FLT3/CD99 Bispecific Antibody-Based Nanoparticles for Acute Myeloid Leukemia.

Cluster of differentiation 99 (CD99) is a receptor that is significantly upregulated in acute myeloid leukemia (AML). FMS-like tyrosine kinase 3 internal tandem duplication mutation in AML (FLT3-ITD AML) exhibits even higher levels of CD99 expression. Our group previously employed a novel peptide platform technology called elastin-like polypeptides and fused it with single-chain antibodies capable of binding to FLT3 (FLT3-A192) or CD99 (CD99-A192). Targeting either FLT3 or CD99 using FLT3-A192 or CD99-A192 led to AML cell death and reduced leukemia burden in AML mouse models. Here, we report on the development of a novel Co-Assembled construct that is capable of binding to both CD99 and FLT3 and the antileukemia activity of the bispecific construct in FLT3-ITD AML preclinical models. This dual-targeting Co-Assembled formulation exhibits cytotoxic effects on AML cells (AML cell lines and primary blasts) and reduced leukemia burden and prolonged survival in FLT3-ITD AML mouse models. Altogether, this study demonstrates the potential of an innovative therapeutic strategy that targets both FLT3 and CD99 in FLT3-ITD AML. SIGNIFICANCE: This study investigates a dual-targeting strategy in acute myeloid leukemia (AML), focusing on FLT3 and CD99. The approach demonstrates enhanced therapeutic potential, presenting a novel option for AML treatment.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

Steric stabilization of bioactive nanoparticles using elastin-like polypeptides.

Elastin-like polypeptides (ELP) are versatile, thermo-responsive polymers that can be conjugated to virtually any therapeutic cargo. Derived from short amino-acid sequences and abundant in humans, certain ELPs display low immunogenicity. Substrates for endogenous proteases, ELPs are biodegradable and thus, are candidate biomaterials. Peptides and proteins can be directly coupled with ELPs through genetic engineering, while other polymers and small molecules can be appended through covalent bioconjugation or non-covalent complexation. ELPs that phase separate at physiological temperatures can form the core of nano assemblies; however, ELPs that remain soluble can sterically stabilize the corona of a variety of nanoparticles. Nanoparticles with ELPs at their corona promote colloids with favorable pharmacokinetic (PK) properties that enables therapeutic efficacy with intermittent administration. This review highlights a comprehensive spectrum of ELP fusions shown to stabilize the solubility, and sometimes bioactivity, of their cargo - with a focus on biophysical properties that underlie their therapeutic effects.

αB-Crystallin Peptide Fused with Elastin-like Polypeptide: Intracellular Activity in Retinal Pigment Epithelial Cells Challenged with Oxidative Stress.

BACKGROUND: Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina. METHODS: The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering 'DLS'. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with H2O2 and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy. RESULTS: The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape. CONCLUSIONS: ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization.

The Effects of an Online Positive Psychology Course on Happiness, Health, and Well-Being.

This study investigated the effects of an 8-week online positive psychology course on happiness, health, and well-being. There were 65 undergraduate students in the course and a comparison group of 63 undergraduates taking other online psychology courses. The participants were assessed on positive mental health (e.g., happiness, positive emotions), negative mental health (e.g., anxiety, depression), general health, and personal characteristics (e.g., hope, resilience) during the first and last week of the courses. The anxiety and depression measures had cut-offs for clinically significant symptoms. The hypotheses were that the positive psychology students would have significant improvements on all measures and a reduction in the percent anxious and depressed relative to the comparison group. The hypotheses were supported with large effect sizes for positive and negative mental health (mean ds = 0.907 and - 0.779, respectively) and medium-to-large effects for general health and personal characteristics (d = 0.674 and mean ds = 0.590, respectively). There was a reduction from 49.2 to 23.1% percent anxious and from 18.6 to 6.2% percent depressed with no change in the comparison group. In addition, improvements in the online positive psychology course were compared with a previous study of a similar face-to-face positive psychology course (Smith et al., 2021) showing the effect sizes for improvements relative to the comparison groups were larger in the online vs. face-to-face course (mean ds = 0.878. vs. 0.593). Possible explanations for these differences are discussed along with the implications for maximizing the benefits of positive psychology courses in the future.

