Incorporation of Three Extracyclic Arginine Residues into a Melanocortin Macrocyclic Agonist (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Arg-Arg-Arg-Ac)-DPro]) Decreases Food Intake When Administered Intrathecally or Subcutaneously Compared to a Macrocyclic Ligand Lacking Extracyclic Arginine Residues (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro)].

Of the three Food and Drug Administration-approved melanocortin peptide drugs, two possess a cyclic scaffold, demonstrating that cyclized melanocortin peptides have therapeutic relevance. An extracyclic Arg residue, critical for pharmacological activity in the approved melanocortin cyclic drug setmelanotide, has also been demonstrated to increase the signal when fluorescently labeled cell-penetrating cyclic peptides are incubated with HeLa cells, with the maximal signal observed with three extracyclic Arg amino acids. Herein, a branching Lys residue was substituted into two macrocyclic melanocortin peptide agonists to incorporate 0-3 extracyclic Arg amino acids. Incorporation of the Arg residues resulted in equipotent or increased agonist potency at the mouse melanocortin receptors in vitro, suggesting that these substitutions were tolerated in the macrocyclic scaffolds. Further in vivo evaluation of one parent ligand (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-Pro]) and the three Arg derivative (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Ac-Arg-Arg-Arg)-Pro)] demonstrated that the three Arg derivative further decreased food intake compared to the parent macrocycle when the compounds were administered either via intrathecal injection or subcutaneous dosing. This suggests that three extracyclic Arg amino acids may be beneficial in the design of cyclic melanocortin ligands and that in vitro pharmacological profiling may not predict the in vivo efficacy of melanocortin ligands.

Emerald ash borer invasion of riparian forests alters organic matter and bacterial subsidies to south Michigan headwater streams.

Emerald ash borer (EAB) has killed millions of ash trees in the United States and Canada, yet impacts on terrestrial-aquatic linkages are largely unknown. Ash tree death along streams creates canopy gaps, increasing light to riparian plants and potentially affecting organic matter subsidies. Six EAB-related canopy gaps along streams across a gradient of timing of EAB invasion in Michigan were characterized for coarse woody material (CWM), terrestrial and aquatic leaf litter and their associated bacterial communities, and macroinvertebrates upstream, downstream, and at the center of the gap. Stream sites downstream of EAB-related canopy gaps had significantly lower dissolved oxygen and macroinvertebrate diversity than sites upstream and at the gaps. Yet there was no difference in CWM or aquatic leaf litter, likely due to downstream movement of organic matter from upstream riparian sources. Low abundance bacterial amplicon sequence variants unique to gap or forest were detected in leaves and leaf litter, suggesting that EAB-related canopy gaps altered leaf-associated bacterial communities. Overall, EAB invasion indirectly impacted some variables, while organic matter dynamics were resistant to change.

Incorporation of Indoylated Phenylalanine Yields a Sub-Micromolar Selective Melanocortin-4 Receptor Antagonist Tetrapeptide.

The melanocortin family is involved in many physiological functions, including pigmentation, steroidogenesis, and appetite. The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R) possess overlapping but distinct roles in energy homeostasis. Herein, the third and fourth positions of a tetrapeptide lead compound [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], previously reported to possess MC3R agonist and MC4R antagonist activities, were substituted with indoylated phenylalanine (Wsf/Wrf) residues in an attempt to generate receptor subtype selective compounds. At the third position, d-amino acids were required for melanocortin agonist activity, while both l- and d-residues resulted in MC4R antagonist activity. These results indicate that l-indoylated phenylalanine residues at the third position of the scaffold can generate MC4R over MC3R selective antagonist ligands, resulting in a substitution pattern that may be exploited for novel MC4R ligands that can be used to probe the in vivo activity of the MC4R without involvement of the MC3R.

Invasive Dreissena Mussel Coastal Transport From an Already Invaded Estuary to a Nearby Archipelago Detected in DNA and Zooplankton Surveys.

