Underlaying Mechanisms Research Articles

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.

Harmine acts as a quorum sensing inhibitor decreasing the virulence and antibiotic resistance of Pseudomonas aeruginosa

BackgroundAs antimicrobial resistance (AMR) has become a global health crisis, new strategies against AMR infection are urgently needed. Quorum sensing (QS), responsible for bacterial communication and pathogenicity, is among the targets for anti-virulence drugs that thrive as one of the promising treatments against AMR infection.MethodsWe identified a natural compound, Harmine, through virtual screening based on three QS receptors of Pseudomonas aeruginosa (P. aeruginosa) and explored the effect of Harmine on QS-controlled and pathogenicity-related phenotypes including pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14. The protective effect of Harmine on Caenorhabditis elegans (C. elegans) and mice infection models was determined and the synergistic effect of Harmine combined with common antibiotics was explored. The underlaying mechanism of Harmine’s QS inhibitory effect was illustrated by molecular docking analysis, transcriptomic analysis, and target verification assay.ResultsIn vitro results suggested that Harmine possessed QS inhibitory effects on pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14, and in vivo results displayed Harmine’s protective effect on C. elegans and mice infection models. Intriguingly, Harmine increased susceptibility of both PA14 and clinical isolates of P. aeruginosa to polymyxin B and kanamycin when used in combination. Moreover, Harmine down-regulated a series of QS controlled genes associated with pathogenicity and the underlying mechanism may have involved competitively antagonizing autoinducers’ receptors LasR, RhlR, and PqsR.ConclusionsThis study shed light on the anti-virulence potential of Harmine against QS targets, suggesting the possible use of Harmine and its derivates as anti-virulence compounds.

The role of non-coding RNAs in neuropathic pain.

Neuropathic pain (NPP) is a refractory pain syndrome, caused by damage or disease of the somatosensory nervous system and characterized by spontaneous pain, hyperalgesia, abnormal pain and sensory abnormality. Non-coding RNAs (ncRNAs), including microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA) and Piwi interacting RNA (piRNA), play a notable role in initiation and maintenance of NPP. In this review, we summarize the role of ncRNAs in NPP and their underlaying mechanism. Generally, ncRNAs are interacted with mRNA, protein or DNA to regulate the molecules and signals assciated with neuroinflammation, ion channels, neurotrophic factors and others, and then involved in the occurrence and development of NPP. Therefore, this review not only contributes to deepen our understanding of the pathophysiological mechanism of NPP, but also provides theoretical basis for the development of new therapy strategies for this disorder.

Inhibition of dengue virus infection in vitro by fucoidan and polysaccharide extract from marine alga Sargassum spp.

Dengue virus (DENV) infection poses a global health threat, leading to severe conditions with the potential for critical outcomes. Currently, there are no specific drugs available whereas the vaccine does not offer comprehensive protection across all DENV serotypes. Therefore, the development of potential antiviral agents is necessary to reduce the severity risk and interrupt the transmission circuit. The search for effective antiviral agents against DENV has predominantly focused on natural resources, particularly those demonstrating diverse biological activities and high safety profiles. Cyanobacteria and algae including Leptolyngbya sp., Spirulina sp., Chlorella sp., and Sargassum spp., which are prevalent species in Thailand, have been reported for their diverse biological activities and high safety profiles. However, their anti-DENV activity has not been documented. In this study, the screening assay was performed to compare the antiviral activity against DENV of crude polysaccharide and ethanolic extracts derived from 4 species of cyanobacteria and algae in Vero cells. Polysaccharide extracts from Sargassum spp. were the most effective in inhibiting DENV-2 infection under co-infection conditions, where the virus was exposed to the extract at the time of infection. Treatment of the extract significantly reduced the ability of DENV to bind to the host cells to 47.87 ± 3.88 % while treatment upon virus binding step had no antiviral effect suggesting the underlaying mechanism of the extract on interfering virus binding step. Fucoidan, a key bioactive substance in Sargassum polysaccharide, showed to reduce DENV-2 infection to 26.59 ± 5.01 %, 20.46 ± 6.58 % under the co-infection condition in Vero and A549 cells, respectively. In accompanied with Sargassum polysaccharide, fucoidan disturbed the virus binding to the host cells. These findings warrant further development and exploration of the Sargassum-derived polysaccharide, fucoidan, as a promising candidate for combating DENV infections.

