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The lack of an appropriate preclinical model of metabolic dysfunction-associated steatotic liver disease (MASLD) that recapitulates the whole disease spectrum impedes exploration of disease pathophysiology and the development of effective treatment strategies. Here, we develop a mouse model (Streptozotocin with high-fat diet, STZ + HFD) that gradually develops fatty liver, metabolic dysfunction-associated steatohepatitis (MASH), hepatic fibrosis, and hepatocellular carcinoma (HCC) in the context of metabolic dysfunction. The hepatic transcriptomic features of STZ + HFD mice closely reflect those of patients with obesity accompanying type 2 diabetes mellitus, MASH, and MASLD-related HCC. Dietary changes and tirzepatide administration alleviate MASH, hepatic fibrosis, and hepatic tumorigenesis in STZ + HFD mice. In conclusion, a murine model recapitulating the main histopathologic, transcriptomic, and metabolic alterations observed in MASLD patients is successfully established. Metabolic dysfunction-associated steatotic liver disease (MASLD) characterizes a spectrum of liver disorders initiated by hepatic lipid accumulation associated with metabolic syndrome. Here, the authors generate a mouse model that recapitulates the main histopathologic, transcriptomics, and metabolic alterations observed in MASLD patients.

Congenital hyperinsulinism (CHI) is a rare genetic condition characterized by uncontrolled insulin secretion, resulting in hypoglycemia. Although glucagon has lately been regarded as a therapeutic option for CHI, its use is severely hampered by its poor solubility and stability at physiological pH, as well as its short duration of action. To address these constraints, we developed HM15136, a novel long-acting glucagon analog composed of a glucagon analog conjugated to the Fc fragment of human immunoglobulin G4 via a polyethylene glycol linker. In this study, we established that HM15136 was more soluble than natural glucagon (≥ 150 mg/mL vs 0.03 mg/mL). Next, we confirmed that HM15136 activated glucagon receptor in vitro and induced glycogenolysis and gluconeogenesis in rat primary hepatocytes. Pharmacokinetics (PK)/Pharmacodynamics (PD) analysis of HM15136 shows that HM15136 has a markedly longer half-life (36 h vs. < 5 min) and increased bioavailability (90%) compared to native glucagon in mice. Further, HM15136 could effectively reverse acute hypoglycemia induced by insulin challenge, and multiple doses of HM15136 could sustain increased blood glucose levels in CHI rats. In conclusion, our findings indicate that HM15136 promotes sustained elevation of blood glucose, demonstrating the potential for development as a once-weekly therapy for CHI.

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (β-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response. Immunity: A call to arms A factor secreted by ‘early responder’ immune cells stimulates a signaling loop that fuels the initial response against infection. These cells belong to the innate immune system, which is the first line of defense and releases diverse signaling molecules to recruit other immune cells against invading pathogens. Researchers led by Yoe-Sik Bae of Sungkyunkwan University in South Korea have demonstrated that an immunostimulatory protein called Wnt5a promotes this initial immune migration. Cells called macrophages ‘consume’ the pathogens they encounter, and subsequently alert other cells by producing Wnt5a and other signals. The researchers showed that Wnt5a in turn attracts innate immune cells known as neutrophils, and triggers multiple signaling cascades in these cells. This causes them to secrete additional signals that attract more neutrophils and other immune cell types, thereby strengthening protection against bacteria and viruses.

Shigella , the agent of bacillary dysentery, invades epithelial cells by locally inducing actin reorganization. Upon cell invasion, Shigella induces calcium (Ca 2+ ) signalling, but its role in invasion has remained unclear. Here we show that components involved in inositol 1, 4, 5- trisphosphate (InsP 3 ) signalling are implicated in Shigella invasion. Although global Ca 2+ responses are dispensable for bacterial invasion, local Ca 2+ responses of unprecedented long duration are associated with invasion sites. Fluorescence recovery after photo-bleaching experiments indicate that diffusion of small solutes is hindered at Shigella -invasion sites and that diffusion hindrance is dependent on bacterially induced actin reorganization. Computational simulations and experimental challenge of the model support the notion that local accumulation of InsP 3 permitted by restricted diffusion and enrichment of InsP 3 receptors account for sustained local Ca 2+ increases at entry sites. Thus, cytoskeletal reorganization through diffusion hindrance shapes the duration of local Ca 2+ signals. Shigella triggers an increase in intracellular calcium during invasion of host cells. Here the authors show that increased actin polymerization at the invasion site slows the diffusion of signalling mediators, thus sustaining localized calcium influx at invasion sites.

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-γ1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-γ1, abolished interactions with translational elongation factor 1-α. Here, we report that the Y509A/F510A mutant PLC-γ1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-γ1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-γ1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-γ1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-γ1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-γ1 activation in vivo .