Biomimetic SARS-CoV-2 Spike Protein Nanoparticles.

COVID-19 is an infectious respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus contains a crucial coat protein that engages with target cells via a receptor binding domain (RBD) on its spike protein. To better study the RBD and its therapeutic opportunities, we genetically engineered a simple fusion with a thermo-responsive elastin-like polypeptide (ELP). These fusions express in Escherichia coli at a high yield in the soluble fraction and were easily purified using ELP-mediated phase separation (79 mg/L culture). Interestingly, they assembled peptide-based nanoparticles (Rh = 71.4 nm), which was attributed to oligomerization of RBDs (25.3 kDa) counterbalanced by steric stabilization by a soluble ELP (73.4 kDa). To investigate their biophysical properties, we explored the size, shape, and binding affinity for the human angiotensin-converting enzyme 2 (hACE2) and cellular uptake. Biomimetic nanoparticles such as these may enable future strategies to target the same cells, tissues, and cell-surface receptors as those harnessed by SARS-CoV-2.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Intergenerational trauma transmission is associated with brain metabotranscriptome remodeling and mitochondrial dysfunction.

Intergenerational trauma increases lifetime susceptibility to depression and other psychiatric disorders. Whether intergenerational trauma transmission is a consequence of in-utero neurodevelopmental disruptions versus early-life mother-infant interaction is unknown. Here, we demonstrate that trauma exposure during pregnancy induces in mouse offspring social deficits and depressive-like behavior. Normal pups raised by traumatized mothers exhibited similar behavioral deficits to those induced in pups raised by their biological traumatized mothers. Good caregiving by normal mothers did not reverse prenatal trauma-induced behaviors, indicating a two-hit stress mechanism comprising both in-utero abnormalities and early-life poor parenting. The behavioral deficits were associated with profound changes in the brain metabotranscriptome. Striking increases in the mitochondrial hypoxia marker and epigenetic modifier 2-hydroxyglutaric acid in the brains of neonates and adults exposed prenatally to trauma indicated mitochondrial dysfunction and epigenetic mechanisms. Bioinformatic analyses revealed stress- and hypoxia-response metabolic pathways in the neonates, which produced long-lasting alterations in mitochondrial energy metabolism and epigenetic processes (DNA and chromatin modifications). Most strikingly, early pharmacological interventions with acetyl-L-carnitine (ALCAR) supplementation produced long-lasting protection against intergenerational trauma-induced depression.

The role of Olfaction in MCH-regulated spontaneous maternal responses.

Melanin concentrating hormone (MCH) is involved in the initiation of maternal behavior during the postpartum period. Virgin females also display some aspects of maternal care independent of the hormonal and neurochemical changes associated with pregnancy and parturition. Maternal behavior in virgin females is triggered by pups-generated chemosensory signals. We therefore examined the role of MCH in maternal-related behaviors in virgin mice and whether it involves chemosensory mechanisms. We used mice with germline knock-out of MCH receptor (MCHR1 KO) and mice with conditional ablation of MCH neurons (MCH cKO) using Cre-inducible diphtheria toxin (iDTR) system. We report that germline deletion of MCHR1 and ablation of MCH neurons impair spontaneous maternal behavior that is induced upon pups' exposure. The latency and duration to retrieve pups by MCHR1 KO and MCH cKO mice are longer than their control littermate mice. In support of this finding, we found that in the three-chamber social test, both MCHR1 KO and MCH cKO mice display a lack of interest in interacting with pups. Strikingly, however, we found that while MCHR1 KO mice were unable to detect pups' chemosensory signals and displayed impairment in general olfactory discrimination, MCH cKO mice exhibited normal olfactory function. Our findings indicate that the lack of MCHR1 or of normal MCH levels causes defects in maternal behavior in non-sensitized virgin mice, and that disruption of the olfactory signaling might not count for these defects.