Social vs. individual age-dependent costs of imperfect vaccination.

In diseases with long-term immunity, vaccination is known to increase the average age at infection as a result of the decrease in the pathogen circulation. This implies that a vaccination campaign can have negative effects when a disease is more costly (financial or health-related costs) for higher ages. This work considers an age-structured population transmission model with imperfect vaccination. We aim to compare the social and individual costs of vaccination, assuming that disease costs are age-dependent, while the disease's dynamic is age-independent. A model coupling pathogen deterministic dynamics for a population consisting of juveniles and adults, assumed to be rational agents, is introduced. The parameter region for which vaccination has a positive social impact is fully characterized and the Nash equilibrium of the vaccination game is obtained. Finally, collective strategies designed to promote voluntary vaccination, without compromising social welfare, are discussed.

Population dynamics and games of variable size.

This work introduces the concept of Variable Size Game Theory (VSGT), in which the number of players in a game is a strategic decision made by the players themselves. We start by discussing the main examples in game theory: dominance, coexistence, and coordination. We show that the same set of pay-offs can result in coordination-like or coexistence-like games depending on the strategic decision of each player type. We also solve an inverse problem to find a d-player game that reproduces the same fixation pattern of the VSGT. In the sequel, we consider a game involving prosocial and antisocial players, i.e., individuals who tend to play with large groups and small groups, respectively. In this game, a certain task should be performed, that will benefit one of the participants at the expense of the other players. We show that individuals able to gather large groups to perform the task may prevail, even if this task is costly, providing a possible scenario for the evolution of eusociality. The next example shows that different strategies regarding game size may lead to spontaneous separation of different types, a possible scenario for speciation without physical separation (sympatric speciation). In the last example, we generalize to three types of populations from the previous analysis and study compartmental epidemic models: in particular, we recast the SIRS model into the VSGT framework: Susceptibles play 2-player games, while Infectious and Removed play a 1-player game. The SIRS epidemic model is then obtained as the replicator equation of the VSGT. We finish with possible applications of VSGT to be addressed in the future.

Precision Mass Measurement of the Proton Dripline Halo Candidate

We report the first mass measurement of the proton-halo candidate ^{22}Al performed with the low energy beam ion trap facility's 9.4 T Penning trap mass spectrometer at facility for rare isotope beams. This measurement completes the mass information for the lightest remaining proton-dripline nucleus achievable with Penning traps. ^{22}Al has been the subject of recent interest regarding a possible halo structure from the observation of an exceptionally large isospin asymmetry [J. Lee et al., Large isospin asymmetry in Si22/O22 Mirror Gamow-Teller transitions reveals the halo structure of ^{22}Al, Phys. Rev. Lett. 125, 192503 (2020).PRLTAO0031-900710.1103/PhysRevLett.125.192503]. The measured mass excess value of ME=18 092.5(3) keV, corresponding to an exceptionally small proton separation energy of S_{p}=100.4(8) keV, is compatible with the suggested halo structure. Our result agrees well with predictions from sd-shell USD Hamiltonians. While USD Hamiltonians predict deformation in the ^{22}Al ground state with minimal 1s_{1/2} occupation in the proton shell, a particle-plus-rotor model in the continuum suggests that a proton halo could form at large quadrupole deformation. These results emphasize the need for a charge radius measurement to conclusively determine the halo nature.

Machine Learning-Based Prediction of Reduction Potentials for PtIV Complexes.

