Extremophile enzyme activity lab: using catalase from Pyrobaculum calidifontis to highlight temperature sensitivity and thermostable enzyme activity.

There are places on earth that are considered to possess extreme physico-chemical characteristics as they relate to life. Surprisingly, there are microbes that have adapted various strategies that enable them to form robust communities in these environments. The microbes that live in these environments, called extremophiles, are described as being thermophilic, psychrophilic, halophilic, acidophilic, alkaliphilic, barophilic, and so on. Given that extremophiles were not discovered until relatively recently due to a view point that the environments in which they inhabited were not conducive to life, it is reasonable to conclude that the concept of extremophiles may be hard to grasp for students. Herein is described a laboratory exercise adapted from laboratory exercises that use mesophilic catalase enzymes to illustrate the influence of physico-chemical parameters on enzyme activity. Catalase is an enzyme that accelerates the degradation of hydrogen peroxide to water and oxygen gas. In addition to mesophilic catalases, the catalase from Pyrobaculum calidifontis, a hyperthermophile with an optimal growth temperature of 90°C, is used to highlight the adaptation of an enzyme to an extreme environment. A visual comparison of bubble production by the hyperthermophilic and mesophilic enzymes after heating at high temperatures dramatically illustrates differences in thermostability that will likely reinforce concepts that are given in a pre-laboratory lecture that discusses not only the extremophiles themselves but also their applications in biotechnology and possible role in the field of astrobiology.

Reversible O-H Bond Activation by Tripodal tris(Nitroxide) Aluminum and Gallium Complexes.

Herein, we report the preparation and characterization of the Group 13 metal complexes of a tripodal tris(nitroxide)-based ligand, designated (TriNOx3-)M (M = Al (1), Ga (2), In (3)). Complexes 1 and 2 both activate the O-H bond of a range of alcohols spanning a ∼10 pKa unit range via an element-ligand cooperative pathway to afford the zwitterionic complexes (HTriNOx2-)M-OR. Structures of these alcohol adduct products are discussed. We demonstrate that the thermodynamic and kinetic aspects of the reactions are both influenced by the identity of the metal, with 1 having higher reaction equilibrium constants and proceeding at a faster rate relative to 2 for any given alcohol. These parameters are also influenced by the pKa of the alcohol, with more acidic alcohols reacting both to more completion and faster than their less acidic counterparts. Possible mechanistic pathways for the O-H activation are discussed.

Combining Donor Strength and Oxidative Stability in Scorpionates: A Strongly Donating Fluorinated Mesoionic Tris(imidazol-5-ylidene)borate Ligand.

Strongly donating scorpionate ligands support the study of high-valent transition metal chemistry; however, their use is frequently limited by oxidative degradation. To address this concern, we report the synthesis of a tris(imidazol-5-ylidene)borate ligand featuring trifluoromethyl groups surrounding its coordination pocket. This ligand represents the first example of a chelating poly(imidazol-5-ylidene) mesoionic carbene ligand, a scaffold that is expected to be extremely donating. The {NiNO}10 complex of this ligand, as well as that of a previously reported strongly donating tris(imidazol-2-ylidene)borate, has been synthesized and characterized. This new ligand's strong donor properties, as measured by the υNO of its {NiNO}10 complex and natural bonding orbital second-order perturbative energy analysis, are at par with those of the well-studied alkyl-substituted tris(imidazol-2-ylidene)borates, which are known to effectively stabilize high-valent intermediates. The good donor properties of this ligand, despite the electron-withdrawing trifluoromethyl substituents, arise from the strongly donating imidazol-5-ylidene mesoionic carbene arms. These donor properties, when combined with the robustness of trifluoromethyl groups toward oxidative decomposition, suggest this ligand scaffold will be a useful platform in the study of oxidizing high-valent transition-metal species.

Supporting adolescents' participation in muscle-strengthening physical activity: protocol for the 'Resistance Training for Teens' (RT4T) hybrid type III implementation-effectiveness trial.

