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Kou, Z.

Publications and source records attributed to Kou, Z..

12 recordsLinked to original sources

Spatial modulation of RAF by RAF/MEK glue enables full-dose combination with pan-RAF inhibitor and potent RAS-mutant tumor-selective MAPK and growth inhibition

The clinical benefit of MAPK-targeted therapies depends on greater pathway inhibition in tumors than normal tissues. Although pan-RAF inhibitors are active in RAS-mutant cancers, combining them with MEK inhibitors requires dose reductions due to toxicity, limiting efficacy. We show the toxicity results from MEK inhibitor-mediated feedback relief, which promotes RAF activation and pan-RAF inhibitor engagement in normal cells, narrowing the therapeutic index. We further demonstrate that MEK is exclusively cytosolic, and RAF/MEK glues overcome this limitation through spatial trapping. By stabilizing cytosolic RAF-MEK complexes, RAF/MEK glues prevent feedback-driven RAF activation in normal cells while maintaining inhibition of oncogenic RAF signaling in RAS-mutant tumors, where RAF is constitutively activated at the plasma membrane. Consequently, this enables full-dose combination with pan-RAF inhibitors, resulting in deeper MAPK suppression and robust tumor regressions in RAS-mutant models. Thus, by spatially controlling wild-type effectors, drug-induced proximity can be harnessed to increase tumor selectivity of pathway-targeted therapies. SignificanceMAPK-targeted therapies rarely achieve durable responses in RAS-mutant cancers due to dose-limiting toxicities. We show that RAF/MEK glues, by spatially trapping RAF, can be combined with pan-RAF inhibitors at full dose, yielding tumor-selective MAPK inhibition and tumor regressions in RAS-mutant models. Thus, drug-induced proximity can be exploited for tumor-selective therapy.

cancer biology↗

Null Subtraction Imaging for Functional Ultrasound Brain Activation Mapping

Null Subtraction Imaging (NSI) is a nonlinear beamforming approach that combines multiple receive apodizations and subtraction to improve spatial resolution in ultrasound imaging. In NSI, a DC offset parameter is introduced in the apodization design to control the sharpening of the effective beam pattern and, therefore, the degree of spatial-resolution enhancement. Here, we investigate the use of NSI in functional ultrasound (fUS) imaging of the mouse brain and compare its performance with conventional delay-and-sum (DAS) beamforming across a range of DC offset values. fUS acquisitions were performed in three anesthetized wild-type mice during periodic vibrissae stimulation. Activation maps were computed by correlating cerebral blood volume (CBV) signals with the stimulation pattern. Activation area, edge gradient, Dice similarity coefficient, and signal-to-noise ratio (SNR) were used to evaluate spatial localization, boundary sharpness, vascular alignment and signal stability, respectively. NSI yielded more spatially confined activation maps than DAS and produced sharper activation boundaries. However, for low DC offsets (DC < 0.5), the CBV signal exhibited increased fluctuations, which reduced temporal stability and limited the reliability of the functional maps. As the DC offset increased, temporal SNR improved, while the spatial-resolution gain progressively decreased. In our imaging configuration, intermediate DC values around DC {approx} 0.5 provided the most favorable compromise between improved spatial localization and sufficient temporal stability for reliable functional activation detection. These results demonstrate the feasibility of applying NSI to functional ultrasound imaging and provide a quantitative framework for selecting the DC parameter in fUS studies.

bioengineering↗

Molecular Architecture and Function Mechanism of Tri-heteromeric GluN1-N2-N3A NMDA Receptors

