This offers a direct avenue towards the mechanochemical investigation of reactions comprising scarce, expensive, or toxic compounds. Our strategy is applied to model systems, including inorganic, metal-organic, and organic mechanosyntheses, resolves previously misinterpreted mechanisms in mechanochemical syntheses, and promises broad, new directions for mechanochemical research.

Tribochemistry, Mechanical Alloying, Mechanochemistry: What is in a Name?

Over the decades, the application of mechanical force to influence chemical reactions has been called by various names: mechanochemistry, tribochemistry, mechanical alloying, to name but a few. The evolution of these terms has largely mirrored the understanding of the field. But what is meant by these terms, why have they evolved, and does it really matter how a process is called? Which parameters should be defined to describe unambiguously the experimental conditions such that others can reproduce the results, or to allow a meaningful comparison between processes explored under different conditions? Can the information on the process be encoded in a clear, concise, and self-explanatory way? We address these questions in this Opinion contribution, which we hope will spark timely and constructive discussion across the international mechanochemical community.

Tailored Mobility in a Zeolite Imidazolate Framework (ZIF) Antibody Conjugate*.

Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral-flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC-MS/MS provided evidence that a covalent link was established between these metal-organic frameworks and lysine residues in IgG antibodies.

Caenorhabditis elegans as an in vivo model to assess fucoidan bioactivity preventing Helicobacter pylori infection.

Currently, Helicobacter pylori is the unique biological carcinogenic agent. The search for antimicrobial alternatives to antibiotics against this pathogen has been categorized as a priority due to the drastic failure associated with current applied antibiotic therapy. The present study assessed the bioactive antimicrobial capability of fucoidan ("Generally Recognized as Safe" approval - European Commission December 2017) from different species of Phaeophyceae algae (Fucus vesiculosus, Undaria pinnatifida, Macrocystis pyrifera) against H. pylori. All the studied fucoidans showed bacteriostatic and bactericidal effects at the studied concentrations [5-100] µg ml-1 and exposure times [0-7 days]. The most effective anti-H. pylori fucoidan was validated in Caenorhabditis elegans as an in vivo model. C. elegans feed was supplemented with Undaria pinnatifida [0-100] µg ml-1 fucoidan, resulting in a significant improvement in lifespan, lowered H. pylori concentration in the digestive tract, and increased egg-laying pattern. New research lines proposing this compound as an active agent in nutraceutical and preventive novel therapies should be opened.

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg.

Understanding the unexpected effect of frequency on the kinetics of a covalent reaction under ball-milling conditions.

We here explore how ball-mill-grinding frequency affects the kinetics of a disulfide exchange reaction. Our kinetic data show that the reaction progress is similar at all the frequencies studied (15-30 Hz), including a significant induction time before the nucleation and growth process starts. This indicates that to start the reaction an initial energy accumulation is necessary. Other than mixing, the energy supplied by the mechanical treatment has two effects: (i) reducing the crystal size and (ii) creating defects in the structure. The crystal-breaking process is likely to be dominant at first becoming less important later in the process when the energy supplied is stored at the molecular level as local crystal defects. This accumulation is taken here to be the rate-determining step. We suggest that the local defects accumulate preferentially at or near the crystal surface. Since the total area increases exponentially when the crystal size is reduced by the crystal-breaking process, this can further explain the exponential dependence of the onset time on the milling frequency.

Pressure promoted low-temperature melting of metal-organic frameworks.

Metal-organic frameworks (MOFs) are microporous materials with huge potential for chemical processes. Structural collapse at high pressure, and transitions to liquid states at high temperature, have recently been observed in the zeolitic imidazolate framework (ZIF) family of MOFs. Here, we show that simultaneous high-pressure and high-temperature conditions result in complex behaviour in ZIF-62 and ZIF-4, with distinct high- and low-density amorphous phases occurring over different regions of the pressure-temperature phase diagram. In situ powder X-ray diffraction, Raman spectroscopy and optical microscopy reveal that the stability of the liquid MOF state expands substantially towards lower temperatures at intermediate, industrially achievable pressures and first-principles molecular dynamics show that softening of the framework coordination with pressure makes melting thermodynamically easier. Furthermore, the MOF glass formed by melt quenching the high-temperature liquid possesses permanent, accessible porosity. Our results thus imply a route to the synthesis of functional MOF glasses at low temperatures, avoiding decomposition on heating at ambient pressure.

Risk Characterization of Antibiotic Resistance in Bacteria Isolated from Backyard, Organic, and Regular Commercial Eggs.