Some of the well-known drawbacks of clinically approved PtII complexes can be overcome using six-coordinate PtIV complexes as inert prodrugs, which release the corresponding four-coordinate active PtII species upon reduction by cellular reducing agents. Therefore, the key factor of PtIV prodrug mechanism of action is their tendency to be reduced which, when the involved mechanism is of outer-sphere type, is measured by the value of the reduction potential. Machine learning (ML) models can be used to effectively capture intricate relationships within PtIV complex data, leading to highly accurate predictions of reduction potentials and other properties, and offering significant insights into their electrochemical behavior and potential applications. In this study, a machine learning-based approach for predicting the reduction potentials of PtIV complexes based on relevant molecular descriptors is presented. Leveraging a data set of experimentally determined reduction potentials and a diverse range of molecular descriptors, the proposed model demonstrates remarkable predictive accuracy (MSE = 0.016 V2, RMSE = 0.13 V, R2 = 0.92). Ab initio calculations and a set of different machine learning algorithms and feature engineering techniques have been employed to systematically explore the relationship between molecular structure and similarity and reduction potential. Specifically, it has been investigated whether the reduction potential of these compounds can be described by combining ML models across different combinations of constitutional, topological, and electronic molecular descriptors. Our results not only provide insights into the crucial factors influencing reduction potentials but also offer a rapid and effective tool for the rational design of PtIV complexes with tailored electrochemical properties for pharmaceutical applications. This approach has the potential to significantly expedite the development and screening of novel PtIV prodrug candidates. The analysis of principal components and key features extracted from the model highlights the significance of structural descriptors of the 2D Atom Pairs type and the lowest unoccupied molecular orbital energy. Specifically, with just 20 appropriately selected descriptors, a notable separation of complexes based on their reduction potential value is achieved.

In vitro studies into establishing therapeutic bioequivalence of complex topical products: Weight of evidence.

Over the past decade, topically applied drug products have experienced extraordinary price increases, due to the shortage of multisource generic drug products. This occurrence is mainly related to the underlying challenges evolved in topical bioequivalence documentation. Although there has been continuing regulatory efforts to present surrogate in vitro methods to clinical endpoint studies, there is still a continued need for cost- and time-efficient alternatives that account for product specificities. Hence, this work intended to expose bioequivalence assessment issues for complex topical formulations, and more specifically those related with product efficacy guidance. As a model drug and product, a bifonazole 10 mg/g cream formulation was selected and two different batches of the commercially available Reference Product (RP) were used: RP1 that displayed lower viscosity and RP4 which presented high, but not the highest, viscosity. In vitro human skin permeation testing (IVPT) was carried out and the results were evaluated by means of the traditional bioequivalence assessment approach proposed by the EMA, as well as by the Scaled Average Bioequivalence assessment approach proposed by the FDA. Based on previous experience, there was an expectation of a high level of variability in the results, thus alternative methods to evaluate local drug skin availability were developed. More specifically, an infected skin disease model, where ex vivo human skin was infected and ATP levels were used as a biological marker for monitoring antifungal activity after product application. The results showed that permeation equivalence could not be supported between the different RP batches. In contrast, this statistical difference between the formulation batches was not indicated in the disease model. Nevertheless, in pivotal IVPT studies, the lowest permeant formulation (RP4) evidenced a higher antifungal in vitro activity as reported by the lower levels of ATP. A critical appraisal of the results is likewise presented, focusing on an outlook of the real applicability of the regulatory guidances on this subject.

Dual-energy X-ray absorptiometry: an effective approach for predicting broiler chicken body composition.

