Correlating the Diffusion of Water to Performance in Model Reverse Osmosis Polyamides with Controlled Crosslink Densities.

We systematically reduce the cross-link density of a PA network based on m-phenylene diamine by substituting a fraction of the trifunctional trimesoyl chloride cross-linking agent with a difunctional isophthaloyl analog that promotes chain extension, in order to elucidate robust design cues for improving the polyamide (PA) separation layer in reverse osmosis (RO) membranes for desalination. Thin films of these model PA networks are fully integrated into a composite membrane and evaluated in terms of their water flux and salt rejection. By incorporating 15 mol % of the difunctional chain extender, we reduce the cross-link density of the network by a factor of two, which leads to an 80 % increase in the free or unreacted amine content. The resulting swelling of the PA network in liquid water increases by a factor of two accompanied by a 30 % increase in the salt passage through the membrane. Surprisingly, this leads to a 30 % decrease in the overall permeance of water through the membrane. This conundrum is resolved by quantifying the microscopic diffusion coefficient of water inside the PA network with quasi-elastic neutron scattering. In the highest and lowest cross-link density networks, water shows strong signatures of confined diffusion. At short length scales, the water exhibits a translational diffusion that is consistent with the jump-diffusion mechanism. This translational diffusion coefficient is approximately five times slower in the lowest cross-linked density network, consistent with the reduced water permeance. This is interpreted as water molecules interacting more strongly with the increased free amine content. Over longer length scales the water diffusion is confined, exhibiting mobility that is independent of length scale. The length scales of confinement from the quasi-elastic neutron scattering experiments at which this transition from confined to translational diffusion occurs is on the order of (5 to 6) Å, consistent with complementary X-ray scattering, small angle neutron scattering, and positron annihilation lifetime spectroscopy measurements. The confinement appears to come from heterogeneities in the average inter-atomic distances, suggesting that diffusion occurs by water bouncing between chains and occasionally sticking to the polar functional groups. The results obtained here are compared with similar studies of water diffusion through both rigid porous silicates and ion exchange membranes, revealing robust design cues for engineering high-performance RO membranes.

Bottlebrush polymers in the melt and polyelectrolytes in solution share common structural features.

Uncharged bottlebrush polymer melts and highly charged polyelectrolytes in solution exhibit correlation peaks in scattering measurements and simulations. Given the striking superficial similarities of these scattering features, there may be a deeper structural interrelationship in these chemically different classes of materials. Correspondingly, we constructed a library of isotopically labeled bottlebrush molecules and measured the bottlebrush correlation peak position [Formula: see text] by neutron scattering and in simulations. We find that the correlation length scales with the backbone concentration, [Formula: see text], in striking accord with the scaling of ξ with polymer concentration cP in semidilute polyelectrolyte solutions [Formula: see text] The bottlebrush correlation peak broadens with decreasing grafting density, similar to increasing salt concentration in polyelectrolyte solutions. ξ also scales with sidechain length to a power in the range of 0.35-0.44, suggesting that the sidechains are relatively collapsed in comparison to the bristlelike configurations often imagined for bottlebrush polymers.

Qualities of the dying experience of persons who access medical assistance in dying: A scoping review.

Defining a "good death" is complex and grounded in diverse cultural, social, and personal factors. Although there is a significant body of literature exploring the broad concepts of death and dying, there is a dearth in literature that has explored what constitutes a good death for persons undergoing assisted dying such as Medical Assistance in Dying (MAiD). In this scoping review of 19 articles, we explore dying experiences and what a good death entails for people accessing MAiD. Understanding personal values and ideas about positive dying experiences can guide patients, care partners, and clinicians in their preparation toward, and facilitation of, a good death experience particularly among persons who access MAiD.

Role of nanoparticle size and surface chemistry on ion transport and nanostructure of perfluorosulfonic acid ionomer nanocomposites.

