What Can Be Learned from the Partitioning Behavior of Proteins in Aqueous Two-Phase Systems?

This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.

Cryptic diversity in southern African kelp.

The southern coast of Africa is one of the few places in the world where water temperatures are predicted to cool in the future. This endemism-rich coastline is home to two sister species of kelps of the genus Ecklonia maxima and Ecklonia radiata, each associated with specific thermal niches, and occuring primarily on opposite sides of the southern tip of Africa. Historical distribution records indicate that E. maxima has recently shifted its distribution ~ 70 km eastward, to sites where only E. radiata was previously reported. The contact of sister species with contrasting thermal affinities and the occurrence of mixed morphologies raised the hypothesis that hybridization might be occurring in this contact zone. Here we describe the genetic structure of the genus Ecklonia along the southern coast of Africa and investigate potential hybridization and cryptic diversity using a combination of nuclear microsatellites and mitochondrial markers. We found that both species have geographically discrete genetic clusters, consistent with expected phylogeographic breaks along this coastline. In addition, depth-isolated populations were found to harbor unique genetic diversity, including a third Ecklonia lineage. Mito-nuclear discordance and high genetic divergence in the contact zones suggest multiple hybridization events between Ecklonia species. Discordance between morphological and molecular identification suggests the potential influence of abiotic factors leading to convergent phenotypes in the contact zones. Our results highlight an example of cryptic diversity and hybridization driven by contact between two closely related keystone species with contrasting thermal affinities.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Solvent polarity and hydrophobicity of solutes are two sides of the same coin.

The hydrophobicity of solutes measures the intensity of a solute's interaction with aqueous environment. The aqueous environment may change with its composition, leading to changes in its solvent properties largely characterized by polarity. As a result, the relative hydrophobicity of a solute is a function of the solute structure and the properties of the water-based solvent determined by the total composition of the aqueous phase. This aspect is commonly ignored by medicinal chemists even though it is essential for drug distribution between different biological tissues. Partitioning of solutes in aqueous two-phase systems provides the relative hydrophobicity estimates for any water-soluble compounds that can be used to improve predictions of the toxicity and other biological effects of these compounds.

Adjuvant Hyperthermic Intravesical Chemotherapy in Intermediate- and High-Risk Non-muscle Invasive Bladder Cancer.

INTRODUCTION: Non-muscle invasive bladder cancer (NMIBC) is a frequently diagnosed neoplasm, which is typically managed with transurethral resection of bladder tumor (TURBT) eventually followed by intravesical therapies. Bacillus Calmette-Guérin (BCG) is used as first-line adjuvant treatment in high- (HR) and intermediate-risk (IR) NMIBC, although, in the latter, mitomycin C (MMC) may also be used. Multiple limitations to the use of BCG encouraged the search for therapeutic alternatives. In this context, hyperthermic intravesical chemotherapy with MMC (HIVEC-MMC) emerged as a promising therapy in the adjuvant setting for NMIBC. The aim of our study was to evaluate the tolerability, compliance, and survival outcomes of HIVEC-MMC in patients with IR- and HR-NMIBC. MATERIAL AND METHODS: This was a single-center retrospective analysis of IR- and HR- NMIBC patients who received HIVEC-MMC after TURBT between August 2018 and August 2022. Levels of risk stratification were defined using the European Association of Urology (EAU) criteria. The protocol consisted of four weekly HIVEC-MMC instillations (induction) followed by six monthly instillations (maintenance). The primary outcomes were to evaluate the tolerability and compliance with the HIVEC-MMC protocol and secondary outcomes were disease-free survival (DFS) and overall survival (OS). For the purpose of statistical analysis, methods of descriptive statistics, survival analysis (Kaplan-Meier estimation), and multivariate analysis (Cox regression, and binary logistic regression) were used. RESULTS: Fifty-seven patients were enrolled with a median age of 67.9 (34.4-83.5) years old. In this cohort, 40 patients (70.2%) had primary tumors. At the time of referral for HIVEC-MMC, the majority of the patients had IR-NMIBC (n= 33, 57.9%). A total of 41 patients (71.9%) completed the HIVEC-MMC protocol. Disease recurrence and adverse events (AEs) were the most common reasons to stop the protocol. After a median follow-up of 31 months (95% CI, 5.0-54.0), 32 patients (61.4%) were disease-free, 22 (38.6%) experienced recurrent disease and six patients (10.5%) died, although only one death was directly attributable to bladder cancer. The median DFS was 42 months (95% CI, 28.0-56.0). Completion of the HIVEC-MMC maintenance phase protocol stood as a predictive factor for DFS (44 months, 95% CI 29.1-58.9 vs. 14 months, 95% CI 0.0-29.6, p < 0.001; HR 4.48, 95% CI 1.65-12.15). The median OS was not reached; the 24- and 48-month OS were 92.6% and 82.7%, respectively. EAU risk group, ECOG-PS, and completion of HIVEC protocol were found to be significant predictive factors of OS but lost their significance on multivariate analysis. However, if we exclude those who experienced recurrence during the maintenance phase protocol, treatment completion had a significant positive impact on OS (HR: 42.8, 95% CI 1.75-1045.072, p= 0.021). CONCLUSIONS: Our study suggests that HIVEC is a secure and well-tolerated treatment with promising efficacy data, making this therapeutic approach a feasible option in IR- and HR-NMIBC patients, mainly in those who cannot tolerate or have contraindications to BCG therapy, but also as an alternative during BCG shortages.

