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The combination of physical equations with deep learning is becoming a promising methodology for bioprocess digitalization. In this paper, we investigate for the first time the combination of long short‐term memory (LSTM) networks with first principles equations in a hybrid workflow to describe human embryonic kidney 293 (HEK293) culture dynamics. Experimental data of 27 extracellular state variables in 20 fed‐batch HEK293 cultures were collected in a parallel high throughput 250 mL cultivation system in an industrial process development setting. The adaptive moment estimation method with stochastic regularization and cross‐validation were employed for deep learning. A total of 784 hybrid models with varying deep neural network architectures, depths, layers sizes and node activation functions were compared. In most scenarios, hybrid LSTM models outperformed classical hybrid Feedforward Neural Network (FFNN) models in terms of training and testing error. Hybrid LSTM models revealed to be less sensitive to data resampling than FFNN hybrid models. As disadvantages, Hybrid LSTM models are in general more complex (higher number of parameters) and have a higher computation cost than FFNN hybrid models. The hybrid model with the highest prediction accuracy consisted in a LSTM network with seven internal states connected in series with dynamic material balance equations. This hybrid model correctly predicted the dynamics of the 27 state variables (R2 = 0.93 in the test data set), including biomass, key substrates, amino acids and metabolic by‐products for around 10 cultivation days. [ABSTRACT FROM AUTHOR]

The development of gene therapies based on recombinant adeno‐associated viruses (rAAVs) has grown exponentially, so the current rAAV manufacturing platform needs to be more efficient to satisfy rising demands. Viral production exerts great demand on cellular substrates, energy, and machinery; therefore, viral production relies heavily on the physiology of the host cell. Transcriptomics, as a mechanism‐driven tool, was applied to identify significantly regulated pathways and to study cellular features of the host cell for supporting rAAV production. This study investigated the transcriptomic features of two cell lines cultured in their respective media by comparing viral‐producing cultures with non‐producing cultures over time in parental human embryonic kidney cells (HEK293). The results demonstrate that the innate immune response signaling pathways of host cells (e.g., RIG‐I‐like receptor signaling pathway, Toll‐like receptor signaling pathway, cytosolic DNA sensing pathway, JAK‐STAT signaling pathway) were significantly enriched and upregulated. This was accompanied by the host cellular stress responses, including endoplasmic reticulum stress, autophagy, and apoptosis in viral production. In contrast, fatty acid metabolism and neutral amino acid transport were downregulated in the late phase of viral production. Our transcriptomics analysis reveals the cell‐line independent signatures for rAAV production and serves as a significant reference for further studies targeting the productivity improvement in the future. [ABSTRACT FROM AUTHOR]

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer‐feeding bark beetles Ips typographus L. ("Ityp") and Dendroctonus ponderosae Hopkins ("Dpon") (Scolytinae), and the pine weevil Hylobius abietis L. ("Habi"; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2‐phenylethanol (2‐PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2‐PE, suggesting a shared evolutionary history of 2‐PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer‐feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2‐PE being important for intraspecific communication and/or playing a putative role in beetle–microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs. [ABSTRACT FROM AUTHOR]

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 10 6  cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (Y T ) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 10 10  TCID 50 /mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells., (© 2024 The Author(s). Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Background: The metabolic flexibility of endothelial cells is linked to their phenotypic plasticity. Frataxin is critical in determining the iron metabolism and fate of endothelial cells. This study aimed to investigate frataxin-mediated metabolic remodeling during the endothelial-to-mesenchymal transition (EndoMT)., Methods and Results: Endothelial cell-specific frataxin knockout and frataxin mutation mice were subjected to angiotensin II to induce hypertension. EndoMT and cardiac fibrosis were assessed using histological and protein expression analyses. Fatty acid oxidation (FAO) in microvascular endothelial cells was measured using a Seahorse XF96 analyzer. We showed that inhibition of FAO accompanies angiotensin II-induced EndoMT. Frataxin knockout mice promote EndoMT, associated with increased cardiac fibrosis following angiotensin II infusion. Angiotensin II reduces frataxin expression, which leads to mitochondrial iron overload and subsequent carbonylation of sirtuin 3. In turn, carbonylated sirtuin 3 contributes to the acetylated frataxin at lysine 189, making it more prone to degradation. The frataxin/sirtuin 3 feedback loop reduces hydroxyl-CoA dehydrogenase α subunit-mediated FAO. Additionally, silymarin is a scavenger of free radicals, restoring angiotensin II-induced reduction of FAO activity and sirtuin 3 and frataxin expression, improving EndoMT both in vitro and in vivo. Furthermore, frataxin mutation mice showed suppressed EndoMT and improved cardiac fibrosis., Conclusions: The frataxin/sirtuin 3 feedback loop has the potential to attenuate angiotensin II-induced EndoMT by improving FAO.