Two trials were carried out to develop and validate linear regression equations for body composition prediction using Dual-energy X-ray absorptiometry (DEXA). In Trial 1, 300 Cobb500 male chickens raised from 1 to 42 d of age were scanned in DEXA to estimate total weight, fat mass, soft lean tissue (SLT) mass, bone mineral content (BMC), and fat percentage. DEXA estimates were compared to body ash, crude fat, SLT (sum of protein and water) and scale body weight. The dataset was split, with 70% used for prediction equations development and 30% for testing, and the 5k-fold cross-validation analysis was used to optimize the equations. The R2, mean absolute error (MAE), and root-mean-squared error (RMSE) were used as precision and accuracy indicators. A negative correlation (ρ = -0.27) was observed for ash content, while no correlation was observed for protein content (P > 0.05). Predictive linear equations were developed to assess broiler weight (R2 = 0.999, MAE = 25.12, RMSE = 38.99), fat mass (R2 = 0.981, MAE = 13.87, RMSE = 21.28), ash mass (R2 = 0.956, MAE = 3.98, RMSE = 5.61), SLT mass (R2 = 0.997, MAE = 35.73, RMSE = 52.45), water mass (R2 = 0.997, MAE = 29.56, RMSE = 43.94), protein mass (R2 = 0.989, MAE = 12.94, RMSE = 19.05), fat content (R2 = 0.855, MAE = 0.81, RMSE = 1.05), SLT content (R2 = 0.658, MAE = 1.01, RMSE = 1.28), and water content (R2 = 0.678, MAE = 0.99, RMSE = 1.27). All equations passed the test. In Trial 2, 395 Cobb500 male chickens were raised from 1 to 42 d of age and used for validation of prediction equations. The equations developed for weight, fat mass, ash mass, SLT mass, water mass, and protein mass were validated. In conclusion, DEXA was found to be an effective approach for measuring the body composition of broilers when using predictive equations validated in this study for estimate calibration.

Fish diversity in the rivers that drain the Baixada Maranhense and the Mearim basin in northeastern Brazil.

The Baixada Maranhense Environmental Protection Area of the Brazilian state of Maranhão encompasses a biologically rich region that includes the basins of four principal rivers, the Mearim, Pericumã, Pindaré, and Turiaçu, which form a complex of wetlands. The present study provides a comprehensive inventory of the fish fauna of the rivers that drain the Baixada Maranhense based on the identification of voucher specimens that were deposited in a scientific collection. Expeditions were conducted between 2014 and 2020. The inventory identified a total of 100 fish species representing 79 genera, 34 families, and 11 orders, which are found in both freshwater and estuarine environments. Six (Potamotrygon orbignyi, Leporinus piau, Moenkhausia loweae, Serrasalmus spilopleura, Pachypops fourcroi and Peckoltia greedoi) of the 100 taxa identified here are considered to be new records for one or more of the basins surveyed during this study, based on the existing data, while four - Colossoma macropomum, Megaleporinus macrocephalus, Cichla sp. and Cichla kelberi - are not native. The most speciose orders were the Siluriformes and Characiformes, while the most diverse families were the Loricariidae, Characidae, Auchenipteridae, Cichlidae, and Serrasalmidae. These data are consistent with the findings of most of the previous studies of freshwater ecosystems in the Neotropical region, in particular those of the rivers of Maranhão. The results of the present study represent an important advance in the understanding of the diversity of the fish fauna of the Mearim, Pericumã, Pindaré, and Turiaçu rivers, which are still poorly studied, and have likely had their diversity underestimated up to now.

Quantification and purification of isotopic contamination at the ReAccelerator of the Facility for Rare Isotope Beams.

At the ReAccelerator within the Facility for Rare Isotope Beams, a combination of an interchangeable aluminum foil and a silicon detector was developed to quantify isobaric contamination in rare isotope beams. The device is simple to operate and is now used routinely. In this article, we describe the system and show an application of the device to determine the level of contamination of an Si-32 rare isotope beam by stable S-32. In addition, we describe how the new diagnostic device helped confirm an enhancement of the beam purity prior to beam delivery to experiments.

Selective, broad-spectrum antiviral photodynamic disinfection with dicationic imidazolyl chlorin photosensitizers.