Evaluation of Plant Introduction Lines of Yellow Squash (Cucurbita pepo) for Resistance against Single Infection of Cucurbit Chlorotic Yellows Virus and Cucurbit Leaf Crumple Virus

Whitefly-transmitted viruses have emerged as a major threat to cucurbit production in the United States during the past several decades. Cucurbit chlorotic yellows virus (CCYV), Cucurbit yellow stunting disorder virus (CYSDV), and Cucurbit leaf crumple virus (CuLCrV) are the main arthropod-borne plant viruses in cucurbit crops, including yellow squash (Cucurbita pepo). Symptoms of these viruses include interveinal chlorosis, chlorotic spots, yellowing, and curling of the leaves. The evaluation of specific viruses affecting a plant is challenging because of the prevalence of mixed infections in naturally infected fields. To devise an efficient breeding-based management approach, two PI lines (PI 171625 and PI 171627) were screened in a greenhouse to assess their resistance to individual infections of CCYV and CuLCrV. These lines were compared against a susceptible cultivar Gentry in two separate trials. PI 171627 displayed delayed symptoms, a reduced virus load, and a smaller area under the disease progress curve (AUDPC) compared with PI 171625 and susceptible cultivar Gentry when the plants were infected with CCYV. However, the AUDPC for CuLCrV was the same for both genotypes and the commercial line. Accession PI 171627, which displayed delayed and milder symptoms, could potentially provide a source for resistance against CCYV for breeding program. Future research is needed to comprehend the underlaying mechanism to understand this response.

Fbxo45 facilitates the malignant progression of breast cancer by targeting Bim for ubiquitination and degradation

BackgroundBreast cancer is one of the common malignancies in women. Evidence has demonstrated that FBXO45 plays a pivotal role in oncogenesis and progression. However, the role of FBXO45 in breast tumorigenesis remains elusive. Exploration of the regulatory mechanisms of FBXO45 in breast cancer development is pivotal for potential therapeutic interventions in patients with breast cancer.MethodsHence, we used numerous approaches to explore the functions of FBXO45 and its underlaying mechanisms in breast cancer pathogenesis, including CCK-8 assay, EdU assay, colony formation analysis, apoptosis assay, RT-PCR, Western blotting, immunoprecipitation, ubiquitination assay, and cycloheximide chase assay.ResultsWe found that downregulation of FBXO45 inhibited cell proliferation, while upregulation of FBXO45 elevated cell proliferation in breast cancer. Silencing of FBXO45 induced cell apoptosis, whereas overexpression of FBXO45 inhibited cell apoptosis in breast cancer. Moreover, FBXO45 interacted with BIM and regulated its ubiquitination and degradation. Furthermore, knockdown of FBXO45 inhibited cell proliferation via regulation of BIM pathway. Notably, overexpression of FBXO45 facilitated tumor growth in mice. Strikingly, FBXO45 expression was associated with poor survival of breast cancer patients.ConclusionOur study could provide the rational for targeting FBXO45 to obtain benefit for breast cancer patients. Altogether, modulating FBXO45/Bim axis could be a promising strategy for breast cancer therapy.