N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in brain development and synaptic function. Previous studies have focused on GluN1-N2 (2A-2D) and GluN1- N3 (3A and 3B) di-heteromeric (di-) NMDARs, leaving the activation mechanism and stoichiometry of GluN1-N2-N3 tri-heteromeric (tri-) NMDARs largely unexplored. In this study, we employed a two-step affinity-tagged chromatography approach to purify recombinantly expressed GluN1-N2A-N3A tri-NMDARs and determined their cryo-EM structure. Based on the proteoliposome single-channel recording, we discovered GluN1-N2A-N3A can be activated upon co-binding of glycine and glutamate, exhibiting two distinct conductance levels. Furthermore, leveraging structural-based click- chemistry, we introduced photo-crosslinker p-azido-phenylalanine (AzF) into the N- terminal domain of GluN2A and GluN2B, enabling the crosslinking with GluN3A subunit both in vitro and in vivo. These findings provide molecular insights into the subunit arrangement, native architecture and activation mechanism of GluN1-N2-N3A tri- NMDARs and also highlight the complexity of NMDAR assembly and function in the brain.

neuroscience↗

RAS mutation-specific signaling dynamics in response to paralog- and state- selective RAS inhibitors

A high therapeutic index (TI), balancing potent oncogenic signaling inhibition in tumor cells with minimal effects on normal cells, is critical for effective cancer therapies. Recent advances have introduced diverse RAS-targeting inhibitors, including mutant-specific inhibitors (e.g., KRAS(G12C) and KRAS(G12D)), as well as paralog- and state-selective inhibitors. Non-mutant-specific RAS inhibition can be accomplished by 1) panRAS-GEF(OFF) inhibitors which inactivate RAS indirectly by inhibiting SHP2 or SOS1, thereby blocking the nucleotide exchange step of RAS activation, 2) direct KRAS(OFF)-selective inhibitors sparing NRAS and HRAS, and 3) panRAS(ON) inhibitors that directly target active RAS, by occluding binding of its effector RAF. However, the signaling inhibition index (SII) - the differential inhibition of oncogenic signaling between RAS-mutant (RAS(MUT)) and normal cells - remains poorly defined for these approaches. In this study, we evaluated the SII of state- and paralog-selective RAS inhibitors across diverse RAS-mutant (RAS(MUT)) and RAS-wild-type (RAS(WT)) models. PanRAS-GEF(OFF) inhibitors exhibited neutral or negative SII, with comparable or reduced MAPK suppression in KRAS(G12X) cells relative to RAS(WT) cells. KRAS(G13D) models showed low sensitivity (negative SII) to panRAS-GEF(OFF) inhibitors, particularly in the context of NF1 loss. Combination treatments with SHP2 and MEK inhibitors resulted in low SII, as pathway suppression was similar in RAS(MUT) and RAS(WT) cells. Furthermore, RAS(Q61X) models were resistant to combined SHP2 inhibitor+MEK inhibitor due to dual mechanisms: MEK inhibitor-induced NRAS(Q61X) reactivation and RAS(MUT)-induced SHP2 conformations impairing inhibitor binding. Overall, panRAS-GEF(OFF) inhibitors exhibited the lowest SII. PanKRAS(OFF) inhibitors demonstrated a higher SII, while panRAS(ON) inhibitors displayed broader activity but relatively narrow SII. We observed that tumors that were sensitive to RAS(MUT)-specific inhibitors, were also sensitive to the state-selective RAS inhibitors (OFF, or ON). In fact, all RAS inhibitors (mutant-specific and state- or paralog-selective) were active in the same portion of RAS(MUT) models, while the majority of RAS(MUT) cell lines were insensitive to all of them. These findings reveal significant SII variability among RAS-targeted inhibitors, depending on the specific RAS driver mutation and cell context and underscore the importance of incorporating SII considerations into the design and clinical application of RAS-targeted therapies to improve therapeutic outcomes. Main pointsO_ST_ABSPanRAS-GEF(OFF) inhibitors have limited SII and effectivenessC_ST_ABSThe Signaling Inhibition Index (SII) - i.e. the differential inhibition of oncogenic signaling between tumor and normal cells - was neutral or negative for panRAS-GEF(OFF) inhibitors, with comparable or reduced MAPK suppression in KRAS(G12X) mutant versus RAS(WT) cells. KRAS(G13D) models showed reduced sensitivity, particularly with NF1 loss. SHP2+MEK inhibitor combinations also had low SII, with RAS(Q61X) models demonstrating resistance due to NRAS(Q61X) reactivation and impaired SHP2 inhibitor binding. PanKRAS(OFF) selective inhibitors have higher SII than panRAS-GEF(OFF) inhibitorspanKRAS(OFF)-selective inhibitors have a higher SII compared to panRAS-GEF(OFF) inhibitors, offering better tumor-versus-normal cell selectivity. PanRAS(ON) inhibitors have broad but modest SIIWhile panRAS(ON) inhibitors displayed a broader activity profile, their ability to selectively inhibit mutant RAS signaling over normal cells remained relatively narrow (low SII). Most KRAS-mutant tumors will be insensitive to any single RAS-targeted inhibitorState- and paralog-selective inhibitors have enhanced activity in the same RAS-MUT cancer models that are also sensitive to RAS-MUT-specific inhibitors, suggesting that most KRAS-MUT tumors will not respond uniformly to any one RAS-targeting inhibitor. SII varies across RAS inhibitors, necessitating tailored therapeutic strategiesThe effectiveness of paralog- and state-selective inhibitors depends on specific RAS mutations and cell context, highlighting the need to integrate SII considerations into the development and clinical application of RAS-targeted therapies.

