The Role of Ionic Liquids in the Pharmaceutical Field: An Overview of Relevant Applications.

Solubility, bioavailability, permeation, polymorphism, and stability concerns associated to solid-state pharmaceuticals demand for effective solutions. To overcome some of these drawbacks, ionic liquids (ILs) have been investigated as solvents, reagents, and anti-solvents in the synthesis and crystallization of active pharmaceutical ingredients (APIs), as solvents, co-solvents and emulsifiers in drug formulations, as pharmaceuticals (API-ILs) aiming liquid therapeutics, and in the development and/or improvement of drug-delivery-based systems. The present review focuses on the use of ILs in the pharmaceutical field, covering their multiple applications from pharmaceutical synthesis to drug delivery. The most relevant research conducted up to date is presented and discussed, together with a critical analysis of the most significant IL-based strategies in order to improve the performance of therapeutics and drug delivery systems.

Valorisation of sea urchin (Paracentrotus lividus) gonads through canning.

Fresh sea urchin (Paracentrotus lividus) gonads are a delicacy with short seasonal availability, very often heterogeneous in size and intrinsic characteristics. This study aimed to valorise this resource through the preparation of canned products (with/without Porphyra spp.) and evaluate their physicochemical and sensory quality (3-12 months). Canning contributed to a decrease in protein, K and most carotenoids contents; and a concentration of lipids, ash, Na and Se levels. A simulated 12-month ageing led to decrease the protein and ß-carotene contents; and the Na and lutein levels concentration. The macroalgae addition resulted in an orange, darker and less soft product, with higher carbohydrates, Na, Se and carotenoids contents. A 25 g-dose contributes to significant daily intakes of protein (8-9%), EPA+DHA (47-53%), I (35-62%) and Se (30-47%). The products were commercially stable/sterile and had good sensory acceptance. Overall, canning constitutes a strategy to provide a nutritionally balanced product available all year-round.

Microbiological responses to depuration and transport of native and exotic clams at optimal and stressful temperatures.

The microbiological responses of two bivalves species from Tagus estuary, Venerupis pullastra (native clam) and Ruditapes philippinarum (exotic clam) were investigated during 48 h of depuration and subsequent simulated transport in semi-dry conditions at two temperatures (4 and 22 °C) until reaching 50% lethal time (LT50). Regardless of temperature and species, the maintenance of clams in water for 48 h (depuration period) did not affect LT50 during transport. R. philippinarum showed higher survival rates than V. pullastra, always reaching LT50 later, especially at 4 °C. Significant differences between clams' species were found in almost all microbiological parameters. This can be related with clams' biological activity and habitat environmental conditions since both clams do not coexist in Tagus estuary. Depuration was efficient to reduce the bacterial load, particularly Escherichia coli, but not efficient to remove Vibrio spp. In both species, the growth of Vibrio spp. was inhibited at 4 °C, whereas exponential growth occurred at 22 °C. Total viable counts significantly increased in most treatments, while E. coli counts significantly decreased to undetected levels, except for non-depurated R. philippinarum simulated transported at 4 °C. Thus, this study highlights the importance of clams depuration for at least 24 h in polluted estuarine areas, followed by transport at low temperatures (4 °C).

Bivalve Shellfish Safety in Portugal: Variability of Faecal Levels, Metal Contaminants and Marine Biotoxins during the Last Decade (2011-2020).

Bivalves are a high-value product whose production has markedly increased, reaching 9863 tonnes in Portugal in 2021. Bivalves' habitats-lagoons, estuaries and coastal waters-are exposed to biological and anthropogenic contaminants, which can bioaccumulate in these organisms and pose a significant public health risk. The need to obtain a safe product for human consumption led to the implementation of standardised hygiene regulations for harvesting and marketing bivalve molluscs, resulting in routine monitoring of bivalve production areas for microbial quality, metal contaminants, and marine biotoxins. While excessive levels of biotoxins and metal contamination lead to temporary harvesting bans, high faecal contamination leads to area reclassification and impose post-harvest treatments. In this study, the seasonal and temporal variability of these parameters were analysed using historical data generated by the monitoring programme during the last decade. Moreover, the impact of the monitoring program on bivalve harvesting from 2011 to 2020 was assessed. This program presented a considerable improvement over time, with an increase in the sampling effort and the overall program representativeness. Finally, contamination risk, revising control measures, and defining recommendations for risk mitigation measures are given in the light of ten years' monitoring.