INTRODUCTION: In Australia, only 22% of male and 8% of female adolescents meet the muscle-strengthening physical activity guidelines, and few school-based interventions support participation in resistance training (RT). After promising findings from our effectiveness trial, we conducted a state-wide dissemination of the 'Resistance Training for Teens' (RT4T) intervention from 2015 to 2020. Despite high estimated reach, we found considerable variability in programme delivery and teachers reported numerous barriers to implementation. Supporting schools when they first adopt evidence-based programmes may strengthen programme fidelity, sustainability, and by extension, programme impact. However, the most effective implementation support model for RT4T is unclear. OBJECTIVE: To compare the effects of three implementation support models on the reach (primary outcome), dose delivered, fidelity, sustainability, impact and cost of RT4T. METHODS AND ANALYSIS: We will conduct a hybrid type III implementation-effectiveness trial involving grade 9 and 10 (aged 14-16 years) students from 90 secondary schools in New South Wales (NSW), Australia. Schools will be recruited across one cohort in 2023, stratified by school type, socioeconomic status and location, and randomised in a 1:1:1 ratio to receive one of the following levels of implementation support: (1) 'low' (training and resources), (2) 'moderate' (training and resources+external support) or 'high' (training and resources+external support+equipment). Training includes a teacher workshop related to RT4T programme content (theory and practical sessions) and the related resources. Additional support will be provided by trained project officers from five local health districts. Equipment will consist of a pack of semiportable RT equipment (ie, weighted bars, dumbbells, resistance bands and inverted pull up bar stands) valued at ~$A1000 per school. Study outcomes will be assessed at baseline (T0), 6 months (T1) and 18 months (T2). A range of quantitative (teacher logs, observations and teacher surveys) and qualitative (semistructured interviews with teachers) methods will be used to assess primary (reach) and secondary outcomes (dose delivered, fidelity, sustainability, impact and cost of RT4T). Quantitative analyses will use logistic mixed models for dichotomous outcomes, and ordinal or linear mixed effects regression models for continuous outcomes, with alpha levels set at p<0.025 for the outcomes and cost comparisons of the moderate and high support arms against the low support arm. ETHICS AND DISSEMINATION: Ethics approval has been obtained from the University of Newcastle (H-2021-0418), the NSW Department of Education (SERAP:2022215), Hunter New England Human Research Ethics Committee (2023/ETH00052) and the Catholic Schools Office. The design, conduct and reporting will adhere to the Consolidated Standards of Reporting Trials statement, the Standards for Reporting Implementation Studies statement and the Template for Intervention Description and Replication checklist. Findings will be published in open access peer-reviewed journals, key stakeholders will be provided with a detailed report. We will support ongoing dissemination of RT4T in Australian schools via professional learning for teachers. TRIAL REGISTRATION NUMBER: ACTRN12622000861752.

Data-driven predictions of complex organic mixture permeation in polymer membranes.

Membrane-based organic solvent separations are rapidly emerging as a promising class of technologies for enhancing the energy efficiency of existing separation and purification systems. Polymeric membranes have shown promise in the fractionation or splitting of complex mixtures of organic molecules such as crude oil. Determining the separation performance of a polymer membrane when challenged with a complex mixture has thus far occurred in an ad hoc manner, and methods to predict the performance based on mixture composition and polymer chemistry are unavailable. Here, we combine physics-informed machine learning algorithms (ML) and mass transport simulations to create an integrated predictive model for the separation of complex mixtures containing up to 400 components via any arbitrary linear polymer membrane. We experimentally demonstrate the effectiveness of the model by predicting the separation of two crude oils within 6-7% of the measurements. Integration of ML predictors of diffusion and sorption properties of molecules with transport simulators enables for the rapid screening of polymer membranes prior to physical experimentation for the separation of complex liquid mixtures.

The International Framework for School Health Promotion: Supporting Young People Through and After the COVID-19 Pandemic.

BACKGROUND: The worldwide COVID-19 government restrictions imposed on young people to limit virus spread have precipitated a growing and long-term educational and health crisis. CONTRIBUTIONS TO THE THEORY: This novel study used Sen's Capabilities Approach as a theoretical framework to examine the current health and educational impacts of COVID-19 on youth, referencing emerging literature. The objective was to inform the design of an internationally relevant framework for school health promotion to support young people through and after the COVID-19 pandemic. Mapping of existing health resources, internal/external conversion factors and capabilities were used to identify classroom, school and system level strategies that will enable young people to flourish. Four central enablers were identified and used in the design of the International Framework for School Health Promotion (IFSHP). IMPLICATIONS FOR SCHOOL HEALTH POLICY, PRACTICE AND EQUITY: The IFSHP can be used by educational institutions, school leaders and teachers to innovate existing health promotion programs, policies and practices to support young people through and after the COVID-19 pandemic. CONCLUSIONS: School systems, schools and teachers are encouraged to utilize the IFSHP to review and innovate existing school health programs to ensure they meet the increased physical and mental health needs of young people.