Disclosure: J. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. Y. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. B. Ye: Employee; Self; Hanmi Pharm. Co., Ltd. W. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. J. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Bae: Employee; Self; Hanmi Pharm. Co., Ltd. D. Kim: Employee; Self; Hanmi Pharm. Co., Ltd. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd. Glucagon plays an essential role in glucose homeostasis, and has been used to treat acute hypoglycemia in diabetic patients. However, due to poor solubility and stability at physiological pH as well as short duration of action, its utility was hampered when managing chronic hypoglycemic diseases. One such example is congenital hyperinsulinism (CHI). CHI is a rare genetic disorder characterized by unregulated insulin secretion which leads to severe hypoglycemia especially during fasting condition. Another example is post-bariatric hypoglycemia (PBH) which can also lead to postprandial neuroglycopenia. Despite this life-threatening condition, no drugs are approved for CHI and/or PBH, and available glucagon therapies are very inefficient and inconvenient due to abovementioned limitations. Thus, to provide more optimal therapy for these chronic hypoglycemic diseases, a novel long-acting glucagon analog (HM15136) was developed. Here, we evaluated HM15136’s 1) solubility and stability at physiologic pH, 2) in vitro properties, and 3) potential therapeutic effect in animal models of CHI or PBH. First, we demonstrated improved solubility of HM15136 (≥150 vs. 0.03 mg/mL) at pH 7.0 and stability at day 20, compared to those of native glucagon. Next, in vitro cAMP assay was performed in CHO cells expressing human glucagon receptor (hGCGR), and HM15136 selectively activated hGCGR as a full agonist (EC50 = 0.024 vs. 0.003 nM; relative activity = ca.12.7%). In line with this, HM15136 promoted glycogenolysis and gluconeogenesis in rat primary hepatocytes in a dose dependent manner (relative potency = ca.20%), confirming glucose producing nature of HM15136. To investigate in vivo therapeutic effect on CHI and PBH, chronic hypoglycemia rats induced by SC infusion of insulin (CHI rats) and postprandial hypoglycemia rats induced by vertical sleeve gastrectomy (VSG rats) were established, respectively. Notably, HM15136 treatment significantly restored FBG by 37.3 ∼ 69.7% (p<0.05 ∼ 0.001) in CHI rats. Consistently, HM15136 treatment significantly prevented a massive BG reduction during MMTT in VSG rats (ΔBG between 0 and 45 min = +21 vs. -30 mg/dL, p<0.001). Furthermore, weekly dosing potential and BG elevation effect of HM15136 were also demonstrated in healthy human subjects (1). Based on these results, HM15136 could be a potential therapeutic option for chronic hypoglycemic diseases including CHI and PBH. Phase 2 clinical study in CHI subjects is on-going to assess the clinical relevance of these findings. Reference: (1) Diabetes Obes Metab. 2022 Mar; 23(3):411-20 Presentation: Saturday, June 17, 2023

CMOS scaling has been the driving force behind the revolution of digital signal processing (DSP) systems, but scaling is slowing down and the CMOS device is approaching its fundamental scaling limit. At the same time, advanced DSP algorithms are continuing to evolve, so there is a growing gap between the increasing complexities of the algorithms and what is practically implementable. The growing gap can be bridged by exploring the synergy between algorithm design and hardware design, using the so-called co-design techniques. In this thesis, algorithm and architecture co-design techniques are applied to X-ray computed tomography (CT) image reconstruction. Analysis of fixed-point quantization and CT geometry identifies an optimal word length, intrinsic parallelism, and a mismatch between the object and projection grids. A water-filling buffer is designed to resolve the grid mismatch, and is combined with parallel fixed-point arithmetic units to improve the throughput. The effects of the fixed-point arithmetic on the image quality are analyzed, and an analytical upper bound on the quantization error variance of the reconstructed image is derived. This investigation eventually leads to an out-of-order sectored processing architecture that reduces the off-chip memory access by three orders of magnitude, allowing for a throughput of 925M voxel projections/s at 200MHz on a Xilinx Virtex-5 FPGA. The co-design techniques are further applied to the design of spiking neural networks for sparse coding. Analysis of the neuron spiking dynamics leads to the optimal tuning of network size, spiking rate, and neuron update step size to keep the neuron spiking sparse and random. The resulting sparsity enables a bus-ring architecture to address routing complexity. The bus and ring sizes are optimized to achieve both high throughput and scalability. The architecture is demonstrated in a 65nm CMOS chip. The test chip demonstrates sparse feature extraction at a high throughput of 1.24G pixel/s at 1.0V and 310MHz. The error tolerance of sparse coding can be exploited to further enhance the energy efficiency. As a natural next step after the sparse coding chip, a neural-inspired inference module (IM) is designed for object recognition. The object recognition chip consists of an IM based on the spiking locally competitive algorithm and a sparse event-driven classifier. A light-weight learning co-processor is integrated on chip to enable on-chip learning. The throughput and energy efficiency are further improved using a number of architectural techniques including sub-dividing the IM network and classifier into modules and optimal pipelining. The result is a 65nm CMOS chip that performs sparse coding at 10.16G pixel/s at 1.0V and 635MHz. The integrated IM and classifier provide extra error tolerance for voltage scaling, allowing the power to be lowered to 3.65mW at a throughput of 640M pixel/s. Algorithm and architecture co-design techniques have been demonstrated in this work to advance the hardware design for CT image reconstruction, neuromorphic sparse coding, and object recognition. The co-design techniques can be applied to the design of other advanced DSP algorithms for emerging applications.

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC- gamma 1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC- gamma 1, abolished interactions with translational elongation factor 1- alpha . Here, we report that the Y509A/F510A mutant PLC- gamma 1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC- gamma 1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC- gamma 1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC- gamma 1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC- gamma 1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC- gamma 1 activation in vivo.

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-γ1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-γ1, abolished interactions with translational elongation factor 1-α. Here, we report that the Y509A/F510A mutant PLC-γ1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-γ1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-γ1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-γ1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-γ1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-γ1 activation in vivo.