Switchable adhesive films of pullulan loaded with a deep eutectic solvent-curcumin formulation for the photodynamic treatment of drug-resistant skin infections.

Antimicrobial photodynamic therapy (aPDT) is a potent tool to surpass the global rise of antimicrobial resistance; still, the effective topical administration of photosensitizers remains a challenge. Biopolymer-based adhesive films can safely extend the residence time of photosensitizers. However, their wide application is narrowed by their limited water absorption capacity and gel strength. In this study, pullulan-based films with a switchable character (from a solid film to an adhesive hydrogel) were developed. This was accomplished by the incorporation of a betaine-based deep eutectic solvent (DES) containing curcumin (4.4 µg.cm-2) into the pullulan films, which tuned the films' skin moisture absorption ability, and therefore they switch into an adhesive hydrogel capable of delivering the photosensitizer. The obtained transparent films presented higher extensibility (elongation at break up to 338.2%) than the pullulan counterparts (6.08%), when stored at 54% of relative humidity, and the corresponding hydrogels a 4-fold higher adhesiveness than commercial hydrogels. These non-cytotoxic adhesives allowed the inactivation (∼5 log reduction), down to the detection limit of the method, of multiresistant strains of Staphylococcus aureus in ex vivo skin samples. Overall, these materials are promising for aPDT in the treatment of resistant skin infections, while being easily removed from the skin.

Injectable Nanocomposite Hydrogels of Gelatin-Hyaluronic Acid Reinforced with Hybrid Lysozyme Nanofibrils-Gold Nanoparticles for the Regeneration of Damaged Myocardium.

Biopolymeric injectable hydrogels are promising biomaterials for myocardial regeneration applications. Besides being biocompatible, they adjust themselves, perfectly fitting the surrounding tissue. However, due to their nature, biopolymeric hydrogels usually lack desirable functionalities, such as antioxidant activity and electrical conductivity, and in some cases, mechanical performance. Protein nanofibrils (NFs), such as lysozyme nanofibrils (LNFs), are proteic nanostructures with excellent mechanical performance and antioxidant activity, which can work as nanotemplates to produce metallic nanoparticles. Here, gold nanoparticles (AuNPs) were synthesized in situ in the presence of LNFs, and the obtained hybrid AuNPs@LNFs were incorporated into gelatin-hyaluronic acid (HA) hydrogels for myocardial regeneration applications. The resulting nanocomposite hydrogels showed improved rheological properties, mechanical resilience, antioxidant activity, and electrical conductivity, especially for the hydrogels containing AuNPs@LNFs. The swelling and bioresorbability ratios of these hydrogels are favorably adjusted at lower pH levels, which correspond to the ones in inflamed tissues. These improvements were observed while maintaining important properties, namely, injectability, biocompatibility, and the ability to release a model drug. Additionally, the presence of AuNPs allowed the hydrogels to be monitorable through computer tomography. This work demonstrates that LNFs and AuNPs@LNFs are excellent functional nanostructures to formulate injectable biopolymeric nanocomposite hydrogels for myocardial regeneration applications.

Integrated Approach to Extract and Purify Proteins from Honey by Ionic Liquid-Based Three-Phase Partitioning.

The purification of value-added compounds by three-phase partitioning (TPP) is a promising alternative to conventional processes since the target compound can be easily recovered from the liquid-liquid interphase. Although this technique has been successfully applied to the recovery of proteins, the minimization of the use of salts and solvents must be pursued to improve the overall process sustainability. Accordingly, we have here investigated the use of biobased glycine-betaine ionic liquids (IL) directly with honey, a carbohydrate-rich matrix, as phase-forming components of TPP systems. These ILTPP systems were applied in the purification of major royal jelly proteins (MRJPs) from honey. The results obtained show that MRJPs mostly precipitate in the ILTPP interphase, with a recovery yield ranging between 82.8% and 97.3%. In particular, MRJP1 can be obtained with a purity level up to 90.1%. Furthermore, these systems allow the simultaneous separation of antioxidants and carbohydrates to different liquid phases. The proposed approach allows the separation of proteins, antioxidants, and carbohydrates from honey in a single step, while using only ILs and a real carbohydrate-rich matrix, thus being sustainable TPP processes.

