Reactor hydrodynamics can play a significant role in antisolvent crystallizations. In this work, the impact of suspension height/clearance ratio (H/C) and power per volume (PV) on the particle size distribution (PSD) parameters Dv10, Dv50, and Dv90 of an active pharmaceutical ingredient (API) were evaluated. The API solution was added near the liquid surface of the antisolvent with a buret, at a rate of approximately 5 mL/min, between the impeller and the reactor’s wall. Statistical models were developed, and it was found that PSD parameters seem to be influenced by the H/C and PV. A relationship between the PSD parameters and the nucleation rate was also witnessed. Furthermore, different mathematical methodologies (indicator function and Monte Carlo simulations) were used to obtain a design space comprising the probability of success of having PSD parameters within specification. An operating region comprising the probability of success was estimated, which can aid in minimizing the risk of failure in antisolvent crystallization processes and consequently help reduce the financial losses caused by out-of-specification batches.

Summary The emergence of bacterial resistance to different antibiotics in clinical use, together with the knowledge on the mechanisms by which bacteria resist the action of aminoglycosides, have contributed to the renewed interest in these molecules as potential antimicrobials. Here, we give an overview on natural and semisynthetic aminoglycosides and their structural features and modes of action, focusing on the structural insight underlying resistance mechanisms. Developments on carbohydrate chemistry and microarray technology are highlighted as powerful approaches toward generation of new aminoglycosides and for screening their interactions with RNAs and proteins. The link between antibiotic uptake and the human gut microbiome is also addressed, focusing on gut microbiome function and composition, antibiotic-induced alterations in host health, and antibiotic resistance. In addition, strategies to modulate human microbiome responses to antibiotics are discussed as novel approaches for aminoglycoside usage and for the effectiveness of antibiotic therapy.

The scale-up of crystallization processes is a challenging step in production of active pharmaceutical ingredients (APIs). When moving from lab to industrial scale, the mixing conditions tend to modify due to the different geometry and agitation performance, which is particularly important in anti-solvent crystallizations where the size of the crystals depends on the mixing and incorporation of the anti-solvent in the solution. In this work, the results obtained in anti-solvent lab-scale crystallization experiments were used to develop multivariate statistical models predicting Particle Size Distribution (PSD) parameters (Dv10, Dv50 and Dv90) in function of predictors such as percentage of volume, power per volume and tip speed. Firstly, the collinearity among the predictors was assessed by Variance Inflation Factor (VIF) diagnosis. Subsequently, least squares method was employed to find correlations among the predictors and output variables. The optimization of the models was executed by testing quadratic, logarithmic and square root terms of the predictors and removing the least statistically significant regression coefficient. The quality of the fitting was evaluated in terms of adjusted R2 (R2adj). The modelled Dv10, Dv50 and Dv90 values presented a good fitting to the experimental data, with R2adj higher than 0.79, either when using power per volume or tip speed along the percentage of volume as predictors. Afterwards, the particle size distribution parameters of industrial scale production were predicted using the previously developed models. The deviations between predicted and experimental values were lower than 17%. This demonstrates that multivariate statistical models developed in lab-scale conditions can be successfully used to predict particle size distribution in industrial-size vessels.