Immune Response to SARS-CoV-2 Vaccination in Cancer Patients: A Prospective Study.

Introduction Cancer patients on active treatment are at increased risk of developing coronavirus disease 2019 (COVID-19), making effective immunization of the utmost importance. However, the effectiveness of vaccination in this population is still unclear. This study aims to evaluate the response against COVID-19 in a cohort of patients with active cancer under immunosuppressive therapy. Methods This was a prospective, cross-sectional, single-center study that included patients with cancer under immunosuppressive therapy vaccinated against COVID-19 between April and September 2021. Exclusion criteria were: previous known severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, single-dose vaccine or incomplete vaccination scheme. Immunoglobulin G (IgG) anti-SARS-CoV-2 antibody levels were assessed using 35.2 binding antibody units (BAU)/mL as the positive cut-off. Assessments were performed 14-31 days after the first and second dose and three months after the second dose. Results A total of 103 patients were included. The median age was 60 years. Most patients were being treated for gastrointestinal cancer (n=38, 36.9%), breast cancer (n=33, 32%) or head and neck cancer (n=18, 17.5%). At evaluation, 72 patients (69.9%) were being treated with palliative intent. The majority were being treated with chemotherapy (CT) alone (57.3%). At the first assessment, levels of circulating SARS-CoV-2 IgG consistent with seroconversion were present in 49 patients (47.6%). At the time of the second assessment, 91% (n=100) achieved seroconversion. Three months after the second dose, 83% (n=70) maintained levels of circulating SARS-CoV-2 IgG consistent with seroconversion. In this study, no SARS-CoV-2 infection was reported in the study population. Conclusions Our findings suggest that this group of patients had a satisfactory COVID-19 immunization response. Although promising, this study should be replicated on a wider scale in order to validate these findings.

Mechanism of Phase Separation in Aqueous Two-Phase Systems.

Liquid-liquid phase separation underlies the formation of membrane-less organelles inside living cells. The mechanism of this process can be examined using simple aqueous mixtures of two or more solutes, which are able to phase separate at specific concentration thresholds. This work presents the first experimental evidence that mesoscopic changes precede visually detected macroscopic phase separation in aqueous mixtures of two polymers and a single polymer and salt. Dynamic light scattering (DLS) analysis indicates the formation of mesoscopic polymer agglomerates in these systems. These agglomerates increase in size with increasing polymer concentrations prior to visual phase separation. Such mesoscopic changes are paralleled by changes in water structure as evidenced by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis of OH-stretch bands. Through OH-stretch band analysis, we obtain quantitative estimates of the relative fractions of four subpopulations of water structures coexisting in aqueous solutions. These estimates indicate that abrupt changes in hydrogen bond arrangement take place at concentrations below the threshold of macroscopic phase separation. We used these experimental observations to develop a model of phase separation in aqueous media.

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

Reviewing Treatment Options for Advanced Renal Cell Carcinoma: Is There Still a Place for Tyrosine Kinase Inhibitor (TKI) Monotherapy?