Sleep health of young adults in Western Australia and associations with physical and mental health: A population-level cross-sectional study.

OBJECTIVES: This article aims to report on the sleep health characteristics of a population-level sample of young Australian adults and examine associations with measures of physical and mental health. METHODS: A cross-sectional study using data from the Raine Study. Data from participants (n = 1234) born into the study (Generation 2) at the 22-year follow-up were used, including data from a self-report questionnaire and polysomnography. RESULTS: The highest prevalence of suboptimal sleep health was seen on measures of sleep duration (30%), onset latency (18%), satisfaction (25%) and regularity (60%). Dissatisfaction with sleep (physical health: ß =0.08; mental health: ß =0.34) and impaired daytime alertness (physical health: ß =0.09; mental health: ß =0.08) were significantly associated with poorer physical and mental health and inadequate polysomnography-measured sleep duration was associated poorer mental health (ß =0.07) (all ps<0.05). CONCLUSIONS: Satisfaction with sleep and daytime alertness, both of which are assessed via self-report, are essential aspects of sleep health for young adults. IMPLICATIONS FOR PUBLIC HEALTH: Findings could inform public health interventions, including screening guidelines, to improve the sleep health and, in turn, the physical and mental health of young adults in Australia.

Coexistence of psoriasis and cutaneous T-cell lymphoma.

It has been reported that individuals with psoriasis are at an increased risk of developing cutaneous T-cell lymphoma (CTCL). However, the increased risk of lymphoma in these patients has been questioned because CTCL in its early stages may be incorrectly labelled as psoriasis, thus introducing potential for misclassification bias. We retrospectively reviewed patients with a confirmed diagnosis of CTCL seen in a tertiary cutaneous lymphoma clinic (n = 115) over a 5-year period and found that 6 (5.2%) patients had clinical evidence of coexisting psoriasis. This demonstrates that there is a small cohort of individuals who develop both psoriasis and CTCL.

An individualized approach to teaching adults with autism to successfully navigate job interviews via remote instruction.

Adults with autism spectrum disorder (ASD) experience challenges securing employment, which may partially explain overall underemployment or unemployment in this population. One of the first steps to obtaining employment is participating in a job interview. However, social communication deficits may interfere with an individual with ASD's participation in a job interview. The current study evaluated the use of behavioral skills training delivered via remote instruction to teach interview skills to seven adults with ASD. Results showed overall improvement during interviews as well as posttraining tests with a career development expert. These data suggest that an individualized approach to teaching may be an effective strategy to help adults with ASD successfully navigate job interviews.

A novel skin cancer prevention strategy: Preservice teachers' perceptions of a sun safety intervention and experiences in schools.

ISSUE ADDRESSED: Teachers play a vital role in developing children's sun protection routines however upskilling preservice teachers (PSTs) while at university has not yet been trialled as a targeted skin cancer prevention strategy. Hence, this study investigated PSTs perceptions and experiences of sun safety following a brief pilot intervention and placement in primary schools in Western Australia. METHODS: This study used a triangulation mixed methods design. Participants (n = 161) completed a post intervention survey which was analysed quantitatively. A random sub-sample was invited to participate in focus groups (three groups, n = 21) and one-on-one interviews (n = 4). This data was transcribed and uploaded in NVIVO software for thematic analysis. RESULTS: Participants felt the intervention increased their awareness of the dangers of overexposure to ultraviolet radiation (UVR) with many feeling more knowledgeable, skilled and confident to teach sun safety in school settings. Most reported clear sun safety messages in their placement schools. However, only 34.4% reported they had been briefed on the school's sun safety procedures. There was consensus among PSTs that sun protection in primary schools needs to be improved to maximise the protection of children from harmful UVR overexposure. Participants supported a need for consistent sun protection messaging across primary schools with greater emphasis on education rather than compliance management to sun protection. CONCLUSION: Enhancing existing teacher education programs to include more rigorous curriculum content and pedagogical approaches to sun protection education is a novel skin cancer prevention strategy and could feasibly support PSTs self-efficacy to effectively deliver sun safety curriculum in Australian schools.