This study was conducted to assess the risk due to antimicrobial-resistant strains of Salmonella spp., Listeria monocytogenes, and Escherichia coli isolated from the eggshell and the contents of eggs bought in markets in Valencia (Spain). Thirty-four samples from three different production styles were analyzed: standard ( n = 34), organic ( n = 16), and backyard ( n = 10) eggs. L. monocytogenes was not isolated in any style of production. Only one strain of Salmonella was isolated from standard production, which was resistant to ciprofloxacin and amoxicillin. E. coli strains were resistant in 22% of the isolates from organic production, 12.25% from standard production, and 11.23% from backyard production. In all cases, the highest resistance was observed for amoxicillin-clavulanate. None of the isolates from standard and backyard eggs were resistant to chloramphenicol, ciprofloxacin, gentamycin, and streptomycin, while only ceftriaxone was found to be effective against all E. coli isolates from organic eggs. ß-Lactamase genes blaTEM , blaSHV, and blaCMY-2 and the resistance genes for tetracycline tetA, tetB, and tetC were tested. The most commonly detected antimicrobial resistance genes among the E. coli isolates were tetA (49.30%), blaTEM (47.89%), and tetB (36.62%). Overall, a maximum public health risk is associated with ß-lactam antibiotics.

Understanding the Influence of Surface Solvation and Structure on Polymorph Stability: A Combined Mechanochemical and Theoretical Approach.

We explore the effect of solvent concentration on the thermodynamic stability of two polymorphs of a 1:1 cocrystal of theophylline and benzamide subjected to ball-mill liquid assisted grinding (LAG) and we investigate how this can be related to surface solvent solvation phenomena. In this system, most stable bulk polymorph form II converts to metastable bulk polymorph form I upon neat grinding (NG), while form I can fully or partially transform into form II under LAG conditions, depending on the amount of solvent used. Careful and strict experimental procedures were designed to achieve polymorph equilibrium under ball-mill LAG conditions for 16 different solvents. This allowed us to determine 16 equilibrium polymorph concentration curves as a function of solvent concentration. Ex-situ powder X-ray diffraction (PXRD) was used to monitor the polymorph concentration and crystallite size. The surface site interactions point (SSIP) description of noncovalent interactions was used in conjunction with the SSIMPLE method for calculating solvation energies to determine which functional groups are more or less exposed on the polymorph crystal surfaces. Our results demonstrate that (i) ball-mill LAG equilibrium curves can be successfully achieved experimentally for a cocrystal system; (ii) the equilibrium curves vary from solvent to solvent in onset values and slopes, thus confirming the generality of the interconversion phenomenon that we interpret here in terms of cooperativity; (iii) the concentration required for a switch in polymorphic outcome is dependent on the nature of the solvent; (iv) the SSIP results indicate that the theophylline π-system face is more exposed on the surface of form I while the theophylline N-methyl groups are more exposed in form II; and (v) for some solvents, form II has a significantly smaller crystal size at equilibrium than form I in the investigated solvent concentration range. Therefore, the free energy of the 1:1 cocrystal of theophylline and benzamide polymorphs studied here must be affected by surface solvation under ball-mill LAG conditions.

Improving the Antimicrobial Power of Low-Effective Antimicrobial Molecules Through Nanotechnology.

The objective of this work was on the one hand to assess the antibacterial activity of amines anchored to the external surface of mesoporous silica particles against Listeria monocytogenes in comparison with the same dose of free amines as well. It was also our aim to elucidate the mechanism of action of the new antimicrobial device. The suitability of silica nanoparticles to anchor, concentrate and improve the antimicrobial power of polyamines against L. monocytogenes has been demonstrated in a saline solution and in a food matrix. Moreover, through microscope observations it has been possible to determine that the attractive binding forces between the positive amine corona on the surface of nanoparticles and the negatively charged bacteria membrane provoke a disruption of the cell membrane. The surface concentration of amines on the surface of the nanoparticles is so effective that immobilized-amines were 100 times more effective in killing L. monocytogenes bacteria than the same amount of free polyamines. This novel approach for the creation of antimicrobial nanodevices opens the possibility to put in value the antimicrobial power of natural molecules that have been discarded because of its low antimicrobial power. PRACTICAL APPLICATION: Consumers demand for high-quality products, free from chemical preservatives, with an extended shelf-life. In this study, a really powerful antimicrobial agent based on a nanomaterial functionalized with a non-antimicrobial organic molecule was developed as a proof of concept. Following this approach it could be possible to develop a new generation of natural and removable antimicrobials based on their anchoring to functional surfaces for food, agricultural or medical purposes.

Dynamic Covalent Chemistry under High-Pressure:A New Route to Disulfide Metathesis.

This work describes, for the first time, the application of combined pressure and temperature stimuli in disulfide metathesis reactions. In the system studied, above a pressure of 0.2 GPa, equimolar amounts of symmetric disulfides bis 4-chlorophenyl disulfide [(4-ClPhS)2 ] and bis 2-nitrophenyl disulfide [(2-NO2 PhS)2 ] react to give the heterodimeric product 4-Cl-PhSSPh-2-NO2 . In contrast to experiments conducted in solution at atmospheric pressure or in mechanochemical experiments under ball-mill grinding conditions, there is no necessity to use a base or thiolate anion as a catalyst for the exchange reaction under investigated conditions. Single-crystal and powder X-ray diffraction revealed also that, despite the high-pressure conditions of this reaction, the heterodimeric-disulfide product unexpectedly crystallizes into the low-density polymorph A. This counterintuitive result contrasts with the high-pressure stability of the higher-density polymorph B, confirmed by its compression up to 2.8 GPa with no signs of a phase transition.