Background: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism., Methods and Results: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice ( P <0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice ( P <0.01 and P <0.05), but not in TLR3 -/- mice, by reinforcing neutrophil recruitment ( P <0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion ( P <0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation ( P <0.05). Finally, eRNA triggered plasma clot formation in vitro ( P <0.01)., Conclusions: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Background: Recent studies suggest that enoxaparin may have therapeutic effects on oral squamous cell carcinoma. We aimed to assess this effect utilizing xenograft mouse model through evaluations of proliferation and angiogenesis markers at the RNA and protein levels., Methods: Mice were divided into enoxaparin treatment (n = 4), positive control (n = 4) and negative control (n = 3) groups. Immunohistochemical analyses were performed utilizing Bcl-2, Bax and Ki-67 antibodies. Expression levels of proliferation and apoptosis related genes were calculated utilizing qRT-PCR. Time-dependent proliferation assays were performed in OSC-19 and HEK293 cell-lines., Results: Bax antibody showed positive staining in the cytoplasm and nuclei of tumor cells, while Bcl-2 antibody displayed staining only in the cytoplasm. A proliferation index of 15%-20% was found in all groups with the Ki-67 marker indicating no metastasis. Enoxaparin treatment caused decrease in BCL2, BAX and CCNB1 genes' expressions. Compared to HEK293, proliferation assays demonstrated higher division rates in OSC-19 with a significant decrease in viability after 96 h., Conclusion: Reduced BCL-2 expression indicates a regression of tumor growth, but reduced BAX expression is not correlated with increased apoptosis. Despite the aggressive nature of OSC-19, our results showed a low cell viability with a high division rate when compared with the control HEK293. This paralleled our in vivo findings that showed absence of lymph node metastasis across all mice groups. This discrepancy with the literature suggests that further investigations of the underlying mechanisms and protein-level analyses are needed to draw definitive conclusions about the effect of enoxaparin on OSC-19 behavior., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Zika virus (ZIKV) infection may lead to severe neurological consequences, including seizures, and early infancy death. However, the involved mechanisms are still largely unknown. TRPC channels play an important role in regulating nervous system excitability and are implicated in seizure development. We investigated whether TRPCs might be involved in the pathogenesis of ZIKV infection. We found that ZIKV infection increases TRPC4 expression in host cells via the interaction between the ZIKV-NS3 protein and CaMKII, enhancing TRPC4-mediated calcium influx. Pharmacological inhibition of CaMKII decreased both pCREB and TRPC4 protein levels, whereas the suppression of either TRPC4 or CaMKII improved the survival rate of ZIKV-infected cells and reduced viral protein production, likely by impeding the replication phase of the viral life cycle. TRPC4 or CaMKII inhibitors also reduced seizures and increased the survival of ZIKV-infected neonatal mice and blocked the spread of ZIKV in brain organoids derived from human-induced pluripotent stem cells. These findings suggest that targeting CaMKII or TRPC4 may offer a promising approach for developing novel anti-ZIKV therapies, capable of preventing ZIKV-associated seizures and death., (© 2024. The Author(s).)

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of autosomal dominant movement disorders. Among the SCAs associated with impaired ion channel function, SCA19/22 is caused by pathogenic variants in KCND3, which encodes the voltage-gated potassium channel Kv4.3. SCA19/22 is clinically characterized by ataxia, dysarthria and oculomotor dysfunction in combination with other signs and symptoms, including mild cognitive impairment, peripheral neuropathy and pyramidal signs. The known KCND3 pathogenic variants are localized either in the transmembrane segments, the connecting loops, or the C-terminal region of Kv4.3. We have identified a novel pathogenic variant, c.455A>G (p.D152G), localized in the N-terminus of Kv4.3. It is located in the immediate neighbourhood of the T1 domain, which is responsible for multimerization with the β-subunit KChIP2b and thus for the formation of functional heterooctamers. Electrophysiological studies showed that p.D152G does not affect channel gating, but reduces the ionic current in Kv4.3, even though the variant is not located in the transmembrane domains. Impaired channel trafficking to the plasma membrane may contribute to this effect. In a patient with a clinical picture corresponding to SCA19/22, p.D152G is the first pathogenic variant in the N-terminus of Kv4.3 to be described to date with an effect on ion channel activity., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine., Methods and Results: Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming., Conclusions: This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel, which is considered a highly validated target for pain perception. Repeated activation with agonists to desensitize receptors or use the antagonists can both exert analgesic effects. In this work, two series of novel phenylpiperazine derivatives were designed, synthesized, and evaluated for the in vitro receptor inhibitory activity and in vivo analgesic activity. Among them, L-21 containing sulfonylurea group was identified with potent TRPV1 antagonistic activity and analgesic activity in various pain models. At the same time, L-21 exhibited low risk of hyperthermia side effect. These results indicated that L-21 is a promising candidate for further development of novel TRPV1 antagonist to treat pain., (© 2024 John Wiley & Sons Ltd.)