Herein, we present a systematic investigation of the impact of silica nanoparticle (SiNP) size and surface chemistry on the nanoparticle dispersion state and the resulting morphology and vanadium ion permeability of the composite ionomer membranes. Specifically, Nafion containing a mass fraction of 5% silica particles, ranging in nominal diameters from 10 nm to >1 µm and with both sulfonic acid- and amine-functionalized surfaces, was fabricated. Most notably, an 80% reduction in vanadium ion permeability was observed for ionomer membranes containing amine-functionalized SiNPs at a nominal diameter of 200 nm. Further, these membranes exhibited an almost 400% increase in proton selectivity when compared to pristine Nafion. Trends in vanadium ion permeability within a particular nominal diameter were seen to be a function of the surface chemistry, where, for example, vanadyl ion permeability was observed to increase with increasing particle size for membranes containing unfunctionalized SiNPs, while it was seen to remain relatively constant for membranes containing amine-functionalized SiNPs. In general, the silica particles tended to exhibit a higher extent of aggregation as the size of the particles was increased. From small-angle neutron scattering experiments, an increase in the spacing of the hydrophobic domains was observed for all composite membranes, though particle size and surface chemistry were seen to have varying impacts on the spacing of the ionic domains of the ionomer.

Examining the Macrosystem Level of Influence on Community Health Worker Effectiveness in the State of Nebraska: A Qualitative Approach.

Community health workers (CHWs) serve as the linkage between community and providers and are stakeholders for bridging services to the public. However, integration of CHWs into health care organizations is often lacking. This study explored macrosystem level barriers faced by CHWs and their ability to do their jobs effectively. Using qualitative interviews from CHWs (n = 28) in Nebraska, we used an abductive approach to derive the following themes: (1) CHWs and client macrosystem barriers, (2) CHW workforce supports, and (3) macrosystem solutions for CHW workforce sustainability. Study results also found various macrosystem barriers affecting CHW workforces including immigration policies, insurance policies, funding sources, supervisor support, and obstacles for health seeking of clients. Moreover, through the lens of CHWs, results revealed the need to provide and advocate for solutions that prioritize the needs of CHWs as they continue to fill a crucial gap in community healthcare systems.

Effect of Carbohydrate Mouth Rinse on Resistance Exercise Performance.

ABSTRACT: Green, MS, Kimmel, CS, Martin, TD, Mouser, JG, and Brune, MP. Effect of carbohydrate mouth rinse on resistance exercise performance. J Strength Cond Res 36(7): 1916-1921, 2022-A carbohydrate mouth rinse (CMR) has been shown to enhance short duration endurance performance and raises the possibility that a similar strategy could improve performance during resistance exercise. Eighteen male and female (N = 36) resistance trained subjects (mean values ± SD; age: 21.5 ± 1.6 years, height: 1.72 ± 0.09 m, body mass: 72.8 ± 13.4 kg, and body fat: 16.7 ± 5.8%) performed 3 experimental visits during which bench press resistance exercise (4 × 10 repetitions at 65% of 1 repetition maximum [1RM] with 120 seconds recovery) and repetitions to failure at 60% 1RM were performed. Subjects rinsed 25 ml of water (WAT), noncaloric placebo (PLA), or 6.4% maltodextrin (CHO) solution for 10 seconds during exercise in a crossover, counter-balanced manner. Rating of perceived exertion (RPE), pleasure-displeasure (FS), number of repetitions to fatigue (REPS), and postexercise blood glucose (GLU) and lactate (LA) were measured. Compared to WAT (17.7 ± 0.8), PLA (19.0 ± 0.7; p = 0.025), and CHO (18.7 ± 0.8; p = 0.039) treatments resulted in higher REPS, with no difference between PLA and CHO treatments (p = 0.310). Rating of perceived exertion progressively increased each set (p < 0.0001), but was not affected by treatment (p = 0.897). Pleasure-displeasure declined during recovery from sets 3 and 4 (p < 0.05) but was also not affected by treatment (p = 0.692). Postexercise GLU (p = 0.103) and LA (p = 0.620) were not different between treatments. Although a placebo effect was present for REPS, this study failed to detect an effect of CMR on REPS, RPE, FS, GLU, or LA on upper-body resistance exercise.

Enhanced photocatalytic hydrogen evolution from organic semiconductor heterojunction nanoparticles.