Renal cell carcinoma (RCC) comprises a highly heterogeneous group of kidney tumours built upon distinct genetic- and epigenetic-driven mechanisms and molecular pathways. Therefore, responsiveness to treatment is considerably variable across patients, adding an extra layer of complexity to the already challenging therapeutic decision process. The last decade brought an unprecedented shift in the medical approach to advanced or metastatic RCC; in fact, immunotherapy-based combinations have significantly transformed the therapeutic arsenal and clinical outcomes of these patients. These strategies were quickly adopted by international guidelines committees as the new standards of care. However, this enhanced efficacy comes at the expense of tolerability, with a predictable negative impact on patients' quality of life. Moreover, subgroup and post hoc analyses of the major clinical trials have shown that not all patients benefit equally from these innovative approaches. In this context, a group of experts on kidney cancer met and discussed the state of the art in the field, with a special emphasis on the appropriateness of using monotherapy with an anti-angiogenesis tyrosine kinase inhibitor (TKI) to treat specific subgroups of patients with RCC. This article reviews the main topics that were considered to be pertinent for that discussion and establishes the profile of patients for whom TKI monotherapy remains a sensible frontline option by avoiding overtreatment and an unnecessary exposure to treatment-related toxicity.

Hydrogen Bond Arrangement Is Shown to Differ in Coexisting Phases of Aqueous Two-Phase Systems.

Analysis by attenuated total reflection-Fourier transform infrared spectroscopy shows that each coexisting phase in aqueous two-phase systems has a different arrangement of hydrogen bonds. Specific arrangements vary for systems formed by different solutes. The hydrogen bond arrangement is shown to correlate with differences in hydrophobic and electrostatic properties of the different phases of five specific systems, four formed by two polymers and one by a single polymer and salt. The results presented here suggest that the arrangement of hydrogen bonds may be an important factor in phase separation.

Prognostic factors for early relapse in non-metastatic triple negative breast cancer - real world data.

BACKGROUND: Triple negative breast cancer (TNBC) has the worst prognosis amongst all subtypes. Studies have shown that the achievement of pathologic complete response in the breast and axilla correlates with improved survival. The aim of this study was to identify clinical or pathological features of real-life TNBC patients with a higher risk of early relapse. MATERIALS AND METHODS: Single-centre retrospective analysis of 127 women with TNBC, stage II-III, submitted to neoadjuvant treatment and surgery between January 2016 and 2020. Multivariate Cox regression analysis for disease free survival (DFS) at 2 years was performed and statistically significant variables were computed into a prognostic model for early relapse. RESULTS: After 29 months of median follow-up, 105 patients (82.7%) were alive and, in total, 38 patients (29.9%) experienced recurrence. The 2-year DFS was 73% (95% CI: 21.3-22.7). In multivariate analysis, being submitted to neoadjuvant radiotherapy [HR 2.8 (95% CI: 1.2-6.4), p = 0.017] and not achieving pathologic complete response [HR 0.3 (95% CI: 0.1-1.7), p = 0.011] were associated with higher risk of recurrence. In our prognostic model, the presence of at least one of these variables defined a subgroup of patients with a worse 2-year DFS than those without these features (59% vs. 90%, p < 0.001, respectively). CONCLUSIONS: In this real-life non-metastatic TNBC cohort, neoadjuvant radiotherapy (performed due to insufficient clinical response to neoadjuvant chemotherapy or significant toxicity) impacted as an independent prognostic factor for relapse along with the absence of pathologic complete response identifying a subgroup of higher risk patients for early relapse that might merit a closer follow-up.

Linear Relationships between Partition Coefficients of Different Organic Compounds and Proteins in Aqueous Two-Phase Systems of Various Polymer and Ionic Compositions.

Analysis of the partition coefficients of small organic compounds and proteins in different aqueous two-phase systems under widely varied ionic compositions shows that logarithms of partition coefficients for any three compounds or proteins or two organic compounds and one protein are linearly interrelated, although for protein(s) there are ionic compositions when the linear fit does not hold. It is suggested that the established interrelationships are due to cooperativity of different types of solute-solvent interactions in aqueous media. This assumption is confirmed by analysis of distribution coefficients of various drugs in octanol-buffer systems with varied ionic compositions of the buffer. Analysis of the partition coefficients characterizing distribution of variety of drugs between blood and different tissues of rats in vivo reported in the literature showed that the above assumption is correct and enabled us to identify the tissues with the components of which the drug(s) may engage in presumably direct interactions. It shows that the suggested assumption is valid for even complex biological systems.

Sex-dependent and -independent transcriptional changes during haploid phase gametogenesis in the sugar kelp Saccharina latissima.