Coastal waters of Lake Superior are generally inhospitable to the establishment of invasive Dreissena spp. mussels (both Dreissena polymorpha and Dreissena bugensis). Dreissena have inhabited the Saint Louis River estuary (SLRE; largest commercial port in the Laurentian Great Lakes) for over three decades, but only in the last few years have small colonies been found in the Apostle Islands National Lakeshore (APIS, an archipelago situated 85 km to the east of SLRE) A 2017 survey determined a low abundance Dreissena spatial distribution in APIS, with the largest colonies on the north and west islands which suggested potential veliger transport from the SLRE via longshore currents. Our objective in this study was to determine if Dreissena veligers are transported by currents at low densities along the south shore of Lake Superior from the SLRE to APIS. To do so, we used both eDNA (water and passive substrate samples) and zooplankton collection methods at eight sites evenly spaced between the SLRE and APIS with three sampling times over five weeks. Dreissena veligers were consistently detected along the south shore, although at low abundances (veligers per m3 range = 0-690, median = 8), and for every 1 km increase in distance from the SLRE, both veliger counts and water eDNA copy numbers decreased on average by 5 and 7%, respectively. D. polymorpha (suited to estuary habitats) was detected two times more than D. bugensis (better suited to deep-lake habitats). There was not a trend in the veliger size distribution along the south shore, and temperature and calcium concentrations fluctuated around the threshold for Dreissena veliger and adult development, averaging 11.0°C and 14.8 ppm, respectively. Three zooplankton taxa representative of the estuary community-Daphnia retrocurva, Diaphanosoma birgei, and Mesocyclops copepodites-decreased as the distance from the SLRE increased mirroring Dreissena veliger abundance patterns. Findings represent multiple sources of evidence of a propagule "conveyor belt" for Dreissena along the south shore of Lake Superior. We conclude that veligers are functioning as a propagule, using coastal currents to spread from the point of invasion, thereby traversing coastal habitat previously reported as inhospitable to distant habitats suitable for colonization.

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist.

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP(86-132)], more potent than miniAGRP(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

The melanocortin pathway and energy homeostasis: From discovery to obesity therapy.

BACKGROUND: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. SCOPE OF REVIEW: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target. MAJOR CONCLUSIONS: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

Multiresidue Tetrapeptide Substitutions Yield a 140-fold Selective Melanocortin-3 over Melanocortin-4 Receptor Agonist.

The five melanocortin receptors regulate numerous physiological functions. Although many ligands have been developed for the melanocortin-4 receptor (MC4R), the melanocortin-3 receptor (MC3R) has been less-well characterized, in part due to the lack of potent, selective tool compounds. Previously an Ac-His-Arg-(pI)DPhe-Tic-NH2 scaffold, inverting the Phe-Arg motif of the native melanocortin signal sequence, was identified to possess mMC3R over mMC4R selective agonist activity. In this study, a library of 12 compounds derived from this scaffold was synthesized and assayed at the mouse melanocortin receptors (MCRs), utilizing substitutions previously shown to increase mMC3R agonist potency and/or selectivity. One compound (8, Ac-Val-Gln-DBip-DTic-NH2) was identified as greater than 140-fold selective for the mMC3R over the mMC4R, possessed 70 nM potency at the mMC3R, and partially stimulated the mMC4R at 100 µM concentrations without antagonist activity. This pharmacological profile may be useful in developing new tool and therapeutic ligands that selective signal through the MC3R.

Psychological drivers of risk-reducing behaviors to limit human-wildlife conflict.

Conflicts between people and wild animals are increasing globally, often with serious consequences for both. Local regulations or ordinances are frequently used to promote human behaviors that minimize these conflicts (risk-reducing behaviors), but compliance with ordinances can be highly variable. While efforts to increase compliance could be improved through applications of conservation psychology, little is known about the relative influence of different factors motivating compliance. Using concepts from psychology and risk theory, we conducted a longitudinal study pairing data from mail surveys with direct observations of compliance with a wildlife ordinance requiring residents to secure residential garbage from black bears (Ursus americanus). We assessed the relative influence of beliefs and attitudes toward bears and bear proofing, perceived behavioral control, perceived risks and benefits assigned to bears, norms, trust in management, previous experience with conflicts, and demographics on compliance behavior (i.e., bear proofing). Data on previous experience were obtained through direct observation and survey reports. We found that higher compliance rates were associated with more observed conflicts on a respondent's block. Counter to expectations, however, residents were less compliant when they were more trusting of the management agency and perceived more benefits from bears. We suggest that messages have the potential to increase compliance when they empower residents by linking successful management of conflicts to individual actions and emphasize how reducing conflicts could maintain benefits provided by wildlife. Modifying existing educational materials to account for these psychological considerations and evaluating their impact on compliance behavior over time are important next steps in changing human behaviors relevant to the globally important problem of human-wildlife conflict.