The impact of a rub and rinse regimen on removal of human coronaviruses from contemporary contact lens materials.

PURPOSE: To assess the influence of contemporary contact lens (CL) materials on human coronavirus attachment and the influence of a rub and rinse step to remove these viruses. METHODS: The binding rates of HCoV-229E and HCoV-OC43 to eight soft CL materials and four rigid gas permeable materials were analyzed. The impact of a rub and rinse step to remove these viruses from all materials was examined. The efficacy of Biotrue (Bausch & Lomb), OPTI-FREE Puremoist (Alcon), Clear Care (Alcon) and cleadew (Ophtecs) to remove virus contamination from two representative soft lens materials (etafilcon A and lotrafilcon B) was also determined. RESULTS: Approximately 102 to 103 infectious viral particles were recovered from each CL material. Although some materials were more prone to coronavirus adhesion, contamination of both viral types was reduced to below the limit of quantification (LQ) from all materials using a simple saline rinse step. Exposure to Clear Care and cleadew reduced the number of infectious viral particles from both etafilcon A and lotrafilcon B to below the LQ, while for Biotrue and OPTI-FREE Puremoist, infectious viral particles were reduced to below the LQ only when additional rub and rinse steps were included. CONCLUSION: Human coronavirus contamination can be easily removed from CL surfaces. Although CL care products containing hydrogen peroxide and povidone-iodine efficiently removed virus contamination from CL surfaces without the need for a rub and rinse step, a full regimen including rub and rinse steps is crucial when using CL care products based on non-oxidative systems.

Antiviral Activity of Contemporary Contact Lens Care Solutions against Two Human Seasonal Coronavirus Strains.

Background: Given that reports have suggested SARS-CoV-2 can be transmitted via conjunctiva, the ability of contact lens (CL) care products to reduce the infectiousness of two seasonal human coronavirus (HCoV) (HCoV-229E and HCoV-OC43) surrogates for SARS-CoV-2 was investigated. Methods: Biotrue and Boston Simplus (Bausch&Lomb), OPTI-FREE Puremoist and Clear Care (Alcon), and cleadew and cleadew GP (Ophtecs) were tested. Their ability to inactivate HCoV was evaluated using contact times of 4 and 6 h as well as 1% and 10% of virus inoculum. Results: Non-oxidative systems (Biotrue, Boston Simplus, and OPTI-FREE) did not exhibit a significant log10 reduction compared to controls for the two viral strains for either incubation time (all p > 0.05) when 10% tests were performed. For the 1% test, while Boston Simplus and OPTI-FREE exhibited a significant log10 reduction of both HCoV-229E (after 6 h) and HCoV-OC43 (after either 4 or 6 h incubation), those products showed less than 1 log10 reduction of the two infectious viruses. Oxidative systems based on hydrogen peroxide or povidone-iodine showed a significant log10 reduction compared with the controls for both HCoV-229E and HCoV-OC43 in all tested conditions (all p < 0.01). Clear Care led to virus inactivation to below the limit of quantification for tests performed with 1% of inoculum after 6 h incubation, while cleadew and cleadew GP led to inactivation of the two viruses to below the limit of quantification in all tested conditions. Conclusion: Oxidative CL disinfection systems showed significant virucidal activity against HCoV-229E and HCoV-OC43, while non-oxidative systems showed minimal ability to inactivate the HCoV species examined.

Association Between Androgen Deprivation Therapy and Mortality Among Patients With Prostate Cancer and COVID-19.