Heat-assisted friction stir incremental sheet forming of thermoplastics

Incremental sheet forming (ISF) has been attracting increasing interests for customised manufacture of thermoplastics, but the application in high-performance and hard-to-form thermoplastics is still a challenge. This paper develops a new heat-assisted friction stir incremental sheet forming (HAFS-ISF) process to form polyether ether ketone (PEEK) and polymethyl methacrylate (PMMA, acrylic). Flexible heating tapes and tool rotational friction heat the thermoplastic sheet on both bottom and top surfaces, which maintain the sheet material at high temperature and reduce the through-thickness temperature gradient. Results are given for the effect of key parameters on forming temperature, force, accuracy and thickness distribution. Spindle speed has the most significant effect on all the studied aspects, followed by the pre-heating temperature, while feed rate and step size have less effect. With lower spindle speed of 100 rpm, the through-thickness temperature gradient is improved. Compared with PEEK parts, more uniform forming temperature and smaller forming force can be observed for PMMA parts. But the material brittleness causes worse formability, and the severer twist and stretching effect affect the forming accuracy of PMMA parts. Forming accuracy, fracture and twist are discussed by developing analytical models. The model for forming accuracy considers the initial bending effect and springback. The fracture is discussed by modelling the stress triaxiality and equivalent strain distribution under contact. While the model for twist considers effect of the applied torque on shear strain energy and continuous accumulation of twist along the tool path. By comparing analytical models and testing observations, the effectiveness of the models is confirmed to understand the underlaying mechanisms and to address a few challenges.

Neuromuscular consequences of spinal cord injury: New mechanistic insights and clinical considerations.

The spinal cord facilitates communication between the brain and the body, containing intrinsic systems that work with lower motor neurons (LMNs) to manage movement. Spinal cord injuries (SCIs) can lead to partial paralysis and dysfunctions in muscles below the injury. While traditionally this paralysis has been attributed to disruptions in the corticospinal tract, a growing body of work demonstrates LMN damage is a factor. Motor units, comprising the LMN and the muscle fibers with which they connect, are essential for voluntary movement. Our understanding of their changes post-SCI is still emerging, but the health of motor units is vital, especially when considering innovative SCI treatments like nerve transfer surgery. This review seeks to collate current literature on how SCI impact motor units and explore neuromuscular clinical implications and treatment avenues. SCI reduced motor unit number estimates, and surviving motor units had impaired signal transmission at the neuromuscular junction, force-generating capacity, and excitability, which have the potential to recover chronically, yet the underlaying mechanisms are unclear. Furthermore, electrodiagnostic evaluations can aid in assessing the health lower and upper motor neurons, identify suitable targets for nerve transfer surgeries, and detect patients with time sensitive injuries. Lastly, many electrodiagnostic abnormalities occur in both chronic and acute SCI, yet factors contributing to these abnormalities are unknown. Future studies are required to determine how motor units adapt following SCI and the clinical implications of these adaptations.

Fiber-interferometric second harmonic generator with dual-color standard quantum-limited noise performance.

We present a novel fiber-interferometric device that achieves dual functionality: simultaneous amplification of the pulsed input signal and generation of its second harmonic while effectively suppressing the intensity noise in both modes, reaching the standard quantum-limit. The underlaying mechanism is based on phase-biased nonlinear polarization rotation coupled with type-I phase-matched second harmonic generation, a concept that is both theoretically investigated and experimentally verified. In the experiment, a fiber-optic system is constructed capable of generating 42 MHz ultra-low noise sub-150 fs output pulse trains simultaneously at 1030 nm and 515 nm, with average powers of 165 mW and 50 mW, respectively. Systematic frequency-resolved intensity noise measurements confirm dual wavelength, quantum-limited noise suppression beyond 100 kHz offset-frequency, with suppression levels up to 14 dB, showing correlation with local maxima in average power in both fundamental and second harmonic mode.

Interactive responses to temperature and salinity in larvae of the Asian brush-clawed crab Hemigrapsus takanoi: relevance for range expansion into the Baltic Sea, in the context of climate change

