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The enzymes encoded by the kbl/tdh operon of Escherichia coli catalyze sequential steps in the conversion of threonine to glycine. These enzymes, threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase, are normally expressed at low levels in the cell, the majority of glycine being synthesized from serine via the action of the glyA gene product, serine hydroxymethyl-transferase. In order to gain further understanding of physiological role of the kbl/tdh operon, its expression during growth and in response to certain inducers was studied. In addition, mutations allowing a glyA mutant to grow in the absence of glycine were isolated. Two of the mutations isolated and characterized yielded a significant increase on kbl/tdh operon expression. These mutations are in the genes for hns and cya. The regulation of the kbl/tdh operon by these proteins and its interaction with the leucine responsive regulatory protein (Lrp), another known regulator of this operon, lend support to the hypothesis that the enzymes encoded by the kbl/tdh operon function during adaptation to stress or major changes in environment such as those that occur when the cell leaves the mammalian gut.

Prevalence of obesity among infants and children below 5 years of age is rising dramatically, and early childhood obesity is a forerunner of obesity and obesity-associated diseases in adulthood. Childhood obesity is hence one of the most serious public health challenges today. Here, we have identified a mother-to-child lipid signaling that protects from obesity. We have found that breast milk-specific lipid species, so-called alkylglycerol-type (AKG-type) ether lipids, which are absent from infant formula and adult-type diets, maintain beige adipose tissue (BeAT) in the infant and impede the transformation of BeAT into lipid-storing white adipose tissue (WAT). Breast milk AKGs are metabolized by adipose tissue macrophages (ATMs) to platelet-activating factor (PAF), which ultimately activates IL-6/STAT3 signaling in adipocytes and triggers BeAT development in the infant. Accordingly, lack of AKG intake in infancy leads to a premature loss of BeAT and increases fat accumulation. AKG signaling is specific for infants and is inactivated in adulthood. However, in obese adipose tissue, ATMs regain their ability to metabolize AKGs, which reduces obesity. In summary, AKGs are specific lipid signals of breast milk that are essential for healthy adipose tissue development.

Abstract Disclosure: S. Krylova: None. A. Landgraf: None. S. Sidoli: None. J. Pessin: None. It is well known that premenopausal females are relatively protected against metabolic dysfunction-associated steatotic liver disease (MASLD) compared to postmenopausal females and males. Recently, N6-methyladenosine (m6A) RNA methylation has been shown to protect against MASLD. Higher m6A abundance on RNA globally has been associated with lower levels of lipogenesis and hepatic steatosis. Furthermore, it has been reported that m6A levels drop in steatosis-susceptible male mice placed on HFD, while in steatosis-protected females on HFD m6A levels remain relatively high. While this data demonstrates that m6A methylation might explain the selective protection of females against hepatic steatosis on HFD, it is currently not known what drives the differences in m6A RNA methylation levels between males and females on HFD. Given that the precursor of m6A is S-adenosylmethionine (SAM), a product of the methionine cycle of one-carbon metabolism, and that certain one-carbon metabolites are known to be affected by estrogen levels, we hypothesized that estrogen-driven differences in one-carbon metabolism between males and females on HFD are responsible for their differences in liver m6A levels. In the livers of young male, female, and ovariectomized female mice on normal chow diet (NCD) and HFD, we measured the levels of m6A RNA methylation, as well as the levels of key enzymes of the methionine cycle and of m6A precursor, SAM. Similarly to m6A, levels of SAM were significantly lower in male compared to female livers on HFD. Furthermore, expression of key methionine cycle enzymes, such as Mtr and Ahcy, was significantly lower in livers of males and ovariectomized females compared to wild type females on HFD. These results demonstrate that in female livers on HFD the methionine cycle, under the regulation of estrogen, is upregulated to drive higher levels of SAM availability and, potentially, m6A RNA methylation. We are currently repeating these experiments in an additional group of male mice supplemented with Folic Acid (known to upregulate SAM) to demonstrate causative, rather than correlative relationship between the methionine cycle and m6A. Completing these experiments will allow me to establish a novel estrogen – SAM – m6A axis, which has strong implications in MASLD protection and can be utilized for development of targeted therapies for males and postmenopausal females in the future. Presentation: Saturday, July 12, 2025

[Display omitted] •Dendrimers with the HLA-DR epitope, PADRE, comprise a vaccine platform (PDD).•PDD platform conjugated to a hapten, cocaine, induces durable anti-hapten immunity.•PDD vaccine generates high-affinity cocaine antibodies without need for an adjuvant.•Adjuvant formulations of PDD vaccine do not increase anti-cocaine titer levels.•PDD-elicited anti-cocaine antibody response attenuates rewarding effects of cocaine. Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.

Structure-guided peptide phage display combined with activity-based sorting results in the identification of zymogen activator peptides (ZAPtides) that selectively bind and activate the serine protease–like pro-HGF zymogen to promote Met signaling. Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease–like β-chain of pro-HGF and display titratable activation of pro-HGF–dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro–macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.

The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, we have uncovered principles underlying the organisation of the motor system. We find that dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. Our results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units.