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

L1 syndrome, a neurological disorder with an X-linked inheritance pattern, mainly results from mutations occurring in the L1 cell adhesion molecule (L1CAM) gene. The L1CAM molecule, belonging to the immunoglobulin (Ig) superfamily of neurocyte adhesion molecules, plays a pivotal role in facilitating intercellular signal transmission across membranes and is indispensable for proper neuronal development and function. This study identified a rare missense variant (c.1759G>C; p.G587R) in the L1CAM gene within a male fetus presenting with hydrocephalus. Due to a lack of functional analysis, the significance of the L1CAM mutation c.1759G>C (p.G587R) remains unknown. We aimed to perform further verification for its pathogenicity. Blood samples were obtained from the proband and his parents for trio clinical exome sequencing and mutation analysis. Expression level analysis was conducted using western blot techniques. Immunofluorescence was employed to investigate L1CAM subcellular localization, while cell aggregation and cell scratch assays were utilized to assess protein function. The study showed that the mutation (c.1759G>C; p.G587R) affected posttranslational glycosylation modification and induced alterations in the subcellular localization of L1-G587R in the cells. It resulted in the diminished expression of L1CAM on the cell surface and accumulation in the endoplasmic reticulum. The p.G587R altered the function of L1CAM protein and reduced homophilic adhesion capacity of proteins, leading to impaired adhesion and migration of proteins between cells. Our findings provide first biological evidence for the association between the missense mutation (c.1759G>c; p.G587R) in the L1CAM gene and L1 syndrome, confirming the pathogenicity of this missense mutation., (© 2024 John Wiley & Sons Ltd.)

Background: Itch is the most common symptom of atopic dermatitis (AD) and significantly decreases the quality of life. Skin microbiome is involved in AD pathogenesis, whereas its role in the regulation of itch remains elusive. In this study, we aimed to investigate the effects of skin microbial metabolite propionate on acute and chronic pruritus and to explore the mechanism., Methods: Using various mouse models of itch, the roles of propionate were explored by behavioral tests and histopathology/immunofluorescent analysis. Primary-cultured dorsal root ganglion neurons and HEK293 cells expressing recombinant human TRP channels were utilized for in vitro calcium imaging/in vivo miniature two-photon imaging in combination with electrophysiology and molecular docking approaches for investigation of the mechanism., Results: Propionate significantly alleviated itch and alloknesis in various mouse models of pruritus and AD and decreased the density of intraepidermal nerve fibers. Propionate reduced the responsiveness of dorsal root ganglion neurons to pruritogens in vitro, attenuated the hyper-excitability in sensory neurons in MC903-induced AD model, and inhibited capsaicin-evoked hTRPV1 currents (IC 50  = 20.08 ± 1.11 μM) via interacting with the vanilloid binding site. Propionate also decreased the secretion of calcitonin gene-related peptide by nerves in MC903-induced AD mouse model, which further attenuated itch and skin inflammation., Conclusion: Our study revealed a protective effect of propionate against persistent itch through direct modulation of sensory TRP channels and neuropeptide production in neurons. Regulation of itch via the skin microbiome might be a novel strategy for the treatment of AD., (© 2024 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