Impulsores Psicológicos de los Comportamientos Reductores de Riesgo para Limitar el Conflicto Humano - Fauna Resumen Los conflictos entre las personas y la fauna cada vez son más a nivel mundial y con frecuencia tienen consecuencias severas para ambos. Las regulaciones o decretos locales se usan frecuentemente para promover comportamientos humanos que minimizan estos conflictos (comportamientos reductores de riesgo), pero el cumplimiento de los decretos puede ser altamente variable. Mientras que los esfuerzos por incrementar el cumplimiento podrían mejorar por medio de la aplicación de la psicología de la conservación, se conoce poco sobre la influencia relativa de los diferentes factores que motivan al cumplimiento. Realizamos un estudio longitudinal mediante conceptos tomados de la psicología y la teoría del riesgo. Este estudio emparejó datos obtenidos de encuestas por correo con observaciones directas del cumplimiento de un decreto de fauna que requiere que los residentes protejan los desechos residenciales de los osos negros (Ursus americanus) (es decir, hacerlas a prueba de osos). Evaluamos la influencia relativa de las creencias y las actitudes hacia los osos y hacia hacer los desechos a prueba de osos, el control del comportamiento percibido, los riesgos percibidos y los beneficios asignados a los osos, las normas, la confianza en el manejo, la experiencia previa con conflictos y la demografía del comportamiento de cumplimiento. Los datos sobre la experiencia previa se obtuvieron mediante observación directa y reportes de las encuestas. Encontramos que las tasas más altas de cumplimiento estuvieron asociadas con un mayor número de conflictos observados en la cuadra del respondiente. Sin embargo, contrario a las expectativas, los residentes fueron menos obedientes cuando tuvieron mayor confianza en la agencia de manejo y percibieron más beneficios de la presencia de osos. Sugerimos que los mensajes tienen el potencial de incrementar el cumplimiento cuando empoderan a los residentes al vincular el manejo exitoso de los conflictos con las acciones individuales y enfatizan cómo la reducción de los conflictos podría mantener los beneficios que proporciona la fauna. La modificación de los materiales educativos existentes para que tomen en cuenta estas consideraciones psicológicas y la evaluación de su impacto sobre el comportamiento de cumplimiento a lo largo del tiempo son los siguientes pasos importantes para modificar el comportamiento humano relevante para el problema mundialmente importante que es el conflicto humano - fauna.

Effects of Recreation on Animals Revealed as Widespread through a Global Systematic Review.

Outdoor recreation is typically assumed to be compatible with biodiversity conservation and is permitted in most protected areas worldwide. However, increasing numbers of studies are discovering negative effects of recreation on animals. We conducted a systematic review of the scientific literature and analyzed 274 articles on the effects of non-consumptive recreation on animals, across all geographic areas, taxonomic groups, and recreation activities. We quantified trends in publication rates and outlets, identified knowledge gaps, and assessed evidence for effects of recreation. Although publication rates are low and knowledge gaps remain, the evidence was clear with over 93% of reviewed articles documenting at least one effect of recreation on animals, the majority of which (59%) were classified as negative effects. Most articles focused on mammals (42% of articles) or birds (37%), locations in North America (37.7%) or Europe (26.6%), and individual-level responses (49%). Meanwhile, studies of amphibians, reptiles, and fish, locations in South America, Asia, and Africa, and responses at the population and community levels are lacking. Although responses are likely to be species-specific in many cases, some taxonomic groups (e.g., raptors, shorebirds, ungulates, and corals) had greater evidence for an effect of recreation. Counter to public perception, non-motorized activities had more evidence for a negative effect of recreation than motorized activities, with effects observed 1.2 times more frequently. Snow-based activities had more evidence for an effect than other types of recreation, with effects observed 1.3 times more frequently. Protecting biodiversity from potentially harmful effects of recreation is a primary concern for conservation planners and land managers who face increases in park visitation rates; accordingly, there is demand for science-based information to help solve these dilemmas.

In vivo detection of extrapancreatic insulin gene expression in diabetic mice by bioluminescence imaging.

BACKGROUND: Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP). METHODS: The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic beta-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis. RESULTS: Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia. CONCLUSIONS: BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.

Comparative study of regenerative potential of beta cells from young and aged donor mice using a novel islet transplantation model.

BACKGROUND: The effect of ageing on beta-cell regeneration under hyperglycemia has not been defined and may best be addressed using a unique islet-transplantation model. METHODS: Streptozotocin-induced diabetic FVB/NJ mice were rendered normoglycemic with a therapeutic mass of syngeneic islets implanted in the epididymal fat pad, followed by a subrenal capsular implantation of a subtherapeutic mass of 25 islets from young (3 months) or old (24 months) mice. Three weeks after the second transplant, the islet containing fat pad was removed to reintroduce hyperglycemia. Bromodeoxyuridine (BrdU) was provided to mice continuously in drinking water. Islet grafts under the kidney capsule were harvested at different time points and examined for markers of beta-cell regeneration by immunohistochemistry. RESULTS: After a 7-day labeling, BrdU was detected in 54.2% or 53.0% beta cells of the young or old islet grafts, respectively, under hyperglycemia when compared with 3.3% in grafts under normoglycemia. Ki67-positive beta cells were enhanced from a baseline level of 0.5% to 5.2% (young islets) or 4.0% (old islets) on day 7 of hyperglycemia, then decreased to 2.4% on day 21, at which time point an accumulative 75.3% or 66.8% BrdU-positive beta cells was detected in the young or old grafts, respectively. No statistic difference in the percent BrdU- or Ki67-positive beta cells was detected between the young and aged grafts at any time point studied. CONCLUSIONS: These data reveal that islet beta cells from aged mice can replicate in response to hyperglycemia after transplantation at a capacity and frequencies not significantly different than that of the young adult ones.