The COVID-19 pandemic exposes our vulnerability to viruses that acquire the ability to infect our cells. Classical disinfection methods are limited by toxicity. Existing medicines performed poorly against SARS-CoV-2 because of their specificity to targets in different organisms. We address the challenge of mitigating known and prospective viral infections with a new photosensitizer for antimicrobial photodynamic therapy (aPDT). Photodynamic inactivation is based on local oxidative stress, which is particularly damaging to enveloped viruses. We synthesized a cationic imidazolyl chlorin that reduced by > 99.999% of the percentage inhibition of amplification of SARS-CoV-2 collected from patients at 0.2 µM concentration and 4 J cm-2. Similar results were obtained in the prevention of infection of human ACE2-expressing HEK293T cells by a pseudotyped lentiviral vector exhibiting the S protein of SARS-CoV-2 at its surface. No toxicity to human epidermal keratinocytes (HaCaT) cells was found under similar conditions. aPDT with this chlorin offers fast and safe broad-spectrum photodisinfection and can be repeated with low risk of resistance.

Reactivity of ethyl nitrosoacrylate toward pyrrole, indole and pyrrolo[3,2-c]carbazole: an experimental and theoretical study.

Nitrosoalkenes react with 8-methyl-1,6-dihydropyrrolo[3,2-c]carbazole to give both 2- and 3-alkylated products via hetero-Diels-Alder reaction followed by the cycloadduct ring-opening. Quantum chemical calculations, at DFT level of theory, were carried out to investigate the regioselectivity of the cycloaddition of ethyl nitrosoacrylate with 1,6-dihydropyrrolo[3,2-c]carbazoles as well as with pyrrole and indole, allowing a more comprehensive analysis of the reactivity pattern of nitrosoalkenes with five-membered heterocycles. Furthermore, theoretical calculations confirmed that ethyl nitrosoacrylate reacts with these heterocycles via a LUMOheterodiene-HOMOdienophile controlled cycloaddition. The reactivity of one of the oxime-functionalized 1,6-dihydropyrrolo[3,2-c]carbazole was explored and a new hexahydropyrido[4',3':4,5]pyrrolo[3,2-c]carbazole system was obtained in high yield via a one-pot, two-step procedure.

Fluorinated Mn(III)/(II)-Porphyrin with Redox-Responsive 1 H and 19 F Relaxation Properties.

A new fluorinated manganese porphyrin, (Mn-TPP-p-CF3 ) is reported capable of providing, based on the Mn(III)/Mn(II) equilibrium, dual 1 H relaxivity and 19 F NMR response to redox changes. The physical-chemical characterization of both redox states in DMSO-d6 /H2 O evidenced that the 1 H relaxometric and 19 F NMR properties are appropriate for differential redox MRI detection. The Mn(III)-F distance (dMn-F =9.7-10 Å), as assessed by DFT calculations, is well tailored to allow for adequate paramagnetic effect of Mn(III) on 19 F T1 and T2 relaxation times. Mn-TPP-p-CF3 has a reversible Mn(II)/Mn(III) redox potential of 0.574 V vs. NHE in deoxygenated aqueous HEPES/ THF solution. The reduction of Mn(III)-TPP-p-CF3 in the presence of ascorbic acid is slowly, but fully reversed in the presence of air oxygen, as monitored by UV-Vis spectrometry and 19 F NMR. The broad 1 H and 19 F NMR signals of Mn(III)-TPP-p-CF3 disappear in the presence of 1 equivalent ascorbate replaced by a shifted and broadened 19 F NMR signal from Mn(II)-TPP-p-CF3 . Phantom 19 F MR images in DMSO show a MRI signal intensity decrease upon reduction of Mn(III)-TPP-p-CF3 , retrieved upon complete reoxidation in air within ~24 h. 1 H NMRD curves of the Mn(III)/(II)-TPP-p-CF3 chelates in mixed DMSO/water solvent have the typical shape of Mn(II)/Mn(III) porphyrins.

Alterations promoted by acid straightening and/or bleaching in hair microstructures.