Boosting antibiotics performance by new formulations with deep eutectic solvents.

The critical scenario of antimicrobial resistance to antibiotics highlights the need for improved therapeutics and/or formulations. Herein, we demonstrate that deep eutectic solvents (DES) formulations are very promising to remarkably improve the solubility, stability and therapeutic efficacy of antibiotics, such as ciprofloxacin. DES aqueous solutions enhance the solubility of ciprofloxacin up to 430-fold while extending the antibiotic stability. The developed formulations can improve, by 2 to 4-fold, the susceptibility of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria to the antibiotic. They also improve the therapeutic efficacy at concentrations where bacteria present resistance, without promoting tolerance development to ciprofloxacin. Furthermore, the incorporation of DES decreases the toxicity of ciprofloxacin towards immortalized human epidermal keratinocytes (HaCat cells). The results herein reveal the pioneering use of DES in fluoroquinolone-based formulations and their impact on the antibiotic's characteristics and on its therapeutic action.

Deep Eutectic Solvent Formulations and Alginate-Based Hydrogels as a New Partnership for the Transdermal Administration of Anti-Inflammatory Drugs.

The transdermal administration of nonsteroidal anti-inflammatory drugs (NSAIDs) is a valuable and safer alternative to their oral intake. However, most of these drugs display low water solubility, which makes their incorporation into hydrophilic biopolymeric drug-delivery systems difficult. To overcome this drawback, aqueous solutions of bio-based deep eutectic solvents (DES) were investigated to enhance the solubility of ibuprofen, a widely used NSAID, leading to an increase in its solubility of up to 7917-fold when compared to its water solubility. These DES solutions were shown to be non-toxic to macrophages with cell viabilities of 97.4% (at ibuprofen concentrations of 0.25 mM), while preserving the anti-inflammatory action of the drug. Their incorporation into alginate-based hydrogels resulted in materials with a regular structure and higher flexibility. These hydrogels present a sustained release of the drug, which is able, when containing the DES aqueous solution comprising ibuprofen, to deliver 93.5% of the drug after 8 h in PBS. Furthermore, these hydrogels were able to improve the drug permeation across human skin by 8.5-fold in comparison with the hydrogel counterpart containing only ibuprofen. This work highlights the possibility to remarkably improve the transdermal administration of NSAIDs by combining new drug formulations based on DES and biopolymeric drug delivery systems.

Prevalence of diabetes mellitus in Spanish primary care setting and its association with cardiovascular risk factors and cardiovascular diseases. SIMETAP-DM study. / Prevalencia de diabetes mellitus en el ámbito de la atención primaria española y su asociación con factores de riesgo cardiovascular y enfermedades cardiovasculares. Estudio SIMETAP-DM.

INTRODUCTION: The aims of this study were to determine the age- and sex-adjusted prevalence rates of DM, type-1 DM (T1DM), and type-2 DM (T2DM), and to compare the relationship with cardiovascular risk factors, cardiovascular diseases, chronic kidney disease, and metabolic diseases between populations with and without DM. METHODS: SIMETAP-DM is a cross-sectional observational study conducted in a Primary Care setting with a random population-based sample of 10,579 adults. Response rate: 66%. The diagnoses of DM, T1DM and T2DM were based on clinical and biochemical criteria and/or the checking of these diagnoses in the medical records. The crude and age- and sex-adjusted (standardised for Spanish population) prevalence rates were calculated. RESULTS: The crude prevalence rates of T1DM, T2DM, and DM were 0.87% (95% confidence interval [95% CI]: 0.67-1.13), 14.7% (95% CI: 13.9-15.6), and 15.6% (95% CI: 14.7-16.5), respectively. The age- and sex-adjusted prevalence rates of T1DM, T2DM, and DM were 1.0% (1.3% for men and 0.7% for women), 11.5% (13.6% for men and 9.7% for women), and 12.5% (14.9% for men and 10.5% for women), respectively. The prevalence of DM in the population≥70 years was double (30.3% [95% CI: 28.0-32.7]) that of the population between 40 and 69 years (15.3% [95% CI: 14.1-16.5]). Hypertension, peripheral arterial disease, increased waist-to-height ratio, albuminuria, coronary heart disease, atherogenic dyslipidaemia and hypercholesterolaemia were associated with DM. CONCLUSIONS: In a Spanish primary care setting, the age-adjusted prevalences of T1DM, T2DM and DM in the adult population were 1.0, 11.5, and 12.5%, respectively. One-third (33%) of the population over 70 years had DM.