Background TSPYL5, a putative tumor suppressor gene, belongs to the nucleosome assembly protein family. The chromosomal location of the TSPYL5 gene is 8Q22.1, and its exact role in prostate cancer etiology remains unclear. Further TSPYL5 gene and protein expression in prostate carcinoma cells and diseased tissues including its susceptibility for epigenetic silencing is unknown. Also, not known is the variation in TSPYL5 protein expression with regards to progression of prostatic carcinoma and its possible role in drug sensitivity. Methods TSPYL5, DNMT-1 and DNMT-B gene expression in DU145, LNCaP and RWPE-1 cells and prostate tumor tissues was analyzed by qRT-PCR and RT-PCR. Demethylation experiments were done by treating DU145 and LNCaP cells with 5-aza-2′-deoxycytidine in vitro. Methylation analysis of TSPYL5 gene was performed by methylation specific PCR and pyrosequencing. TSPYL5 protein expression in benign and diseased prostate tumor tissues was performed by immunohistochemistry and in the cells by Western blotting. Results TSPYL5 was differentially expressed in non-tumorigenic prostate epithelial cells (RWPE-1), androgen independent (DU145), dependent (LNCaP) prostate carcinoma cells and tissues. Methylation-specific PCR and pyrosequencing analysis identified an inverse relationship between DNA methylation and expression leading to the silencing of TSPYL5 gene. Treatment of prostate carcinoma cells in which TSPYL5 was absent or low (DU145 and LNCaP) with the demethylating agent 5-aza-2′-deoxycytidine upregulated its expression in these cells. Immunohistochemical studies clearly identified TSPYL5 protein in benign tissue and in tumors with Gleason score (GS) of 6 and 7. TSPYL5 protein levels were very low in tumors of GS ≥ 8. TSPYL5 overexpression in LNCaP cells increased the cell sensitivity to chemotherapy drugs such as docetaxel and paclitaxel, as measured by the cellular viability. Furthermore, the cells also exhibited reduced CDKN1A expression with only marginal reduction in pAKT. Conclusions Decrease in TSPYL5 protein in advanced tumors might possibly function as an indicator of prostate tumor progression. Its absence due to methylation-induced silencing can lead to reduced drug sensitivity in prostate carcinoma.

J. Okada: None. A. Landgraf: None. A.M. Xiaoli: None. Y. Qiu: None. L. Liu: None. V.L. Schuster: None. F. Yang: None. I.J. Kurland: None. C. Eliscovich: None. K. Shinoda: None. J.E. Pessin: None. The liver generates glucose for systemic glucose requirements such as gluconeogenesis in the fasted state, but can shut down glucose release and switch to storage upon feeding. It is also known that hepatocytes across the liver display specific aspects of liver function in a zonation dependent manner. Because gene expression plays a pivotal role in the long-term regulation of metabolism in the liver, we focused on the temporal and spatial regulation of gluconeogenic gene and gluconeogenic activity of hepatocytes across the liver lobule during the metabolic transitions between the fed, fasted, and starvation states in male C57BL/6J mice.We examined gluconeogenic genes (such as Pck1 and G6pc) under various feeding/fasting conditions using single cell RNA sequencing (scRNA-seq) for mRNA expression in combination with quantification of nuclear transcription sites by single molecule Fluorescence In Situ Hybridization (smFISH). We also measured protein expression by western blotting as well as gluconeogenic activity by stable isotope flux analyses in primary isolated pericentral and periportal hepatocytes.Targeted scRNA-seq showed a basal gluconeogenic gene expression in a subset of periportal hepatocytes in the fed state, and became activated primarily in the periportal hepatocytes in the fasted state. However, in the starvation state pericentral hepatocytes also increased gluconeogenic gene expression. Analyses by smFISH not only confirmed the scRNA-seq data, but showed that while few periportal hepatocytes expressed gluconeogenic transcription sites in the fed state, over time the transcription sites expanded outwards to the pericentral hepatocytes in the starvation state. Consistent with gene expression, gluconeogenic protein expression as well as gluconeogenic activity was approximately 7-10 times higher in the periportal hepatocytes during the fed and initial fasted state, but was only 1.5-2 times different between the pericentral and periportal hepatocytes in the starvation state.In summary, these data demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity is dynamic and can vary substantially depending upon the fasted and fed states. Understanding the regulation of the liver under normal physiological conditions will provide the molecular basis for which we can determine the dysregulation that occurs in states of insulin resistance. Saturday, June 1, 2024

Juggling patterns can be described by a closed walk in a (directed) state graph, where each vertex (or state) is a landing pattern for the balls and directed edges connect states that can occur consecutively. The number of such patterns of length n is well known, but a long-standing problem is to count the number of prime juggling patterns (those juggling patterns corresponding to cycles in the state graph). For the case of b = 2 balls we give an expression for the number of prime juggling patterns of length n by establishing a connection with partitions of n into distinct parts. From this we show the number of two-ball prime juggling patterns of length n is ( γ - o ( 1 ) ) 2 n where γ = 1.32963879259 … . For larger b we show there are at least b n - 1 prime cycles of length n .