Human hair is a biopolymer constituted mainly of keratin intermediate filaments, lipids, pigments and water. Cosmetic treatments usually interact with the hair at the molecular level, inducing changes in its components and modifying the physicochemical and mechanical properties of the fibers. Here, the effect of acid straightening on the morphology and ultrastructure of Caucasian hair was investigated by a group of complementary experimental methods: wide-, small- and ultra-small-angle X-ray scattering; high-resolution 3D X-ray microscopy; quasi-elastic neutron scattering and inelastic neutron scattering; thermogravimetry-mass spectrometry; and differential scanning calorimetry (DSC). X-ray diffraction patterns showed that acid straightening associated with a flat iron (∼180°C) changed the cortex of the fiber, shown by denaturation of the intermediate filaments (measured by DSC). The increase in the spacing of the lipid layers and the observation of the dehydration behavior of the fiber provided indications that water may be confined between these layers, while neutron spectroscopy showed alterations in the vibration mode of the CH2 groups of the lipids and an increase of the proton (H+) mobility in the hair structure. The latter may be associated with the extremely low pH of the formulation (pH ≃ 1). Additionally, this investigation showed that bleached hair (one-time bleached) is more damaged by the action of acid straightening than virgin hair, which was shown by a threefold increase in the percentage of total porosity of the tresses. The obtained results demonstrate that the investigation approach proposed here can provide very important thermodynamic and structural information on induced changes of hair structure, and certainly can be applied for the evaluation of the action mode and efficiency of cosmetic treatments.

Effectiveness of adhesive containing MDPB: A practice-based clinical trial.

OBJECTIVES: This prospective practice-based trial assessed the longevity of composite restorations made with an adhesive containing an antibacterial monomer compared to a conventional adhesive. METHODS: 9 general practices in the Netherlands were provided with two composite resin adhesives, each for a period of 9 months. Adhesive P contained the quaternary ammonium salt MDPB, and Adhesive S was a control. Patient's age and caries risk, as well as tooth type/number, reason for restoration placement, used restorative material and adhesive, and restored surfaces were recorded. All interventions carried out on these teeth in the 6 years after restoration were extracted from the electronic patient records, along with their date, type, reason, and surfaces. Two dependent variables were defined: general failure, and failure due to secondary caries. All data handling and multiple Cox regression analysis were carried out in R 4.0.5. RESULTS: 11 dentists from 7 practices made 10,151 restorations over a period of two years in 5102 patients. 4591 restorations were made with adhesive P, whereas 5560 were made with adhesive S. The observation period was up to 6.29 years, median observation time was 3.74 years. Cox regression showed no significant difference between the two adhesive materials when corrected for age, tooth type and caries risk, for general failure nor failure due to caries. SIGNIFICANCE: No difference in restoration survival could be shown between composite restorations made using an adhesive containing MDPB and control. Restorations made with the adhesive containing MDPB also did not fail more or less frequently due to secondary caries. This trial is registered on clinicaltrials.gov with identifier NCT05118100.

Clinical standards for the management of adverse effects during treatment for TB.

BACKGROUND: Adverse effects (AE) to TB treatment cause morbidity, mortality and treatment interruption. The aim of these clinical standards is to encourage best practise for the diagnosis and management of AE.METHODS: 65/81 invited experts participated in a Delphi process using a 5-point Likert scale to score draft standards.RESULTS: We identified eight clinical standards. Each person commencing treatment for TB should: Standard 1, be counselled regarding AE before and during treatment; Standard 2, be evaluated for factors that might increase AE risk with regular review to actively identify and manage these; Standard 3, when AE occur, carefully assessed and possible allergic or hypersensitivity reactions considered; Standard 4, receive appropriate care to minimise morbidity and mortality associated with AE; Standard 5, be restarted on TB drugs after a serious AE according to a standardised protocol that includes active drug safety monitoring. In addition: Standard 6, healthcare workers should be trained on AE including how to counsel people undertaking TB treatment, as well as active AE monitoring and management; Standard 7, there should be active AE monitoring and reporting for all new TB drugs and regimens; and Standard 8, knowledge gaps identified from active AE monitoring should be systematically addressed through clinical research.CONCLUSION: These standards provide a person-centred, consensus-based approach to minimise the impact of AE during TB treatment.