Deep eutectic solvents comprising active pharmaceutical ingredients in the development of drug delivery systems.

INTRODUCTION: Deep eutectic solvents comprising or acting as solvents of active pharmaceutical ingredients (API-DES) emerged as promising alternatives to improve therapeutic efficiency, with the additional possibility to integrate them in (bio)polymer-based systems to enhance their delivery. AREAS COVERED: A critical review of the API-DES field evolution is herein presented, namely on the capacity of DES to integrate APIs in their composition and on the use of DES as solvents for APIs. These strategies avoid a current major concern related to drugs and APIs, i.e. polymorphism, and increase the solubility and bioavailability of the target API which leads to increased bioavailability. Owing to their composition versatility, polymerizable API-DES can also be prepared. Finally, the incorporation of API-DES in (bio)polymer-based systems to improve drug delivery is presented and discussed. EXPERT OPINION: The relatively easy preparation of API-DES and their capacity to tune the API's release profile when incorporated in (bio)polymer-based systems represent an effective alternative to improve the APIs therapeutic action and to develop controlled drug delivery systems. Given the potential and progress demonstrated so far, the authors foresee further research on novel API-DES and on their delivery routes, envisaging the development of alternative therapies and final approval as therapeutics.

Enhanced separation performance of aqueous biphasic systems formed by carbohydrates and tetraalkylphosphonium- or tetraalkylammonium-based ionic liquids.

Aiming at establishing more effective and sustainable separation processes, herein we propose the use of carbohydrates combined with tetralkylphosphonium- and tetralkylammonium-based ionic liquids (ILs) to form aqueous biphasic systems (ABS). The formation of ABS composed of non-aromatic and non-fluorinated ILs with carbohydrates is here shown for the first time. These novel systems are competive extraction platforms when compared against more conventional ABS formed by ILs and salts or fluorinated ILs and carbohydrates. Finally, it is shown that these systems can be efficiently recovered and reused.

Population and methodology of the SIMETAP study: Prevalence of cardiovascular risk factors, cardiovascular diseases, and related metabolic diseases. / Población y metodología del estudio SIMETAP: Prevalencia de factores de riesgo cardiovascular, enfermedades cardiovasculares y enfermedades metabólicas relacionadas.

The prevention of cardiovascular disease is based on the detection and control of cardiovascular risk factors (CVRF). In Spain there are important geographical differences both in the prevalence and in the level of control of the CVRF. In the last decade there has been an improvement in the control of hypertension and dyslipidaemia, but a worsening of cardio-metabolic risk factors related to obesity and diabetes. The SIMETAP study is a cross-sectional descriptive, observational study being conducted in 64 Primary Care Centres located at the Community of Madrid. The main objective is to determine the prevalence rates of CVRF, cardiovascular diseases, and metabolic diseases related to cardiovascular risk. A report is presented on the baseline characteristics of the population, the study methodology, and the definitions of the parameters and diseases under study. A total of 6,631 study subjects were selected using a population-based random sample. The anthropometric variables, lifestyles, blood pressure, biochemical parameters, and pharmacological treatments were determined. The highest crude prevalences were detected in smoking, physical inactivity, obesity, prediabetes, diabetes, hypertension, dyslipidaemias, and metabolic syndrome. A detailed analysis needs to be performed on the prevalence rates, stratified by age groups, and prevalence rates adjusted for age and sex to assess the true epidemiological dimension of these CVRF and diseases.

Molecular characterization of a rare analphoid supernumerary marker chromosome derived from 7q35 → qter: a case report.