Abstract Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

The triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant under several growth conditions and is important for extracellular electron transfer. PpcA plays a central role in transferring electrons resulting from the cytoplasmic oxidation of carbon compounds to the cell exterior. This cytochrome is designed to couple electron and proton transfer at physiological pH, a process achieved via the selection of dominant microstates during the redox cycle of the protein, which are ultimately regulated by a well-established order of oxidation of the heme groups. The three hemes are covered only by a polypeptide chain of 71 residues and are located in the small hydrophobic core of the protein. In this work, we used NMR and X-ray crystallography to investigate the structural and functional role of a conserved valine residue (V13) located within van der Waals contact of hemes III and IV. The residue was replaced by alanine (V13A), isoleucine (V13I), serine (V13S), and threonine (V13T) to probe the effects of the side chain volume and polarity. All mutants were found to be as equally thermally stable as the native protein. The V13A and V13T mutants produced crystals and their structures were determined. The side chain of the threonine residue introduced in V13T showed two conformations, but otherwise the two structures did not show significant changes from the native structure. Analysis of the redox behavior of the four mutants showed that for the hydrophobic replacements (V13A and V13I) the redox properties, and hence the order of oxidation of the hemes, were unaffected in spite of the larger side chain, isoleucine, showing two conformations with minor changes of the protein in the heme core. On the other hand, the polar replacements (V13S and V13T) showed the presence of two more distinctive conformations, and the oxidation order of the hemes was altered. Overall, it is striking that a single residue with proper size and polarity, V13, was naturally selected to ensure a unique conformation of the protein and the order of oxidation of the hemes, endowing the cytochrome PpcA with the optimal functional properties necessary to ensure effectiveness in the extracellular electron transfer respiratory pathways of G. sulfurreducens.

Abstract Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo-dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates. Some of these nsSNPs have been biochemically and structurally characterized but the lack of a systematic and comprehensive study regarding all described and validated nsSNPs is urgent, due to the increasing importance of the enzyme in drug development, personalized medicine and therapy, as well as in pharmacogenetic studies. The objective of the present work was to collect all described nsSNPs of hAOX1 and utilize a series of bioinformatics tools to predict their effect on protein structure stability with putative implications on phenotypic functional consequences. Of 526 nsSNPs reported in NCBI-dbSNP, 119 are identified as deleterious whereas 92 are identified as nondeleterious variants. The stability analysis was performed for 119 deleterious variants and the results suggest that 104 nsSNPs may be responsible for destabilizing the protein structure, whereas five variants may increase the protein stability. Four nsSNPs do not have any impact on protein structure (neutral nsSNPs) of hAOX1. The prediction results of the remaining six nsSNPs are nonconclusive. The in silico results were compared with available experimental data. This methodology can also be used to identify and prioritize the stabilizing and destabilizing variants in other enzymes involved in drug metabolism.

The control of morphology and crystallinity of solution-processed perovskite thin-films for solar cells is the key for further enhancement of the devices’ power conversion efficiency and stability. Improving crystallinity and increasing grain size of perovskite films is a proven way to boost the devices’ performance and operational robustness, nevertheless this has only been achieved with high-temperature processes. Here, we present an unprecedented low-temperature (<80 °C) and ultrafast microwave (MW) annealing process to yield uniform, compact, and crystalline FA0.83Cs0.17Pb(I(1–x)Brx)3 perovskite films with full coverage and micrometer-scale grains. We demonstrate that the nominal composition FA0.83Cs0.17PbI1.8Br1.2 perovskite films annealed at 100 W MW power present the same band gap, similar morphology, and crystallinity of conventionally annealed films, with the advantage of being produced at a lower temperature (below 80 °C vs 185 °C) and during a very short period of time (∼2.5 min versus 60 min). These results open new avenues to fabricate band gap tunable perovskite films at low temperatures, which is of utmost importance for mechanically flexible perovskite cells and monolithic perovskite based tandem cells applications.

Olive pomace is a semi-solid paste resulting from the two-phase olive oil production, being the most significant waste generated by this agro-industry. Olive pomace is reported as an environmental hazard due to its high content in phenolic compounds (phytotoxic). Nevertheless, these compounds, when recovered, can have impactful actions in different human physiological conditions, namely, skin protection, dysfunction treatment or diseases prevention. Therefore, their recovery from olive pomace is crucial for environmental and economical sustainability, without forgetting the functional challenge. In a previous work, lipid and aqueous fractions of olive pomace were studied regarding its major bioactive compounds. The present research aims to describe an environmentally friendly integrated approach to extract and concentrate (by a pressure-driven membrane processing) the phytotoxic compounds of olive pomace. Three types of polymeric composite membranes (NF90, NF270 and BW30) were tested. The composition of the resulting streams (permeates and concentrates) were compared and the process efficiency assessed based on: (1) antioxidant activity and total phenolic and flavonoid contents; (2) inorganic elemental composition (by Inductively Coupled Plasma Atomic Emission Spectroscopy); (3) pH, conductivity and total organic carbon; and (4) permeate flux, membranes' apparent target solutes rejection and fouling index. The BW30 membrane presented the lowest fouling index and was the most effective for extracts concentration, with no phenolic compounds in the permeates, preventing completely the loss of such compounds.