cancer biology↗

Functional characterization of Lipid storage droplets 1 (LSD1) in growth and lipolysis of Hermetia illucens

As intracellular organelles in adipose tissue, lipids droplets manage the balance between triglyceride accumulation and energy consumption in animals. Perilipin family members, associated with surface of lipid droplets, participate the regulation of lipid metabolism. Lipid storage droplet-1 (LSD1)/Perilipin-1 acts as a gatekeeper for adipose lipid storage in animals. Despite extensive studies in fruit fly, the function of LSD1 in insect larval stage remain indistinct. In this study, we characterized the function of LSD1 in black soldier fly Hermetia illucens, a nova resource insect to recycle organic wastes. We found that LSD1 was broadly present in dipteran species and evolved with divergence between mosquitos and flies. We further constructed in vivo mutagenesis mediated by CRISPR/Cas9 and found that mutations in LSD1 increased the larval weight and did not bring any defects in development. Raw fat content was also not significantly influenced in late larval stage and new-emerged adults. Our results not only extend our knowledge of LSD1 in insects, but also help for better understanding of the lipid homeostasis in BSF.

zoology↗

Functional characterization of fatty acid synthase in Hermetia illucens

Fatty acid synthase (FAS) is a key role in de novo lipogenesis (DNL) in most lives. In insects, type I FAS functions in homodimer, activated by a phosphopantetheine transferase (PPT), to de novo produce essential saturated fatty acid for cellular processes. In a nova insect species Hermetia illucens (Diptera: Stratiomyidae), different from most animals and insects, lauric acid (LA) accumulates to a noticeable level, near content of that in coconut oil or palm kernel oil. However, the origin of LA, from de novo biosynthesis or as degraded products from long chain fatty acid, was still ambiguous. Here, in this study, we mined a FAS genes (HiOGS16234) from H. illucens genome and initially characterized its abilities of LA production in a free-fatty-acid producible yeast. After clear the yeast metabolites background of fatty acid by deletion of ScFAS1 and ScELO1 sequentially, which responsible for fatty acid genesis and elongation respectively, functions of HiOGS16234 from H. illucens was in vitro elucidated by analyzing its product portfolio. Through protein domain swap in HiOGS16234, we found the ketoacyl synthase domain and thioesterases domain were responsible for the different product portfolio of HiOGS16234, compared with DmFASN1. Through in vitro assay in yeast, the origin of LA in BSF was firstly elucidated at molecular level, which support DNL origin hypothesis in BSF.