We studied the potential of a recently introduced species, the Asian brush-clawed crab (Hemigrapsus takanoi), to expand its distribution range further into the Baltic Sea. H. takanoi has been documented in the southwestern Baltic Sea since 2014. The ability to persist and further expand into the Baltic Proper will depend on their potential to sustain all stages of their complex life cycle, including pelagic larvae, under the Baltic Sea's conditions. Range limits may be established by the tolerance to low salinity, which in addition may be affected by water temperature. A key question is whether local populations at the distribution limit (within the Baltic Sea) show increased tolerance to low salinities and hence promote further expansion. We quantified the combined effects of salinity (10–33 PSU) and temperature (15–24 °C) on larval development in four populations of H. takanoi (two from the Baltic and two from the North Sea). We found substantial differences in larval performance between the populations from the Baltic and North Seas. Larvae from the North Sea populations always showed higher survival and faster development compared with those from the Baltic Sea. Only weak evidence of elevated tolerance towards low salinity was found in the larvae from the Baltic Sea populations. In addition, larvae from the population located near the range limit showed very low survival under all tested salinity-temperature combinations and no evidence of increased tolerance to low salinity. There was no apparent genetic differentiation among the studied populations in the mitochondrial cytochrome c oxidase subunit one gene (COI) implying high connectivity among the populations. In conclusion, the weak evidence of low salinity tolerance in Baltic Sea populations, and poor larval performance for the population located near the range limit, coupled with limited genetic differentiation suggest that subsidies are needed for populations to persist near the range limit. Alternatively, ontogenetic migrations would be required to sustain those populations. Monitoring efforts are needed to elucidate the underlaying mechanisms and document potential future range expansions.

Identifying Thermal Decomposition Mechanisms of the Solid Electrolyte Interphase with in-Situ Gas Analysis of Lithium-Ion Batteries

The global shift away from fossil fuels has generated an unprecedented demand for electric vehicles and a steady increase in the prevalence of lithium-ion batteries. Additionally, increases in the energy density of storage technologies has led to a corresponding increase in safety concerns regarding thermal runaway reactions. The components of a lithium-ion battery can self-react when exposed to an external heat source, which can cause a thermal runaway reaction process and large-scale fires of the cell. The initial reactions in this process involve the decomposition of the Solid Electrolyte Interphase (SEI) breakdown and conductive salt. The underlaying mechanisms behind these reactions are unclear; in-depth investigations are needed in order to develop a detailed understanding of the thermal runaway process and guide the development of strategies to mitigate the safety hazards posed by these battery systems. In the past, differential/online electrochemical mass spectrometry (DEMS/OEMS) was utilized to analyze the buildup of the SEI as well as decomposition due to overcharge. Our new methodology of high temperature (HT-)OEMS, with a maximum temperature of 132 °C, can simulate heating events with thermal ramps on an in-situ test cell battery. These measurements reveal information regarding reaction product gases and cell voltage drop during first formation and thermal stress tests, and provide novel insight into the electrochemical and thermodynamic phenomena during thermal abuse of lithium-ion batteries.In this study, we use (HT-)OEMS to investigate the behavior of lithium-ion cells with varied additive concentrations and formation currents. This new methodology allowed us to clearly determine that both of the testing parameters impact the formation gas evolution and thus influence the SEI-composition. Systems with high formation rates, as well as with lower concentrations of additives, exhibited an increase in the fraction of CO product gases, which can be explained with a higher presence of lithium alkoxides in the SEI. Additionally, the evolution of H2, CO2 and POF3 gases were observed at temperatures around 80 °C for cells during the thermal stress test after formation. These results allow us to establish a correlation between the thermal conductive salt decomposition and the formation of the SEI-components Li2CO3 and (CH2OLi)2.The identification of the underlying degradation mechanisms and process correlations via (HT-)OEMS provides a novel and important understanding of the phenomena that give rise to thermal runaway in lithium-ion batteries. This cell chemistry-based insight can be used to guide future prevention-focused safety assessments. Figure 1

N-Acetylneuraminic Acid Induces Vascular Endothelial Dysfunction through Targeting Mcu/SQSTM1 Pathway and Attenuates Atherosclerosis