Introduction: An update on the uses and applications of the non-cross-linking (NCL) hybridization assay based on the spectral modulation of gold nanoparticles (AuNPs) are presented, emphasizing DNA and RNA detection. Areas covered: Nanotechnology is strongly impacting the way we address diagnostics and therapeutics. In fact, nanoscale devices and particles have been used in a variety of platforms for improved biosensing and, more interestingly, for molecular diagnostics. AuNPs have been used in a great diversity of DNA and RNA detection strategies that are based on their nanoscale properties. Their unique optical properties have put them at the forefront of colorimetric sensing platforms. Among these, those relying on the NCL mechanism using DNA-modified AuNPs have shown remarkable versatility and simplicity for molecular detection of human pathogens, identification of single base alterations at the basis of human disease, gene expression, among others. Application of the NCL assay to molecular diagnostics will be discussed considering the challenges for validation and clinically relevant targets. Expert commentary: Integration of the NCL approach using AuNPs into chip biosensing platforms, projecting miniaturization and portability, will be addressed in terms of the future, i.e. clinical validation and translation to market.

A series of 4-ethynylaniline gold(I) complexes containing monophosphane (1,3,5-triaza-7-phosphaadamantane (pta; 2), 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (3), and PR3 , with R=naphthyl (4), phenyl (5), and ethyl (6)) and diphosphane (bis(diphenylphosphino)acetylene (dppa; 7), trans-1,2-bis(diphenylphosphino)ethene (dppet; 8), 1,2-bis(diphenylphosphino)ethane (dppe; 9), and 1,3-bis(diphenylphosphino)propane (dppp; 10)) ligands have been synthesized and their efficiency against tumor cells evaluated. The cytotoxicity of complexes 2-10 was evaluated in human colorectal (HCT116) and ovarian (A2780) carcinoma as well as in normal human fibroblasts. All the complexes showed a higher antiproliferative effect in A2780 cells, with the cytotoxicity decreasing in the following order 5>6=9=10>8>2>4>7>3. Complex 4 stands out for its very high selectivity towards ovarian carcinoma cells (IC50 =2.3 μm) compared with colorectal carcinoma and normal human fibroblasts (IC50 >100 μm), which makes this complex very attractive for ovarian cancer therapy. Its cytotoxicity in these cells correlates with the induction of the apoptotic process and an increase of intracellular reactive oxygen species (ROS). The effects of the nuclearity, rigidity, and solubility of these complexes on their biological activity were also analyzed. X-ray crystal structure determination allowed the identification of short N-H⋅⋅⋅π contacts as the main driving forces for the three-dimensional packing in these molecules.

POxylated polyurea dendrimer (PUREG4OOx48)-based nanoparticles were loaded with paclitaxel (PTX) and doxorubicin (DOX) and micronized with chitosan (CHT) by using supercritical CO2-assisted spray drying (SASD). Respirable, biocompatible, and biodegradable dry powder formulations (DPFs) were produced to effectively transport and deliver the chemotherapeutics with a controlled rate to the deep lung. In vitro studies performed with the use of the lung adenocarcinoma cell line showed that DOX@PUREG4OOx48 nanoparticles were much more cytotoxic than the free drug. Additionally, the DPFs did not show higher cytotoxicity than the respective nanoparticles, and DOX-DPFs showed a higher chemotherapeutic effect than PTX formulations in adenocarcinoma cells.