Photocatalysts formed from a single organic semiconductor typically suffer from inefficient intrinsic charge generation, which leads to low photocatalytic activities. We demonstrate that incorporating a heterojunction between a donor polymer (PTB7-Th) and non-fullerene acceptor (EH-IDTBR) in organic nanoparticles (NPs) can result in hydrogen evolution photocatalysts with greatly enhanced photocatalytic activity. Control of the nanomorphology of these NPs was achieved by varying the stabilizing surfactant employed during NP fabrication, converting it from a core-shell structure to an intermixed donor/acceptor blend and increasing H2 evolution by an order of magnitude. The resulting photocatalysts display an unprecedentedly high H2 evolution rate of over 60,000 µmol h-1 g-1 under 350 to 800 nm illumination, and external quantum efficiencies over 6% in the region of maximum solar photon flux.

Nurtured in Nature: a Pilot Randomized Controlled Trial to Increase Time in Greenspace among Urban-Dwelling Postpartum Women.

Spending time in nature is associated with numerous mental health benefits, including reduced depression and improved well-being. However, few studies examine the most effective ways to nudge people to spend more time outside. Furthermore, the impact of spending time in nature has not been previously studied as a postpartum depression (PPD) prevention strategy. To fill these gaps, we developed and pilot tested Nurtured in Nature, a 4-week intervention leveraging a behavioral economics framework, and included a Nature Coach, digital nudges, and personalized goal feedback. We conducted a randomized controlled trial among postpartum women (n = 36) in Philadelphia, PA between 9/9/2019 and 3/27/2020. Nature visit frequency and duration was determined using GPS data. PPD was measured using the Edinburgh Postnatal Depression Scale (EPDS). Participants were from low-income, majority Black neighborhoods. Compared to control, the intervention arm had a strong trend toward longer duration and higher frequency of nature visits (IRR 2.6, 95%CI 0.96-2.75, p = 0.059). When analyzing women who completed the intervention (13 of 17 subjects), the intervention was associated with three times higher nature visits compared to control (IRR 3.1, 95%CI 1.16-3.14, p = 0.025). No significant differences were found in the EPDS scores, although we may have been limited by the study's sample size. Nurture in Nature increased the amount of time postpartum women spent in nature, and may be a useful population health tool to leverage the health benefits of nature in majority Black, low-resourced communities.

H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery.

The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a ΔH and a TΔS component. In the scenario in which TΔS completely compensates ΔH, no allosteric influence of effector binding on substrate affinity is observed. However, in this "silent coupling" scenario, the presence of effector causes a change in the ΔH associated with substrate binding. A suggestion has now been made that "silent modulators" are ideal drug leads because they can be modified to act as either allosteric activators or inhibitors. Any attempt to rationally design the effector to be an allosteric activator or inhibitor is likely to be benefitted by knowledge of the mechanism that gives rise to coupling. Hydrogen/deuterium exchange with mass spectrometry detection has now been used to identify regions of proteins that experience conformational and/or dynamic changes in the allosteric regulation. Here, we demonstrate the expected temperature dependence of the allosteric regulation of rabbit muscle pyruvate kinase by Ala to demonstrate that this effector reduces substrate (phosphoenolpyruvate) affinity at 35°C and at 10°C but is silent at intermediate temperatures. We then explore the use of hydrogen/deuterium exchange with mass spectrometry to evaluate the areas of the protein that are modified in the mechanism that gives rise to the silent coupling between Ala and phosphoenolpyruvate. Many of the peptide regions of the protein identified as changing in this silent system (Ala as the effector) were included in changes previously identified for allosteric inhibition by Phe.

Identification of biochemically neutral positions in liver pyruvate kinase.

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions-high sequence entropy, high surface exposure, and alanine scanning-lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a "neutrality" score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

Effect of Caffeine Supplementation on Quadriceps Performance After Eccentric Exercise.