Importance: Androgen deprivation therapy (ADT) has been theorized to decrease the severity of SARS-CoV-2 infection in patients with prostate cancer owing to a potential decrease in the tissue-based expression of the SARS-CoV-2 coreceptor transmembrane protease, serine 2 (TMPRSS2). Objective: To examine whether ADT is associated with a decreased rate of 30-day mortality from SARS-CoV-2 infection among patients with prostate cancer. Design, Setting, and Participants: This cohort study analyzed patient data recorded in the COVID-19 and Cancer Consortium registry between March 17, 2020, and February 11, 2021. The consortium maintains a centralized multi-institution registry of patients with a current or past diagnosis of cancer who developed COVID-19. Data were collected and managed using REDCap software hosted at Vanderbilt University Medical Center in Nashville, Tennessee. Initially, 1228 patients aged 18 years or older with prostate cancer listed as their primary malignant neoplasm were included; 122 patients with a second malignant neoplasm, insufficient follow-up, or low-quality data were excluded. Propensity matching was performed using the nearest-neighbor method with a 1:3 ratio of treated units to control units, adjusted for age, body mass index, race and ethnicity, Eastern Cooperative Oncology Group performance status score, smoking status, comorbidities (cardiovascular, pulmonary, kidney disease, and diabetes), cancer status, baseline steroid use, COVID-19 treatment, and presence of metastatic disease. Exposures: Androgen deprivation therapy use was defined as prior bilateral orchiectomy or pharmacologic ADT administered within the prior 3 months of presentation with COVID-19. Main Outcomes and Measures: The primary outcome was the rate of all-cause 30-day mortality after COVID-19 diagnosis for patients receiving ADT compared with patients not receiving ADT after propensity matching. Results: After exclusions, 1106 patients with prostate cancer (before propensity score matching: median age, 73 years [IQR, 65-79 years]; 561 (51%) self-identified as non-Hispanic White) were included for analysis. Of these patients, 477 were included for propensity score matching (169 who received ADT and 308 who did not receive ADT). After propensity matching, there was no significant difference in the primary end point of the rate of all-cause 30-day mortality (OR, 0.77; 95% CI, 0.42-1.42). Conclusions and Relevance: Findings from this cohort study suggest that ADT use was not associated with decreased mortality from SARS-CoV-2 infection. However, large ongoing clinical trials will provide further evidence on the role of ADT or other androgen-targeted therapies in reducing COVID-19 infection severity.

Investigating primary preservice teachers' ultraviolet radiation awareness and perceived ability to teach sun safety.

ISSUE ADDRESSED: Sun protection practices in Australian primary schools remain inconsistent. Therefore, this study investigates primary PSTs sun protective sun behaviours, ultraviolet (UV) radiation awareness and perceived ability to teach sun safety. METHODS: A convenience sample of undergraduate PSTs (N = 275; mean age = 23.13 years) enrolled at one Western Australian university completed an online survey. Descriptive analyses provided features of the data. Factors associated with sun protection behaviours and perceived knowledge and skill to teach sun safety were explored using multivariable logistic regression models. RESULTS: Lesser than 10% of participants reported using sun protective measures daily (midday shade use: 6.5%; sunscreen: 7.6%; hat: 4.4%). Only 56.3% reported they understand the UV index, with 68.0% rarely/never using it to aid sun protection. Under half the participants reported they felt they had the knowledge (38.5%) or skills (40%) to effectively teach sun safety in primary schools. Regression analysis revealed gender, undergraduate, year and skin sensitivity were not predictors of UV index use (P > .05) or perceived knowledge of sun safety (P > .05). Skin sensitivity was the strongest predictor for shade usage (P = .02), hat usage (P = .05) and perceived skill to teach sun safety (P = .02). CONCLUSIONS: Survey data indicate UV radiation is inconsistently understood by PSTs. Many felt that they did not have the required knowledge or skill to teach sun safety effectively. SO WHAT?: Improving PSTs UV radiation knowledge while at university is a potential opportunity to improve sun safety delivery in primary schools. A targeted intervention for PSTs is warranted.

Incidence of uveitis and macular edema among patients taking fingolimod 0.5 mg for multiple sclerosis.

BACKGROUND: Patients with multiple sclerosis (MS) have a higher incidence of uveitis compared with the general population. Fingolimod, a first line disease modifying drug used in multiple sclerosis, may cause macular edema and thus requires ophthalmic examination. However, murine models and anecdotal reports suggest fingolimod may reduce the incidence of uveitis. PURPOSE: To report the incidence of uveitis and macular edema among those on fingolimod 0.5 mg (Gilenya®) therapy for multiple sclerosis (MS). METHODS: Retrospective review of patients on fingolimod who developed uveitis and/or macular edema. RESULTS: No patients had an occurrence or history of uveitis. Four of the 188 (2.13%) patients developed macular edema without ocular inflammation. One of the 188 (0.53%) patients developed Acute Macular Neuroretinopathy. CONCLUSION: Patients taking fingolimod have a lower incidence of uveitis than expected in a population of MS patients.