synthetic biology↗

Proteolytic Cleavage of the ENaC gamma Subunit: Impact Upon Na+ and K+ Handling

The ENaC gamma subunit is essential for homeostasis of Na+, K+, and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (Q4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.

physiology↗

Potent Immunogenicity and Broad-Spectrum Protection Potential of Microneedle Array Patch-Based COVID-19 DNA Vaccine Candidates Encoding Dimeric RBD Chimera of SARS-CoV and SARS-CoV-2 Variants

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to pandemic control, and the development of more effective vaccines of broadspectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding heterodimeric receptor-binding domain (RBD) chimera of SARS-CoV and SARS-CoV-2 Omicron BA.1 (RBDSARS/BA1), SARS-CoV and SARS-CoV-2 Beta (RBDSARS/Beta), or Omicron BA.1 and Beta (RBDBA1/Beta) in secreted form. When i.m. injected in mice, RBDSARS/BA1 and RBDSARS/Beta encoding plasmids (pAD1002 and pAD131, respectively) were by far more immunogenic than RBDBA1/Beta plasmid (pAD1003). Dissolvable microneedle array patches (MAP) laden with these DNA plasmids were fabricated. All 3 resulting MAP-based vaccine candidates, namely MAP-1002, MAP1003 and MAP-131, were comparable to i.m. inoculated plasmids with electroporation assistance in eliciting strong and durable IgG responses in BALB/c and C57BL/6 mice as well as rabbits, while MAP-1002 was comparatively the most immunogenic. More importantly, MAP-1002 significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-{gamma}+ T cells. Moreover, MAP-1002 antisera effectively neutralized pseudoviruses displaying spike proteins of SARS-CoV, prototype SARS-CoV-2 or Beta, Delta, Omicron BA1, BA2 and BA4/5 variants. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

immunology↗

Distinct structure and gating mechanism in diverse NMDA receptors with GluN2C and GluN2D subunits

N-Methyl-D-Aspartate (NMDA) receptors are essential for many brain functions. These receptors are heterotetramers typically comprising two GluN1 subunits and two GluN2 subunits. The latter could alternate among four subtypes (N2A-N2D) and determine the functional diversity of NMDA receptors1, 2. For example, receptors containing N2C or N2D exhibit 50-fold lower channel open probability (Po) than those containing N2A (ref.3-5). Structures of N2A- and N2B-containing receptors have been extensively characterized, providing molecular basis for understanding NMDA receptor function6-14. Here we report the cryo-EM structures of N1-N2D and N1-N2C di-heterotetramers (di-receptors), and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a resolution up to 3.0 [A]. Structural analysis showed that the bilobate N-terminal domain (NTD) in N2D adopted an intrinsic closed conformation, leading to a compact NTD tetramer in N1-N2D receptor. Functional studies further demonstrated that, in di-receptors containing N2D but not N2A or N2B, crosslinking NTD at the tetrameric interface had no effect on channel activity, while crosslinking ligand-binding domain (LBD) of two N1 protomers significantly elevated Po. Surprisingly, we found that the N1-N2C di-receptors spontaneously oscillated between symmetric and asymmetric conformation. The later one occupied a predominant population, whereby two N2C protomers exhibited distinct conformation. This asymmetry, which was also found to a lesser extent in N1-N2A di-receptor10, was further locked by the binding of an N2C-specific allosteric potentiator PYD-106 to a unique binding pocket between NTD and LBD in only one N2C protomer. Finally, N2A and N2C in the N1-N2A-N2C tri-receptor displayed the conformation close to that found in one protomer of N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse functional properties of major NMDA receptor subtypes.

neuroscience↗

Transcriptome analysis of SARS-CoV-2 naive and recovered individuals vaccinated with inactivated vaccine