Green, MS, Martin, TD, and Corona, BT. Effect of caffeine supplementation on quadriceps performance after eccentric exercise. J Strength Cond Res 32(10): 2863-2871, 2018-Caffeine use is common among athletes seeking to capitalize on its potential ergogenic effects. Limited research has examined caffeine's effects when used after activities that resulted in exercise-induced muscle damage (EIMD). This study examined the effect of caffeine supplementation on uninjured and injured muscle. Eight men and women (N = 16) who were physically active individuals participated in this study (age: 24.3 ± 4.3 years; height: 173.0 ± 7.0 cm, mass: 75.2 ± 11.5 kg; body fat: 18.2 ± 15.9%). One leg was assessed under uninjured and injured (100 eccentric quadriceps contractions) conditions after caffeine supplementation (6 mg·kg), with the other leg assessed under both conditions after placebo supplementation. Compared with the placebo, caffeine increased peak isokinetic torque by 6.8 ± 2.3 and 9.4 ± 2.5% in uninjured and injured muscle, respectively, but had no effect on maximal voluntary isometric torque or fatigue index in uninjured or injured muscle, with treatments exhibiting similar (p > 0.05) alterations in isometric torque (-11.9 ± 2.2%), fatigue index (-13.9 ± 3.4%), and soreness (+44.0 ± 4.7) after eccentric contractions. The results of this study suggest that caffeine possesses a similar ergogenic effect on isokinetic torque in both uninjured and injured states, but no effect on the production of isometric torque, perception of soreness, or degree of relative fatigue. Athletes should consider the potential caffeine supplementation possesses during recovery from activities that resulted in EIMD.

Effect of conjugation on phase transitions in thermoresponsive polymers: an atomistic and coarse-grained simulation study.

Using atomistic and coarse-grained molecular dynamics (MD) simulations, we explain the shifts in lower critical solution temperature (LCST)-like phase transitions exhibited by elastin-like peptides (ELPs) upon conjugation to other macromolecules (e.g. collagen-like peptides or CLPs). First, using atomistic simulations, we study ELP oligomers with the sequence (VPGFG)6 in explicit water, and characterize the LCST-like transition temperature as one at which the ELP oligomers undergo a change in "hydration state". In agreement with past experimental observations of Luo and Kiick, upon anchoring ELP oligomers to a point to mimic ELP oligomers conjugated to another macromolecule, there is an apparent slight shift in the transition temperature to lower values compared to free (unconjugated) ELP oligomers. However, these atomistic simulations are limited to small systems of short ELPs, and as such do not capture the multiple chain aggregation/phase separation observed in experiments of ELPs. Therefore, we utilize phenomenological coarse-grained (CG) MD simulations to probe how conjugating a block of generic-LCST polymer to another rigid unresponsive macromolecular block impacts the transition temperatures at concentrations and length scales larger than atomistic simulations. We find that when multiple LCST polymer chains are conjugated to a rigid unresponsive polymer block, the increased local crowding of the LCST polymers shifts the transition marked by onset of chain aggregation to smaller effective polymer-polymer attraction energies compared to the free LCST polymer chains. The driving force needed for aggregation is reduced in the conjugates compared to free LCST polymer due to reduction in the loss of polymer configurational entropy upon aggregation.

Wetting-Dewetting and Dispersion-Aggregation Transitions Are Distinct for Polymer Grafted Nanoparticles in Chemically Dissimilar Polymer Matrix.

Simulations and experiments are conducted on mixtures containing polymer grafted nanoparticles in a chemically distinct polymer matrix, where the graft and matrix polymers exhibit attractive enthalpic interactions at low temperatures that become progressively repulsive as temperature is increased. Both coarse-grained molecular dynamics simulations, and X-ray scattering and neutron scattering experiments with deuterated polystyrene (dPS) grafted silica and poly(vinyl methyl ether) PVME matrix show that the sharp phase transition from (mixed) dispersed to (demixed) aggregated morphologies due to the increasingly repulsive effective interactions between the blend components is distinct from the continuous wetting-dewetting transition. Strikingly, this is unlike the extensively studied chemically identical graft-matrix composites, where the two transitions have been considered to be synonymous, and is also unlike the free (ungrafted) blends of the same graft and matrix homopolymers, where the wetting-dewetting is a sharp transition coinciding with the macrophase separation.

Conceptualizing Educational Comparability in Distributed Health Professions Education: A Scoping Review.