BACKGROUND: Analphoid supernumerary marker chromosomes (aSMC) constitute one of the smallest groups of SMC, and are characterized by a centromeric constriction but no detectable alpha-satellite DNA. These marker chromosomes cannot be properly identified by conventional banding techniques alone, and molecular cytogenetic methods are necessary for a detailed characterization. Analphoid SMC derived from chromosome 7 are extremely rare, with only five cases reported so far. CASE PRESENTATION: In this work we report an aSMC involving the terminal long arm of chromosome 7 in a 10-year-old boy with multiple dysmorphic features and severe development delay. Cytogenetic analysis revealed a mosaic karyotype with the presence of an extra SMC, de novo, in 20% of lymphocytes and 73% of fibroblast cells. Next, we performed FISH analysis with multiple DNA probes and cCGH analysis. This identified the origin of the SMC as an analphoid marker resulting of invdup rearrangement of 7q35-qter region. Affimetrix CytoScan HD array analysis redefined the aSMC as a 15.42 Mb gain at 7q35-q36.3 (minimum tetraplicated region-chr7: 143,594,973-159,119,707; GRCh37/hg19) of maternal origin that encloses 67 OMIM genes, 16 of which associated to disease. Uniparental disomy of chromosome 7 (UPD 7) has been excluded. CONCLUSIONS: We report the first patient with an aSMC(7) derived from the terminal 7q region who has been molecularly and clinically full characterized. The use of SNParray in the characterization of SMC reveals to be a powerful tool, giving information not only about copy number variation but also about loss-of-heterozygosity and parental origin. We conclude that an integrated genome-wide copy number variation analysis, if possible associated to FISH and gene expression studies, could facilitate in the future the difficult task of establishing accurate genotype-phenotype correlations and help to improve genetic counselling.

Enhanced extraction of bovine serum albumin with aqueous biphasic systems of phosphonium- and ammonium-based ionic liquids.

Novel aqueous biphasic systems (ABS) composed of phosphonium- or ammonium-based ionic liquids (ILs), combined with a buffered aqueous solution of potassium citrate/citric acid (pH=7.0), were investigated for the extraction of proteins. For that purpose, the phase diagrams, tie-lines and tie-line lengths were determined at 25 °C, and the performance of these ABS for the extraction of bovine serum albumin (BSA) was then evaluated. The obtained results reveal that, with the exception of the more hydrophobic ILs, most of the systems investigated allow the complete extraction of BSA for the IL-rich phase in a single-step. These remarkable extraction efficiencies are far superior to those afforded by more conventional extraction systems previously reported. The composition of the biphasic systems, i.e., the amount of phase-forming components, was also investigated aiming at reducing the overall costs of the process without losing efficiency on the protein extraction. It is shown that the extraction efficiencies of BSA are maintained at 100% up to high protein concentrations (at least up to 10 g L(-1)). The recovery of the BSA from the IL-rich phase by dialysis is also shown in addition to the demonstration of the IL recyclability and reusability, at least for 3 times. In the sequential three-step extractions (BSA recovery/IL reusability), the extraction efficiencies of BSA for the IL-rich phase were maintained at 100%. For the improved ABS, the preservation of the protein native conformation was confirmed by Size Exclusion High-Performance Liquid Chromatography (used also as the quantification method) and by Fourier Transform Infra-Red spectroscopy. According to the results herein reported, ABS composed of phosphonium- or ammonium-based ILs and a biodegradable organic salt represent an alternative and remarkable platform for the extraction of BSA and may be extended to other proteins of interest.

Microbiological composition of native and exotic clams from Tagus estuary: effect of season and environmental parameters.

The influence of seasonal and environmental parameters on the occurrence of bacteria was investigated in two clam species (Venerupis pullastra and Ruditapes philippinarum), water and sediment from the Tagus estuary. Total viable counts (TVC), Escherichia coli, Salmonella spp. and Vibrio spp. were evaluated during one-year. Overall, significant seasonal variations were found in both sampling sites, especially for E. coli and Vibrio spp. levels. In summer, significantly higher Vibrio spp. levels were found in R. philippinarum and sediment samples, but not in V. pullastra clams and water samples. In contrast, significantly higher TVC and E. coli levels were observed in winter months in water and sediment samples. Salmonella spp. was generally isolated when higher levels of E. coli were detected, particularly in R. philippinarum. This study is useful for authorities to develop monitoring strategies for coastal contamination and to estimate human health risks associated with the consumption of bivalves.