In haplodiplontic lineages, sexual reproduction occurs in haploid parents without meiosis. Although widespread in multicellular lineages such as brown algae (Phaeophyceae), haplodiplontic gametogenesis has been little studied at the molecular level. We addressed this by generating an annotated reference transcriptome for the gametophytic phase of the sugar kelp, Saccharina latissima. Transcriptional profiles of microscopic male and female gametophytes were analysed at four time points during the transition from vegetative growth to gametogenesis. Gametogenic signals resulting from a switch in culture irradiance from red to white light activated a core set of genes in a sex-independent manner, involving rapid activation of ribosome biogenesis, transcription and translation related pathways, with several acting at the post-transcriptional or post-translational level. Additional genes regulating nutrient acquisition and key carbohydrate-energy pathways were also identified. Candidate sex-biased genes under gametogenic conditions had potentially key roles in controlling female- and male-specific gametogenesis. Among these were several sex-biased or -specific E3 ubiquitin-protein ligases that may have important regulatory roles. Females specifically expressed several genes that coordinate gene expression and/or protein degradation, and the synthesis of inositol-containing compounds. Other female-biased genes supported parallels with oogenesis in divergent multicellular lineages, in particular reactive oxygen signalling via an NADPH-oxidase. Males specifically expressed the hypothesised brown algal sex-determining factor. Male-biased expression mainly involved upregulation of genes that control mitotic cell proliferation and spermatogenesis in other systems, as well as multiple flagella-related genes. Our data and results enhance genome-level understanding of gametogenesis in this ecologically and economically important multicellular lineage.

The Automatic Assessment of Strength and Mobility in Older Adults: A Test-Retest Reliability Study.

Background: Simple field tests such as the Timed Up and Go test (TUG) and 30 s Chair Stand test are commonly used to evaluate physical function in the elderly, providing crude outcome measures. Using an automatic chronometer, it is possible to obtain additional kinematic parameters that may lead to obtaining extra information and drawing further conclusions. However, there is a lack of studies that evaluate the test-retest reliability of these parameters, which may help to judge and interpret changes caused by an intervention or differences between populations. Thus, the aim of this study was to evaluate the test-retest reliability of the Timed Up and Go test (TUG) and 30 s Chair Stand test in healthy older adults. Methods: A total of 99 healthy older adults participated in this cross-sectional study. The TUG and the 30 s Chair Stand test were performed five times and twice, respectively, using an automatic chronometer. The sit-to-stand-to-sit cycle from the 30 s Chair Stand test was divided into two phases. Results: Overall, reliability for the 30 s Chair Stand test was good for almost each variable (intraclass correlation coefficient (ICC) >0.70). Furthermore, the use of an automatic chronometer improved the reliability for the TUG (ICC >0.86 for a manual chronometer and ICC >0.88 for an automatic chronometer). Conclusions: The TUG and the 30 s Chair Stand test are reliable in older adults. The use of an automatic chronometer in the TUG is strongly recommended as it increased the reliability of the test. This device enables researchers to obtain relevant and reliable data from the 30 s Chair Stand test, such as the duration of the sit-to-stand-to-sit cycles and phases.

Solvatochromism as a new tool to distinguish structurally similar compounds.

It is here reported a new concept based on solvatochromism to distinguish structurally similar compounds in aqueous solutions by the analysis of the stabilization of electronic excited states. The sensitivity of this approach to differentiate similar organic compounds, such as structural isomers or compound differing in the number of methylene groups, or proteins with conformational changes induced by being or not bound to cofactors, differing in two amino acids substitutions, or differing in their glycosylation profile, is demonstrated. The sensitivity of the proposed approach, based on the solvatochromic method, opens the path to its use as an auxiliary analytical tool in biomedical diagnosis/prognosis or in quality control of biologic-based drugs.

Invasion genetics of the mummichog (Fundulus heteroclitus): recent anthropogenic introduction in Iberia.

Human activities such as trade and transport have increased considerably in the last decades, greatly facilitating the introduction and spread of non-native species at a global level. In the Iberian Peninsula, Fundulus heteroclitus, a small euryhaline coastal fish with short dispersal, was found for the first time in the mid-1970s. Since then, F. heteroclitus has undergone range expansions, colonizing the southern region of Portugal, southwestern coast of Spain and the Ebro Delta in the Mediterranean Sea. Cytochrome b sequences were used to elucidate the species invasion pathway in Iberia. Three Iberian locations (Faro, Cádiz and Ebro Delta) and 13 other locations along the native range of F. heteroclitus in North America were sampled. Results revealed a single haplotype, common to all invasive populations, which can be traced to the northern region of the species' native range. We posit that the origin of the founder individuals is between New York and Nova Scotia. Additionally, the lack of genetic structure within Iberia is consistent with a recent invasion scenario and a strong founder effect. We suggest the most probable introduction vector is associated with the aquarium trade. We further discuss the hypothesis of a second human-mediated introduction responsible for the establishment of individuals in the Ebro Delta supported by the absence of adequate muddy habitats linking Cádiz and the Ebro Delta. Although the species has a high tolerance to salinity and temperature, ecological niche modelling indicates that benthic habitat constraints prevent along-shore colonisation suggesting that such expansions would need to be aided by human release.