Atherosclerosis (AS) is a major cause of various fatal diseases, such as stroke, and ischemic heart failure worldwide. The risk factors of AS include hypertension, diabetes mellitus, dyslipidemia, smoking and obesity. Recently, several investigations have shown that metabolic disorder within plaque was characterized as one of the main causes for those risk factors. The increase of N-acetylneuraminic acid (Neu5Ac), a metabolite produced by hexosamine-sialic acid pathway branching from glucose metabolism, in blood was identified as a metabolic biomarker for AS progression. Our previous study revealed that Neu5Ac could promote endothelial inflammatory response and attenuates AS plaque progression in apoE -/- mice. Mitochondrial dysfunction was suggested to be involved for endothelial injury after Neu5Ac accumulation in circulation. However, the underlaying mechanisms have not been fully understood yet. Therefore, the aim of the study was to understand the mechanism of mitochondrial injury induced by Neu5Ac accumulation in vivo using apoE-/- mouse model and in vitro using the endothelial cell line, HUVECs. Investigations have shown that impaired Ca 2+ homeostasis plays an interactive role in the progression of mitochondrial dysfunction. In our present study, we found that Neu5Ac induced Ca 2+ overload, increased ROS levels and promoted the expression of mitochondrial injury makers such as Parkin, TOMM20 and TIMM23. While pretreatment of HUVECs with BAPTA-AM in vitro could inhibit the increase of the intracellular Ca 2+ concentration and reversed the mitochondrial fragment induced by Neu5Ac, suggesting that Neu5Ac in circulation promoted mitochondrial Ca 2+ uptake and induced the mitochondrial homeostasis disorder. Based on the reported results that mitochondrial Ca 2+ uniporter (MCU) was the key regulator of impaired Ca 2+ homeostasis, we then detected the expression of MCU both in vivo and in vitro. We found an increase of MCU both at protein and mRNA levels in HUVEC cell lines. Meanwhile, we also observed that MCU expression was upregulated in aortic arch of apoE -/- mice after Neu5Ac treatment. Silencing MCU in vitro, mitochondrial Ca 2+ concentrations were downregulated and the ability of endothelial adhesion to monocytes was reversed as well. These results suggested that Neu5Ac specifically targeted MCU and induced Ca 2+ uptake. Targeting MCU might be a potential strategy for preventing AS progression induced by Neu5Ac. Furthermore, mitochondrial Ca 2+ uptake was considered as an important determinant of cell fate and mitophagy/autophagy pathway. Our previous study has confirmed that Neu5Ac could activate SQSTM1/p62-mediated excessive autophagy and subsequently induced endothelial injury. To investigate whether MCU silencing could attenuate the autophagy mediated AS progression, we investigated the SQSTM1/p62 expression after silencing MCU in HUVECs. The present results confirmed that Neu5Ac increased mitochondrion-dependent autophagy, and the effects of which were largely relieved by MCU silencing. In summary, impaired Ca 2+ homeostasis was involved in the endothelial injury induced by accumulated Neu5Ac in blood. Furthermore, MCU upregulation is responsible for Neu5Ac-induced pathological AS disorder. Our findings reveal a novel molecular mechanism regulating mitochondrial dysfunction and may open a new window for therapeutic targeting in the treatment of AS.

Mucoadhesive polymers in substance-based medical devices: functional ingredients or what else?

The paper is intended to deal with the regulatory status of the family of substance-based medical devices (SB-MD) which contain mucoadhesive polymers. Mucoadhesive formulations are mainly intended for oral/buccal, gastro-esophageal, nasal, or vaginal administration routes. They contain one or more substances/polymers of either natural, synthetic or semi-synthetic origin endowed with mucoadhesive properties. These are complex substances whose chemical-physical properties are in general well characterized. Hydration and water retaining properties, gel formation, lubricating properties are example of functional characteristics that may be involved in mucosal interaction. However, there are still uncertainties as to the underlaying mechanisms. The idea is to provide support, to the understanding of the prevailing mechanisms of action of the family of SB-MD that exploit mucoadhesion phenomenon to exert the intended therapeutic action. A case study on Hyaluronic acid as a typical representative of mucoadhesive polymers, is presented. The correct understanding of the mechanism of action of the substances/polymers involved in SB-MD is pivotal to a smooth and successful submission to the involved regulatory bodies to a positive assessment and to the final approval.