Prolonging islet allograft survival using in vivo bioluminescence imaging to guide timing of antilymphocyte serum treatment of rejection.

BACKGROUND: Bioluminescence imaging (BLI) is a sensitive and noninvasive method for tracking the fate of transplanted islets. The aim of this study was to investigate whether early detection of rejection by BLI can aid in the timing of antilymphocyte serum (ALS) treatment for prolonging islet graft survival. METHODS: Transgenic islets (200 per recipient) expressing the firefly luciferase from FVB/NJ strain (H-2q) mice were transplanted under the kidney capsule of streptozotocin-induced diabetic allogeneic Balb/c strain (H-2q) mice. BLI signals and serum glucose levels were measured daily after transplant. Four groups of mice were transplanted: group 1 recipients were untreated controls (n=12), group 2 (n=8) received ALS before transplant, group 3 (n=10) received ALS at a time after transplant when normoglycemic but prompted by a reduction (approximately 30%) in BLI signal intensity for 2 consecutive days, and group 4 (n=5) received ALS after transplant when prompted by blood glucose levels increasing approximately 20% from the normoglycemic baseline (BLI reduction approximately 70%). RESULTS: The incidence of graft loss from rejection in groups 1, 2, 3, and 4 was 92.3%, 88%, 40%, and 100%, respectively. The mean (+/-SE) time to graft loss in groups 1, 2, 3 and 4 was 22.5+/-4.8, 29.2+/-9.9, 53.5+/-17.9, and 22.1+/-2.4 days, respectively. CONCLUSIONS: Noninvasive imaging modalities of functional islet mass, such as BLI (but not blood glucose levels), can prompt the appropriate timing of ALS treatment of islet allograft rejection and significantly prolong graft survival or protect the grafts from permanent loss.

The epididymal fat pad as a transplant site for minimal islet mass.

The epididymal fat pad was evaluated as a site of islet transplantation in a syngeneic murine model of diabetes by comparing the transplant outcomes to that of islets transplanted intraportal. Mouse islets engrafted on the intra-abdominal epididymal fat pad ameliorated streptozotocin-induced hyperglycemia with similar efficacy as grafts implanted intraportally. Mice that received as few as 50 islets, either intraportal or in the epididymal fat pad, displayed similar glucose tolerance curves. Bioluminescence imaging and glucose measurement showed stable luminescence signals and blood glucose levels for over 5 months in both transplant sites using transgenic luciferase-positive islets. Prompt recurrent hyperglycemia occurred in all mice after removal of the epididymal fat pad bearing the islet graft. Histological examination of the grafts showed well-granulated insulin containing cells surrounded by healthy adipocytes. This study indicates that the epididymal fat pad maybe a useful islet transplant site in the mouse model for effective glycemic control.

In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection.

BACKGROUND: Bioluminescence imaging (BLI) modalities are being developed to monitor islet transplant mass and function in vivo. The aim of this study was to use the BLI system to determine how the change in functional islet mass correlated to metabolic abnormalities during the course of alloimmune rejection in a murine transplant model. METHODS: Islets obtained from a transgenic mouse strain (FVB/NJ-luc) that constitutively expressed firefly luciferase were transplanted to various implantation sites of syngeneic wild-type FVB/NJ or allogeneic Balb/C streptozotocin-induced diabetic recipients. In vivo graft luminescent signals were repeatedly measured after transplantation using the BLI system and related to blood glucose levels and graft site histologic findings. RESULTS: The BLI signals were detected in as few as 10 islets implanted in the renal subcapsular space, intrahepatic, intraabdominal, and subcutaneous locations. There was a linear relationship between the number of islets transplanted and luminescence intensity. In isografts, stable luminescence intensity signals occurred within 2 weeks of transplantation and remained consistent on a long-term basis (18 months) after transplantation. In allografts, after normoglycemia was achieved and stable luminescence intensity occurred, graft bioluminescent intensity progressively decreased several days before permanent recurrence of hyperglycemia as a result of histologically proven rejection ensued. CONCLUSIONS: Bioluminescence imaging is a sensitive method for tracking the fate of islets after transplantation and is a useful method to detect early loss of functional islet mass caused by host immune responses even before overt metabolic dysfunction is evident. Bioluminescence imaging holds promise for use in designing and testing interventions to prolong islet graft survival.