The Use of Procalcitonin as a Sepsis Marker in a Community Hospital.

BACKGROUND: Procalcitonin (PCT) is a biomarker that shows good sensitivity and specificity in identifying septic patients. METHODS: This study investigated the diagnostic accuracy of PCT in a community hospital setting and how it compared to that of lactic acid. It explored the impact on patient care before and after PCT implementation regarding costs and length of stay. Two comparative groups were analyzed using an exploratory descriptive case-control study with data from a 19-month period after PCT implementation and a retrospective quasi-experimental study using a control group of emergency department patients diagnosed with sepsis using data before PCT implementation. RESULTS: Post-procalcitonin implementation samples included 165 cases and pre-procalcitonin implementation sample included 69 cases. From the 165 sepsis cases who had positive blood cultures, PCT had a sensitivity of 89.7%. In comparison, lactic acid's sensitivity at the current cutoff of 18.02 mg/dL (2.0 mmol/L) was 64.9%. There was a 32% decrease in median cost before and after PCT implementation, even with the length of stay remaining at 5 days in both time periods. CONCLUSIONS: There was a significant decrease after the implementation of PCT in cost of hospitalization compared to costs before implementation. This cost is highly correlated with length of stay; neither the hospital nor the intensive care unit length of stay showed a difference with before and after implementation. There was a positive correlation between lactic acid and PCT values. PCT values had a higher predictive usefulness than the lactic acid values.

Multinational comparative cross-sectional survey of views of medical students about acceptable terminology and subgroups in schizophrenia.

AIM: The aim of this study was to inform thinking around the terminology for 'schizophrenia' in different countries. OBJECTIVES: The objective of this study was to investigate: (1) whether medical students view alternative terminology (psychosis subgroups), derived from vulnerability-stress models of schizophrenia, as acceptable and less stigmatising than the term schizophrenia; (2) if there are differences in attitudes to the different terminology across countries with different cultures and (3) whether clinical training has an impact in reducing stigma. DESIGN: This is a cross-sectional survey that examined the attitudes of medical students towards schizophrenia and the alternative subgroups. SETTING: The study was conducted across eight sites: (1) University of Southampton, UK; (2) All India Institute of Medical Science, India; (3) Rowan University, USA; (4) Peshawar Medical College, Pakistan; (5) Capital Medical University, China; (6) College of Medicine and Medical sciences, Bahrain; (7) Queens University, Kingston, Canada and (8) University of Cape Town, South Africa. METHOD: This study extended an initial pilot conducted by the Royal College of Psychiatrists on the term schizophrenia and psychosis subgroups to assess whether the subgroup terminology might have an effect on the attitudes of a convenience sample of medical students from eight different countries and potentially play a role in reducing stigmatisation. RESULTS: 1873 medical students completed a questionnaire recording their attitudes to schizophrenia and the psychosis subgroups. A reduction in negative perceptions were found for the psychosis subgroups, especially for the stress sensitivity psychosis and anxiety psychosis subgroups. Negative perceptions were found for drug-related psychosis. Participants who had undergone clinical training had overall positive attitudes. Differences across different countries were found. CONCLUSION: The attitudes towards psychosis subgroups used in this study have shown mixed results and variation across countries. Further research is warranted to investigate acceptability of terminology. Methods of reducing stigma are discussed in line with the findings. ETHICS: The study received ethical approval from ERGO (Ethics and Research Governance Online; ID: 15972) and subsequently from the ethics committee at each site.

Discovery of Melanocortin Ligands via a Double Simultaneous Substitution Strategy Based on the Ac-His-dPhe-Arg-Trp-NH2 Template.

The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His1-dPhe2-Arg3-Trp4-NH2 tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1-5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His1-dPhe2-Arg3-Trp4-NH2 tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4, as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at positions 1 and 2, (C) double simultaneous substitution at positions 1 and 4, and (D) double simultaneous substitution at positions 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency or selectivity or converting agonists into antagonists and vice versa.