PURPOSE: This study aimed to create greater clarity about the current understanding and formulate a model of how educational comparability has been used in the literature to inform practice. METHOD: The authors conducted a literature search of 9 online databases, seeking articles published on comparability in distributed settings in health professions education before August 2021, with an updated search conducted in May 2023. Using a structured scoping review approach, 2 reviewers independently screened articles for eligibility with inclusion criteria and extracted key data. All authors participated in the descriptive analysis of the extracted data. RESULTS: Twenty-four articles published between 1987 and 2021 met the inclusion criteria. Most articles were focused on medical education programs (n = 21) and located in North America (n = 18). The main rationale for discussing comparability was accreditation. These articles did not offer definitions or discussions about what comparability means. The program logic model was used as an organizing framework to synthesize the literature on practices that schools undertake to facilitate and demonstrate comparability in the design (inputs), implementation (activities), and evaluation (outcomes) of distributed education. Inputs include common learning objectives, identical assessment tools and policies, governance models that enable clear communication, and reporting structure that is supported by technological infrastructure. Activities include faculty planning meetings and faculty development training. Outcomes include student experiences and academic performances. CONCLUSIONS: This study demonstrated that a more complex understanding of the dynamics of educational processes and practices is required to better guide the practice of educational comparability within distributed education programs. In addition to highlighting the need to develop an accepted definition of educational comparability, further elucidation of the underlying dynamics among input, activities, and outcomes would help to better determine what drivers should be prioritized when considering educational change with attention to context within distributed education.

Saponin TQL1055 adjuvant-containing vaccine confers protection upon Mycobacterium tuberculosis challenge in mice.

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), affects the lungs of infected individuals (pulmonary TB) but can also affect other sites (extrapulmonary TB). The only licensed vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) protects infants and young children but exhibits variable efficacy in protecting against adult pulmonary TB. Poor compliance and prolonged treatment regimens associated with the use of chemotherapy has contributed to the development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. The development of safe and novel adjuvants for human use is critical. In this study, we demonstrate that saponin-based TQL1055 adjuvant when formulated with a TLR4 agonist (PHAD) and Mtb specific immunodominant antigens (ESAT-6 and Ag85B) and delivered intramuscularly in mice, the SA-TB vaccine induced potent lung immune responses. Additionally, the SA-TB vaccine conferred significant protection against Mtb infection, comparable with levels induced by BCG. These findings support the development of a SA-TB vaccine comprising TQL1055, as a novel, safe and effective TB vaccine for potential use in humans.

The impact of implementation of rapid blood culture identification panels on antimicrobial optimization: a retrospective cohort study.

Objective: Determine the impact of limited implementation of a rapid blood culture identification (BCID) panel. Design: Retrospective cohort study. Methods: From February to April 2022, positive blood cultures identified via e-Plex BCID (Roche, Carlsbad, CA) were compared to those identified using standard microbial identification techniques. The primary outcomes assessed were time to optimal therapy, time to de-escalation of anti-MRSA (methicillin-resistant Staphylococcus aureus) agents, and time to de-escalation of anti-pseudomonal agents. Additional analysis investigated the impact of the availability of antimicrobial stewardship program support. This study was conducted at Grady Health System, a large metropolitan safety-net hospital in the southeastern United States. Results: A total of 253 blood cultures were included in this study (153 BCID and 100 standard). Blood culture identification use was associated with a reduction in median time to optimal antimicrobial therapy (43.4 vs 72.1 h, P < .001) and median time to de-escalation of anti-MRSA agents (27.7 vs 46.7 h, P = .006), and a trend towards reduction of median time to de-escalation of anti-pseudomonal agents (38.8 vs 54.8 h, P = .07). These reductions persisted when controlling for patient age, sex, intensive care unit status, Charlson Comorbidity Index, and antimicrobial stewardship program availability. Conclusions: Despite restricted use and lack of 24/7 antimicrobial stewardship program availability, BCID panel utilization was associated with earlier initiation of optimal therapy and pathogen identification with subsequent de-escalation of broad-spectrum antimicrobials, as compared to standard antimicrobial techniques. This suggests the potential for benefit from adopting novel diagnostic technologies outside of idealized fully-resourced settings.

Polydispersity for tuning the potential of mean force between polymer grafted nanoparticles in a polymer matrix.