Pre-estrus progesterone does not affect post-estrus luminal metabolome in cross-bred beef cows.

In brief: The concentration of progesterone through the estrous cycle modulates uterine function to affect the luminal metabolome. This paper reports that the dynamic changes in the bovine uterine luminal metabolome during diestrus are independent of the concentration of progesterone in the previous cycle. Abstract: In cattle, the concentration of sex steroids modulates uterine function, which is reflected in the composition of the luminal metabolome. Ultimately, the uterine luminal metabolome influences embryonic growth and development. Our objectives were (i) to compare the luminal metabolome 4, 7, and 14 days after estrus of cows that were exposed to greater (HP4; n = 16) vs lower (LP4; n = 24) concentrations of progesterone before displaying estrus and ovulating spontaneously and (ii) to identify changes in the luminal concentration of metabolites across these time points. Luminal epithelial cells and fluid were collected using a cytology brush, and gene expression and metabolite concentrations were assessed by RNAseq and targeted mass spectrometry, respectively. Metabolome profile was similar between treatments within each of days 4, 7, and 14 (false discovery rate (FDR): ≥ 0.1). Concentrations of 53 metabolites changed, independent of treatment, across the diestrus. Metabolites were mostly lipids (40 out 53) and the greatest concentrations were at day 14 (FDR: ≤ 0.1). On day 7, the concentration of putrescine and the gene expression of ODC1, PAOX, SLC3A2, and SAT1 increased (P ≤ 0.05). On day 14, the concentration of 3 ceramides, 4 glucosylceramides, and 12 sphingomyelins and the expression of SGMS2 were increased, in addition to the concentration of choline and 20 phosphatidylcholines. Collectively, the post-estrus concentration of luminal metabolites changed dynamically, independent of the concentration of sex steroids on the previous cycle, and the greatest magnitude changes were on day 14 when lipid metabolism was the most enriched pathway.

Vulnerable newborn types: analysis of subnational, population-based birth cohorts for 541 285 live births in 23 countries, 2000-2021.

OBJECTIVE: To examine prevalence of novel newborn types among 541 285 live births in 23 countries from 2000 to 2021. DESIGN: Descriptive multi-country secondary data analysis. SETTING: Subnational, population-based birth cohort studies (n = 45) in 23 low- and middle-income countries (LMICs) spanning 2000-2021. POPULATION: Liveborn infants. METHODS: Subnational, population-based studies with high-quality birth outcome data from LMICs were invited to join the Vulnerable Newborn Measurement Collaboration. We defined distinct newborn types using gestational age (preterm [PT], term [T]), birthweight for gestational age using INTERGROWTH-21st standards (small for gestational age [SGA], appropriate for gestational age [AGA] or large for gestational age [LGA]), and birthweight (low birthweight, LBW [<2500 g], nonLBW) as ten types (using all three outcomes), six types (by excluding the birthweight categorisation), and four types (by collapsing the AGA and LGA categories). We defined small types as those with at least one classification of LBW, PT or SGA. We presented study characteristics, participant characteristics, data missingness, and prevalence of newborn types by region and study. RESULTS: Among 541 285 live births, 476 939 (88.1%) had non-missing and plausible values for gestational age, birthweight and sex required to construct the newborn types. The median prevalences of ten types across studies were T+AGA+nonLBW (58.0%), T+LGA+nonLBW (3.3%), T+AGA+LBW (0.5%), T+SGA+nonLBW (14.2%), T+SGA+LBW (7.1%), PT+LGA+nonLBW (1.6%), PT+LGA+LBW (0.2%), PT+AGA+nonLBW (3.7%), PT+AGA+LBW (3.6%) and PT+SGA+LBW (1.0%). The median prevalence of small types (six types, 37.6%) varied across studies and within regions and was higher in Southern Asia (52.4%) than in Sub-Saharan Africa (34.9%). CONCLUSIONS: Further investigation is needed to describe the mortality risks associated with newborn types and understand the implications of this framework for local targeting of interventions to prevent adverse pregnancy outcomes in LMICs.