The urgent approval of the use of the inactivated COVID-19 vaccine is essential to reduce the threat and burden of the epidemic on global public health, however, our current understanding of the host immune response to inactivated vaccine remains limited. Herein, we performed serum IgG antibody detection and transcriptomics analysis on 20 SARS-CoV-2 naive individuals who received multiple doses of inactivated vaccine and 5 SARS-CoV-2 recovered individuals who received single dose of inactivated vaccine. Our research revealed the important role of many innate immune pathways after vaccination, identified a significant correlation with the third dose of booster vaccine and proteasome-related genes, and found that SARS-CoV-2 recovered individuals can produces a strong immune response to a single dose of inactivated vaccine. These results help us understand the reaction mechanism of the hosts molecular immune system to the inactivated vaccine, and provide a basis for the choice of vaccination strategy.

microbiology↗

Improved Ultrasound Localization Microscopy based on Microbubble Uncoupling via Transmit Excitation (MUTE)

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on the robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on microbubble uncoupling via transmit excitation (MUTE). MUTE "silences" MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then an in vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

bioengineering↗

Analysis on epidemiological characteristics of 4 species natural-focal diseases in Shandong Province, China in 2009-2017: a descriptive analysis

BackgroundNatural-focal diseases have always been a kind of serious disease that endangers human health. It threatens about 100 million people in Shandong Province, and causes illness in thousands of people each year. However, information on the epidemiological characteristics of natural-focal diseases in Shandong Province has been limited. The purpose of the study is to describe and analyze the epidemiological characteristics of natural-focal diseases in Shandong Province, 2009-2017. MethodsWe describe the incidence and distribution of 4 species natural-focal diseases in Shandong Province using surveillance data from 2009-2017. ResultsFrom 2009-2017, 11123 cases of 4 species natural-focal diseases including 257 deaths were reported in Shandong Province, China. The 4 species natural-focal diseases were severe fever with thrombocytopenia syndrome (SFTS), human granulocytic anaplasmosis (HGA), typhus and scrub typhus respectively. The high-risk groups of the 4 species diseases all were farmers and the elderly. The incidence rate of scrub typhus was significantly higher in females, however, this difference was not seen in the other 3 diseases. The 4 species diseases were mainly clustered in middle-southern part of Shandong (Mount Yimeng) and Shandong Peninsula (Laoshan Mountain). The annual incidence of SFTS and scrub typhus had increased in the mass, typhus had been relatively stable, and HGA had declined. However, the popular range of SFTS had been expanding, HGA had been shrinking, and typhus and scrub typhus were unchanged. The epidemic period of SFTS and HGA was from May to October, typhus was from October to November, scrub typhus was from September to November in Shandong Province. The fatality rates of SFTS, typhus, scrub typhus, HGA were 9.19%, 0%, 0.01%, 2.24%, respectively. ConclusionsOur study described and analyzed the prevalence of natural-focal diseases in Shandong, and confirmed that age was closely related to the SFTS fatality rate. This study may be applicable to an improved understanding of the prevalence of natural-focal diseases in Shandong Province in recent years and the better development of the accurate prevention and control strategies for natural-focal diseases in Shandong Province. Author SummaryNatural-focal diseases are a serious public health problem in Shandong Province, China. It threatens about 100 million people in Shandong Province, and causes illness in thousands of people each year. This study used the monitoring data from Shandong Province for 2009-2017 to describe and analyze the epidemiological characteristics of the 4 species natural-focal diseases. The results showed that the risk population of natural-focal diseases in Shandong Province was farmers and the elderly, the epidemic season was mainly in summer and autumn, the middle-southern part of Shandong Province and Shandong Peninsula were more seriously affected compared with other regions. In addition, the epidemic area of SFTS was expanded, with a fatality rate of 9.19%. These findings indicated that public awareness of natural-focal diseases should be raised in the epidemic focus, especially for farmers, and further efforts should be strengthen specially in high-risk areas and during the epidemic season.

epidemiology↗