Structural distortion induced Dzyaloshinskii–Moriya interaction in monolayer CrI3 at heterostructures

The magnetic heterostructures provide flexible ways to realize particular magnetic properties that possess both scientific and practical significance. Here, by first-principles calculation, we predict strong Dzyaloshinskii–Moriya interactions (DMIs) by constructing CrI3/Metal heterostructures. The underlaying mechanisms are ascribed to the large spin-orbital coupling (SOC) of the I atom and the structural distortion in CrI3 layer caused by substrates. This is different from the traditional way that deposit magnetic films on substrate to generate DMI, wherein DMI is dominated by interlayer hybridization and large SOC of substrates. In addition, both Heisenberg exchange and magnetic anisotropy are modulated dramatically, such as Heisenberg exchange is nearly doubled on Au(111), and the out-of-plane magnetism is enhanced by 88% on Ir(111). Our work may provide a experimentally accessible strategy to induce DMI in layered magnetic materials, which will be helpful to the design of spintronics devices.

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2–2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

Generation of two induced pluripotent stem cell lines from catecholaminergic polymorphic ventricular tachycardia patients carrying RYR2 mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a congenital arrhythmic syndrome caused by the RYR2 gene encoded ryanodine receptor. Mutations on RYR2 are commonly associated with ventricular tachycardia after adrenergic stimulation, leading to lethal arrhythmias and sudden cardiac death. We generated two human induced pluripotent stem cell (iPSC) lines from CPVT affected patients carrying single missense heterozygote RYR2 mutations, c.1082 G > A and c.100 A > C. Pluripotency and differentiation capability into derivatives of three germ layers were evaluated along with karyotype stability in the report. The generated patient-specific iPSC lines provide a reliable tool to investigate the CPVT phenotype and understand underlaying mechanisms.

Abstract 3520: Single cell-derived spheroids capture functional and genetic heterogeneity of head and neck cancer

Abstract Introduction: Single cell transcriptome is used for mapping the genetic heterogeneity of a tumour; however, it does not reflect the functional heterogeneity. Currently, no model is available to provide a genotype to phenotype correlations for the heterogeneity. We developed the HNSCC spheroids from single cell that showed hyperproliferation/hypomigration or hypoproliferation/hypermigration properties, and performed transcriptome and proteomics approach to identify the underlaying mechanisms. Materials and methods: Two cells/well from HNSCC cell line UMSCC-22B were cultured in ultra-low attachment plate to develop spheroids, which were identified for their functionality by monitoring growth and cell migration. Hyperproliferative/hypomigratory or hypoproliferative/hypermigratory spheroids were pooled to perform single cell transcriptome. sSpheroid were used for proteomic analysis. Results: Single cell analysis showed four distinct clusters of cells in hyperproliferative/hypomigratory spheroids and six clusters in hypoproliferative/hypermigratory spheroids. Each cluster showed specific gene expression signature enriching a specific pathways. The hyperproliferative/hypomigratory spheroids clusters showed enrichment of cell cycle, anti-apoptosis, and pro-survival genes associated with EGFR, Wnt and Notch signalling pathways. Whereas hypoproliferative/hypermigratory spheroids cluster showed enrichment of TGF-beta, VEGF, and wound healing related pathways. Cell ontology tracing with gene signatures revealed the dominance of neoplastic cells with enrichment of EGFR, MAP Kinase, Wnt signalling pathways in the hyperproliferative/hypomigratory spheroids. The fibroblast cell cluster showed expression of stem cell markers and enrichment of ERK and EGFR related pathways. Bayesians and non-bayesians network analyses identified the surface proteins, ITGA6, Met, CD63, ICAM, and Kit as unique driver genes for hyperproliferative/hypomigratory spheroids whereas ITGB3, CD44, and EGFR were the driver genes for hypoproliferative/hypermigratory spheroids. Proteomic analysis also conformed the enrichment of antiapoptosis and cell survival related pathways in hyperproliferative/hypomigratory spheroids. While epithelial cell differentiation to keratinized cells and wound healing pathways were enriched in hypoproliferative/hypermigratory spheroids. TCGA data shows correlation with our analysis for survival/stage/progression of the disease. Conclusions: Our results suggest that single cell-derived clonal spheroid represents the genetic heterogeneity of cell population and correlates with functionality of the spheroids. These spheroids can be used as tools and the identification of cell clusters in head and neck cancer patients can be used for deciding the optimal therapeutics as well as to predict the progression of the cancer. Citation Format: Jyoti Pandey, Md Jubbair Malik, Ritis K. Shyanti, Jee Min Lee, Dhanir Tailor, Sanjay V. Malhotra, Rupesh Chaturvedi, Rana P. Singh. Single cell-derived spheroids capture functional and genetic heterogeneity of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3520.