Hormonal profile prior to luteolysis modulates the uterine luminal transcriptome in the subsequent cycle in beef cross-bred cows†.

Sex steroid concentrations modulate endometrial function and fertility in cattle. Our objective was to compare the post-estrus luminal transcriptome of cows that were exposed to contrasting concentrations of progesterone (P4) before luteolysis that displayed estrus and ovulated spontaneously. Cross-bred beef cows received either (1) a new CIDR and GnRH (day -9; high progesterone treatment; HP4; n = 16) or (2) a previously used CIDR, PGF2α, and GnRH (low progesterone treatment; LP4; n = 24). All cows received PGF2α at CIDR removal (day -2). Ovarian ultrasonography and blood collections were performed on days -9, -2, -0.5, and 0 (day of observed estrus), and days 4, 7, and 14 for measurement of ovarian structures, P4, and estradiol (E2). Luminal epithelial cells were collected using a cytology brush on days 4, 7, and 14 for RNAseq. On day -2, CL area and concentrations of P4 were greater, while on day -0.5, concentrations of E2 were decreased in HP4. Ovarian structures and hormonal concentrations were similar on days 4, 7, or 14 (P > 0.05). There were enriched pathways in HP4 related to activation and signaling of the innate immune system at day 4, downregulation in the network involved in the extracellular matrix remodeling at day 7, and exacerbated inflammatory response as well as differentiation and activation of macrophages at day 14 (Benjamini-Hochberg P-value ≤ 0.05). In conclusion, manipulation of pre-luteolysis sex steroid concentrations altered the post-estrus luminal transcriptome even though all cows showed estrus and ovulated spontaneously.

Drilling down the bioequivalence assessment of topical antifungal products: Microstructure and release.

In recent years, the regulatory mechanisms for topical generic product bioequivalence (BE) assessment have been subjected to noteworthy changes, with the FDA issuing product specific guidances, and the EMA adopting a more universal approach with the quality and equivalence of topical products draft guideline. The agencies advise on a modular strategy for BE documentation. Nevertheless, their scope, data analysis and criteria are rather distinct. This study aims to tackle bioequivalence assessment issues of complex topical formulations starting by statistical implications of the EMA/FDA approaches concerning the documentation of qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance requirements (Q4). As a model drug product, a bifonazole 10 mg/g cream formulation was selected. For this specific formulation, the commercially available Reference Product (RP) was compared with two comparator products, also commercially available, referred to as comparator product A (CPA) and comparator product B (CPB). The former displays Q1 sameness and Q2 differences, whilst CPB is categorically considered as Q1/Q2 different. Furthermore, intending to establish a regulatory rationale for the submission of a generic product according to the updated regulatory requirements, the RP was likewise compared with a Test Product (TP). This formulation was designed to display equal Q1/Q2 profile to that of the RP. Validated rheology and in vitro release test (IVRT) methods were used to infer on Q3/Q4 characteristics. During rheology studies, statistically significant RP batch to batch differences were observed. Therefore, in an attempt to surpass this heterogeneity, the initial pool of RP batches was expanded to include RP product batches at different lifecycle stages. Despite this effort, it was not possible to classify the RP/TP, RP/CPA or RP/CPB as rheologically equivalent products. Nevertheless, product performance results, retrieved from IVRT, were able to sustain equivalence between the RP and the formulations exhibiting Q1 sameness (TP and CPA). It should however be mentioned, that for some RP batch combinations, the IVRT results failed to demonstrate equivalence according to the EMA requirements. Enlarging the RP batch pool was then a critical step in further understanding an optimum statistical approach for establishing equivalence in product performance. This study highlights the need to that a 'one-fits-all approach' may not be an optimum path way for establishing the regulatory strategy and requirements to support generic product bioequivalence.