Mechanisms ruling the partition of solutes in ionic-liquid-based aqueous biphasic systems - the multiple effects of ionic liquids.

In the past decade, the remarkable potential of ionic-liquid-based aqueous biphasic systems (IL-based ABSs) to extract and purify a large range of valued-added biocompounds has been demonstrated. However, the translation of lab-scale experiments to an industrial scale has been precluded by a poor understanding of the molecular-level mechanisms ruling the separation or partition of target compounds between the coexisting phases. To overcome this limitation, we carried out a systematic evaluation of specific interactions, induced by ILs and several salts used as phase-forming components, and their impact on the partition of several solutes in IL-based ABSs. To this end, the physicochemical characterization of ABSs composed of imidazolium-based ILs, three salts (Na2SO4, K2CO3 and K3C6H5O7) and water was performed. The ability of the coexisting phases to participate in different solute-solvent interactions (where "solvent" corresponds to each ABS phase) was estimated based on the Gibbs free energy of transfer of a methylene group between the phases in equilibrium, ΔG(CH2), and on the Kamlet-Taft parameters - dipolarity/polarizability (π*), hydrogen-bonding donor acidity (α) and hydrogen-bonding acceptor basicity (ß) - of the coexisting phases. Relationships between the partition coefficients, the phase properties expressed as Kamlet-Taft parameters and COSMO-RS descriptors were established, highlighting the ability of ILs to establish specific interactions with given solutes. The assembled results clearly support the idea that the partition of solutes in IL-based ABSs is due to multiple effects resulting from both global solute-solvent and specific solute-IL interactions. Solute-IL specific interactions are often dominant in IL-based ABSs, explaining the higher partition coefficients, extraction efficiencies and selectivities observed with these systems when compared to more traditional ones majorly composed of polymers.

Plastic ingestion in aquatic-associated bird species in southern Portugal.

Excessive use of plastics in daily life and the inappropriate disposal of plastic products are severely affecting wildlife species in both coastal and aquatic environments. Birds are top-predators, exposed to all threats affecting their environments, making them ideal sentinel organisms for monitoring ecosystems change. We set a baseline assessment of the prevalence of marine plastic litter affecting multi-species populations of aquatic birds in southern Portugal. By examining 160 stomach contents from 8 species of aquatic birds, we show that 22.5% were affected by plastic debris. Plastic was found in Ciconia ciconia, Larus fuscus and L. michahellis. Ciconia ciconia ingested the highest amount (number of items and total mass) of plastic debris. Polydimethylsiloxane (PDMS, silicones) was the most abundant polymer and was recorded only in C. ciconia. Plastic ingestion baseline data are of crucial importance to evaluate changes through time and among regions and to define management and conservation strategies.

Alternative probe for the determination of the hydrogen-bond acidity of ionic liquids and their aqueous solutions.

Although highly relevant to a priori select adequate solvents for a given application, the determination of the hydrogen-bond acidity or proton donor ability of aqueous solutions of ionic liquids is a difficult task due to the poor solubility of the commonly used probes in aqueous media. In this work, we demonstrate the applicability of the pyridine-N-oxide probe to determine the hydrogen-bond acidity of both neat ionic liquids and their aqueous solutions, based on 13C NMR chemical shifts, and the suitability of these values to appraise the ability of ionic liquids to form aqueous two-phase systems.

High unexpected genetic diversity of a narrow endemic terrestrial mollusc.

The Iberian Peninsula has an extensive record of species displaying strong genetic structure as a result of their survival in isolated pockets throughout the Pleistocene ice ages. We used mitochondrial and nuclear sequence data to analyze phylogeographic patterns in endemic land snails from a valley of central Portugal (Vale da Couda), putatively assigned to Candidula coudensis, that show an exceptionally narrow distributional range. The genetic survey presented here shows the existence of five main mitochondrial lineages in Vale da Couda that do not cluster together suggesting independent evolutionary histories. Our results also indicate a departure from the expectation that species with restricted distributions have low genetic variability. The putative past and contemporary models of geographic distribution of Vale da Couda lineages are compatible with a scenario of species co-existence in more southern locations during the last glacial maximum (LGM) followed by a post-LGM northern dispersal tracking the species optimal thermal, humidity and soil physical conditions.