Review of Malaysian Medicinal Plants with Potential Anticancer Activity

The global cancer incidence and its high mortality rate indicate limitations in its current treatment and chemotherapeutic strategies. This sparked a worldwide interest in the demand for chemical diversity in searching for therapeutic drugs derived from natural products. Natural products from medicinal plants, whether as pure compounds or crude extracts, offer inexhaustible sources of new drugs because of their unparalleled chemical diversity. This review aims to disseminate detailed information on the anticancer potential of Malaysian medicinal plants, focusing on the bioactive phytochemicals and mechanisms of action against cancer development in both in vitro and in vivo studies. A comprehensive search of PubMed, Google Scholar, Scopus, and ScienceDirect databases was conducted to find relevant articles on the anticancer activity of Malaysian medicinal plants. A total of hundred and twenty-two (122) articles on the anticancer activity of Malaysian medicinal plants was identified and reviewed. Eighty-five (85) plants (in vitro) and 16 plants (in vivo) have been identified to possess anticancer activity. The activity reported was attributed primarily to diverse chemical groups of naturally occurring phytochemicals such as flavonoids, phenolics, glycosides, quercetin, and gallic acid. Henceforth, the findings will hope to aid further research in understanding the underlaying mechanism and the efficiency of the isolation of the bioactive compounds.

Modelling the formation of detrimental phases in cemented carbides

Integrated Computational Materials Engineering (ICME) has proved to be an efficient tool for understanding the process-structure–property relationships and helping us to design materials. For instance, in cemented carbides manufacturing, one of the most critical parameters is the C-window. It is defined as the C content range for which phases detrimental to the mechanical properties are avoided. This processing window has been traditionally defined using applied thermodynamics methods. However, the deviation between equilibrium calculations and real manufacturing conditions requires big additional empirical efforts to precisely define the C-window. In this work, an ICME-based approach is proposed to redefine the processability limits of cemented carbides taking the cooling rate and the material’s initial powder size into consideration. The method relies on the interactive coupling of several adapted models and tools, to not only set the processability boundaries, but also to study the complex mechanisms interplay happening along microstructural evolution. A better understanding of these underlaying mechanisms leads to new inputs that can be used in the design of cemented carbides. In this regard, it is observed that faster cooling rates or coarser WC grades could be effectively used to prevent nucleation of the detrimental phases enlarging the C-window towards lower C contents.

Kinetically Accelerating Elementary Steps via Bridged Ru‐H State for the Hydrogen‐Evolution in Anion‐Exchange Membrane Electrolyzer

AbstractDesigning hydrogen evolution reaction (HER) electrocatalysts for facilitating its sluggish adsorption kinetics is crucial in generating green hydrogen via sustainable water electrolysis. Herein, a high‐performance ultra‐low Ruthenium (Ru) catalyst is developed consisting of atomically‐layered Ru nanoclusters with adjacent single Ru sites, which executs a bridging‐Ru‐H activation strategy to kinetically accelerate the HER elementary steps. Owing to its optimal electronic structure and unique adsorption configuration, the hybrid Ru catalyst simultaneously displayed a drastically reduced overpotential of 16 mV at 10 mA cm−2 as well as a low Tafel slope of 35.2 mV dec−1 in alkaline electrolyte. When further coupled with a commercial IrO2 anode catalyst, the ensembled anion‐exchange membrane water electrolyzer achievs a current density of 1.0 A cm−2 at a voltage of only 1.70 Vcell. In situ spectroscopic analysis verified that Ru single atom and atomically‐layered Ru nanoclusters in the hybrid materials play a critical role in facilitating water dissociation and weakening *H adsorption, respectively. Theoretical calculations further elucidate the underlaying mechanism, suggesting that the dissociated proton at the single atom Ru site orients itself adjacently with Ru nanoclusters in a bridged structure through targeted charge transfer, thus promoting Volmer‐Heyrovsky dynamics and boosting the HER activity.