We present an integrated theory and simulation study of polydisperse polymer grafted nanoparticles in a polymer matrix to demonstrate the effect of polydispersity in graft length on the potential of mean force between the grafted nanoparticles. In dense polymer solutions, increasing polydispersity in graft length reduces the strength of repulsion at contact and weakens the attractive well at intermediate interparticle distances, completely eliminating the latter at high polydispersity index. The reduction in contact repulsion is attributable to polydispersity relieving monomer crowding near the particle surface, especially at high grafting densities. The elimination of the midrange attractive well is attributable to the longer grafts in the polydisperse graft length distribution that introduce longer range steric repulsion and alter the wetting of the grafted layer by matrix chains. Dispersion of the grafted particles is stabilized by increased penetration or wetting of the polydisperse grafted layer by the matrix chains. This work demonstrates that at high grafting densities, polydispersity in graft length can be used to stabilize dispersions of grafted nanoparticles in a polymer matrix at conditions where monodisperse grafts would cause aggregation.

Crossvalidation of two 20-m shuttle-run tests for predicting VO2max in female collegiate soccer players.

The aim of this study was twofold: (a) to compare the maximal attained speed (MAS) from the 20-m shuttle (MST) and 20-m square-shuttle (SST) tests and (b) to crossvalidate 2 equations for predicting maximal oxygen consumption (VO2max) that were previously developed from MST and SST in a group of female collegiate soccer players. Thirty-nine subjects (age: 20.1 ± 1.5 years) participated in the study. A maximal graded exercise treadmill test was used to measure VO2max. In addition, VO2max was predicted from the MAS obtained during MST ((pred)VO2maxMST) and SST ((pred)VO2maxSST) using previously developed equations. Measured VO2max for the group was 44.2 ± 3.3 ml·kg(-1)·min(-1). The MAS was 12.5 ± 0.6 km·h(-1) for MST and 13.3 ± 0.8 km·h(-1) for SST (p < 0.05). The prediction methods yielded a (pred)VO2maxMST of 49.6 ± 3.9 ml·kg(-1)·min(-1) and predVO2maxSST of 41.8 ± 3.1 ml·kg(-1)·min(-1), which were significantly different from measured VO2max (p < 0.05). The validity statistics revealed the following constant error (CE), correlation coefficient (r), standard error of estimate (SEE), and total error (TE) for (pred)VO2maxMST and (pred)VO2maxSST: CE = 5.35 ± 3.83, r = 0.45 (p < 0.05), SEE = 2.97 ml·kg(-1)·min(-1), TE = 6.39 ml·kg(-1)·min(-1); and CE = -2.43 ± 2.49, r = 0.69 (p < 0.05), SEE = 2.39 ml·kg(-1)·min(-1), TE = 3.43 ml·kg(-1)·min(-1), respectively. Residual plots indicated no proportional bias for either prediction model. The results of this study suggest that female collegiate soccer players had a higher MAS from SST compared with that from MST. In addition, SST appeared to be a more accurate predictor of VO2max than MST in the group of athletes.

Divalent cations in human liver pyruvate kinase exemplify the combined effects of complex-equilibrium and allosteric regulation.

There is growing recognition that the functional outcome of binding of an allosteric regulator to a protein/enzyme is influenced by the presence of other ligands. Here, this complexity is exemplified in the allosteric regulation of human liver pyruvate kinase (hLPYK) that is influenced by the presence of a range of divalent cation types and concentrations. For this system, fructose-1,6-bisphosphate (activator) and alanine (inhibitor) both influence the protein's affinity for the substrate, phosphoenolpyruvate (PEP). Mg2+, Mn2+, Ni2+, and Co2+ were the primary divalent cations evaluated, although Zn2+, Cd2+, V2+, Pb2+, Fe2+, and Cu2+also supported activity. Allosteric coupling between Fru-1,6-BP and PEP and between Ala and PEP varied depending on divalent cation type and concentration. Due to complicating interactions among small molecules, we did not attempt the fitting of response trends and instead we discuss a range of potential mechanisms that may explain those observed trends. Specifically, observed "substrate inhibition" may result from substrate A in one active site acting as an allosteric regulator for the affinity for substrate B in a second active site of a multimer. We also discuss apparent changes in allosteric coupling that can result from a sub-saturating concentration of a third allosteric ligand.