Silver nanoparticles (AgNPs) were prepared by GREEN chemistry relying on the reduction of AgNO3 by phytochemicals present in black tea extract. AgNPs were fully characterized by transmission electron microscopy (TEM), ultraviolet-visible spectroscopy ((UV-vis)), X-ray diffraction (XRD) and energy dispersive absorption spectroscopy (EDS). The synthesized AgNPs induced a decrease of the cell viability in a dose-dependent manner with a low IC50 (0.5 ± 0.1 μM) for an ovarian carcinoma cell line (A2780) compared to primary human fibroblasts (IC50 5.0 ± 0.1 μM). The DNA binding capability of CT (calf thymus) DNA was investigated using electronic absorption and fluorescence spectroscopies, circular dichroism and viscosity titration methods. Additionally, the AgNPs strongly quench the intrinsic fluorescence of BSA, as determined by synchronous fluorescence spectra.

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.

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Infectious diseases remain one of the leading causes of morbidity and mortality worldwide. The WHO and CDC have expressed serious concern regarding the continued increase in the development of multidrug resistance among bacteria. Therefore, the antibiotic resistance crisis is one of the most pressing issues in global public health. Associated with the rise in antibiotic resistance is the lack of new antimicrobials. This has triggered initiatives worldwide to develop novel and more effective antimicrobial compounds as well as to develop novel delivery and targeting strategies. Bacteria have developed many ways by which they become resistant to antimicrobials. Among those are enzyme inactivation, decreased cell permeability, target protection, target overproduction, altered target site/enzyme, increased efflux due to over-expression of efflux pumps, among others. Other more complex phenotypes, such as biofilm formation and quorum sensing do not appear as a result of the exposure of bacteria to antibiotics although, it is known that biofilm formation can be induced by antibiotics. These phenotypes are related to tolerance to antibiotics in bacteria. Different strategies, such as the use of nanostructured materials, are being developed to overcome these and other types of resistance. Nanostructured materials can be used to convey antimicrobials, to assist in the delivery of novel drugs or ultimately, possess antimicrobial activity by themselves. Additionally, nanoparticles (e.g., metallic, organic, carbon nanotubes, etc.) may circumvent drug resistance mechanisms in bacteria and, associated with their antimicrobial potential, inhibit biofilm formation or other important processes. Other strategies, including the combined use of plant-based antimicrobials and nanoparticles to overcome toxicity issues, are also being investigated. Coupling nanoparticles and natural-based antimicrobials (or other repurposed compounds) to inhibit the activity of bacterial efflux pumps; formation of biofilms; interference of quorum sensing; and possibly plasmid curing, are just some of the strategies to combat multidrug resistant bacteria. However, the use of nanoparticles still presents a challenge to therapy and much more research is needed in order to overcome this. In this review, we will summarize the current research on nanoparticles and other nanomaterials and how these are or can be applied in the future to fight multidrug resistant bacteria.

Conventional cancer chemotherapy presents several bottlenecks, such as lack of specificity that impacts healthy tissues, rapid drug metabolism, and both intrinsic/acquired drug resistances varying in patient status, which altogether lead to reduction of efficacy. To overcome these issues and improve efficacy, combination with novel nanotechnology approaches-cancer nanomedicine-in the areas of imaging, diagnosis, and drug delivery are being proposed. These developments have been focused upon the preparation and application of nanoparticles for cancer therapy. Gold nanoparticle (AuNP) applications have been projected for improved imaging, diagnosis, and therapy, due to their exquisite physicochemical and optical properties showing potential applications as drug/gene carriers, photothermal and contrast agents. All these features may potentiate selective drug delivery, thus improving efficacy and reducing side effects. In this chapter, we shall discuss applications of nanoparticles with focus on AuNPs as efficient targeted (drug) delivery systems in cancer therapy.