Genomic signatures and antiviral drug susceptibility profile of A(H1N1)pdm09.

BACKGROUND: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management. OBJECTIVE: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence. STUDY DESIGN: A(H1N1)pdm09 viruses circulating in Portugal during pandemic and post-pandemic periods and 2009/2010 season. Viruses were isolated in MDCK-SIAT1 cell culture and subjected to mutation analysis of surface and internal proteins, and to antiviral drug susceptibility profiling. RESULTS: The A(H1N1)pdm09 strains circulating during the epidemic period in Portugal were resistant to amantadine. The majority of the strains were found to be susceptible to oseltamivir and zanamivir, with five outliers to neuraminidase inhibitors (NAIs) identified. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, M1 and NS2/NEP, which were common to all isolates and also some cluster-specific. DISCUSSION: Modification of viral genome transcription, replication and apoptosis kinetics, changes in antigenicity and antiviral drug susceptibility are known determinants of virulence. We report several point mutations with putative roles in viral fitness and virulence, and discuss their potential to result in more virulent phenotypes. Monitoring of specific mutations and genetic patterns in influenza viral genes is essential for risk assessing emergent strains, disease epidemiology and public health implications.

[Influenza activity 2000/2001]. / Actividade gripal em Portugal no inverno de 2000/2001: análise antigénica e genética das estirpes de vírus influenza.

Influenza viral infections are an important cause of morbidity in all age groups and are associated with a high mortality rate amongst the elderly and in risk groups. The current study analyses data from the epidemiological surveillance of influenza during the 2000/2001-winter season. Clinical, epidemiological and virological data relative to cases of influenza syndrome were collected via the National Influenza Surveillance Scheme, in collaboration with the General Directory of Health and integrates the information obtained by the Network of Sentinel Medical Practitioners and Emergency Units. The data analysis hereby obtained shows a low influenza activity during the 2000/2001-winter season, with an epidemiological period characterized by small duration and intensity. The incidence rate of influenza-like illnesses increased above the base line during three weeks and didn't surpass 74 cases per 1,000,000 inhabitants. Influenza B viruses were predominant with the simultaneous presence of influenza A (H1 and H3). The antigenic and genetic characterisation of the isolates allowed the confirmation of the similarity between these viral strains and the vaccine strains as well as the extent of the antigenic drift. Despite the antigenic similarity between the majority of influenza B isolates and the vaccine strains it's noticeable that the genetic characteristics showed an evolution directed towards the B/Sichuan/379/99 strain, which would later be incorporated in the 2001/2002 influenza vaccine. The co-circulation of two different lineages was consequently observed by the phylogenetic analysis of the B strains isolated in our country.

Molecular characterization of the HA gene of influenza type B viruses.

Nucleotide sequences of the HA1 subunit of influenza B viruses isolated in Portugal between 1994 and 2003 influenza winter seasons were analyzed by the Neighbor-Joining algorithm and rates of HA1 evolution estimated by linear regression. From 1994 to 2002, all influenza B viruses studied were of the Yamagata lineage. Strains isolated from 1994 to 1996, 1996 to 1999, and 1999 to 2002 revealed a high similarity with B/Beijing/184/93, B/Yamanashi/166/98, and B/Sichuan/379/99, respectively, and strains isolated during 1994-1995, 1996-1997, and 1998-1999 clustered in more than one branch of the phylogenetic tree. Victoria-related strains reappeared during 2002/2003 and formed only one branch in the phylogenetic tree revealing a closer relationship to B/Shandong/7/97. Evolutionary rates for strains from the Yamagata lineage were estimated as 3.82x10(-3) nucleotides/site/year and 2.62x10(-3) nucleotides/site/year for Victoria-related strains. In order to identify putative influenza B HA1 codons under selective pressure, a codon-substitution model for heterogeneous selective pressure at amino acid sites was used. A percentage of 97.3% of codons under negative selective pressure and 2.7% of codons under positive selective pressure (omega=dN/dS=2.65) were estimated, with posterior probability higher than 0.90. Amino acid sites 75, 197, and 199 were found more likely to be under positive selective pressure.