In our previous study, intranuclear cardiac troponin I (cTnI) may function as a co-factor of Yin Yang 1(YY1). Here, we aimed to explore the role of intranuclear cTnI in ageing hearts. Nuclear translocation of cTnI was demonstrated using Western blot and immunofluorescence. The potential nuclear localization sequences (NLSs) of cTnI were predicted by a web server and then verified in 293T cells by putative NLS-eGFP-GST and NLS-mutant transfection. The ratio of Nuclear cTnI/ Total cTnI (Nu/T) decreased significantly in ageing hearts, accompanied with ATG5-decline-related impaired cardiac autophagy. RNA sequencing was performed in cTnI knockout hearts. The differential expressed genes (DEGs) were analysed by overlapping with YY1 ChIP-sequencing data. cTnI gain and loss experiments in vitro determined those filtered DEGs' expression levels. A strong correlation was found between expression patterns cTnI and FOS. Using ChIP-q-PCR, we demonstrated that specific binding DNA sequences of cTnI were enriched in the FOS promoter -299 to -157 region. It was further verified that pcDNA3.1 (-)-cTnI could increase the promoter activity of FOS by using luciferase report assay. At last, we found that FOS can regulate the ATG5 (autophagy-related gene 5) gene by using a luciferase report assay. Taken together, our results indicate that decreased intranuclear cTnI in ageing hearts may cause impaired cardiac autophagy through the FOS/ATG5 pathway., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Recombinant adeno-associated viral vectors (rAAVs) are one of the most used vehicles for gene therapy, with five rAAV therapeutics commercially approved by the FDA. To improve product yield, we optimized the suspension production process of rAAV8 vectors carrying a proprietary transgene using a commercially available transfection reagent, FectoVIR-AAV. Using a miniaturized automated 250 mL scale bioreactor system, we generated models of vector genome (vg) titer, capsid (cp) titer, and Vg:Cp percentage from two multivariate design of experiment studies, one centered around bioreactor operating parameters, and another based on the transfection conditions. Using the optimized process returned from these models, the vector genome titer from the bioreactor was improved to beyond 1 × 10 12  vg/mL. Five critical parameters were identified that had large effects on the pre-purification vector quantity-the transfection pH, production pH, complexation time, viable cell density at transfection, and transfection reagent to DNA ratio. The optimized process was further assessed for its performance extending to six AAV serotypes, namely AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 carrying a transgene encoding for green fluorescent protein (GFP). Five of the six serotypes returned higher vector genome titers than the control condition. These data suggest that the choice of transfection reagent is a major factor in improving vector yield. The multivariate design of experiment approach is a powerful way to optimize production processes, and the optimized process from one AAV vector can to some extent be generalized to other serotypes and transgenes to accelerate development timelines of new programs., (© 2024 Regeneron Pharmaceuticals, Inc. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galβ-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19., (© 2024 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM&#95;024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Yellow fever (YF) is a life‐threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000–60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg‐derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus‐like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence‐activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken‐tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3‐fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one‐step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D‐structure of the purified VLPs. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection., (© 2024 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

We designed and synthesized thirty novel quinoxaline aryl ethers as anticancer agents, and the structures of final compounds were confirmed with various analytical techniques like Mass, 1 H NMR, 13 C NMR, FTIR, and elemental analyses. The compounds were tested against three cancer cell lines: colon cancer (HCT-116), breast cancer (MDA-MB-231), prostate cancer (DU-145), and one normal cell line: human embryonic kidney cell line (HEK-293). The obtained results indicate that two compounds, FQ and MQ, with IC 50 values < 16 μM, were the most active compounds. Molecular docking studies revealed the binding of FQ and MQ molecules in the active site of the c-Met kinase (PDB ID: 3F66, 1.40 Å). Furthermore, QikProp ADME prediction and the MDS analysis preserved those critical docking data of both compounds, FQ and MQ. Western blotting was used to confirm the impact of the compounds FQ and MQ on the inhibition of the c-Met kinase receptor. The apoptosis assays were performed to investigate the mechanism of cell death for the most active compounds, FQ and MQ. The Annexin V/7-AAD assay indicated apoptosis in MDA-MB-231 cells treated with FQ and MQ, with FQ (21.4%) showing a higher efficacy in killing MDA-MB-231 cells than MQ (14.25%). The Caspase 3/7 7-AAD assay further supported these findings, revealing higher percentages of apoptotic cells for FQ-treated MDA-MB-231 cells (41.8%). The results obtained from the apoptosis assay conclude that FQ exhibits better anticancer activity against MDA-MB-231 cells than MQ., (© 2024 John Wiley & Sons Ltd.)

Recent studies found that non-coding RNAs (ncRNAs) played crucial roles in drug addiction through epigenetic regulation of gene expression and underlying drug-induced neuroadaptations. In this study, we characterized lncRNA transcriptome profiles in the nucleus accumbens (NAc) of mice exhibiting morphine-conditioned place preference (CPP) and explored the prospective roles of novel differentially expressed lncRNA, lncLingo2 and its derived miR-876-5p in the acquisition of opioids-associated behaviours. We found that the lncLingo2 was downregulated within the NAc core (NAcC) but not in the NAc shell (NAcS). This downregulation was found to be associated with the development of morphine CPP and heroin intravenous self-administration (IVSA). As Mfold software revealed that the secondary structures of lncLingo2 contained the sequence of pre-miR-876, transfection of LV-lncLingo2 into HEK293 cells significantly upregulated miR-876 expression and the changes of mature miR-876 are positively correlated with lncLingo2 expression in NAcC of morphine CPP trained mice. Delivering miR-876-5p mimics into NAcC also inhibited the acquisition of morphine CPP. Furthermore, bioinformatics analysis and dual-luciferase assay confirmed that miR-876-5p binds to its target gene, Kcnn3, selectively and regulates morphine CPP training-induced alteration of Kcnn3 expression. Lastly, the electrophysiological analysis indicated that the currents of small conductance calcium-activated potassium (SK) channel was increased, which led to low neuronal excitability in NAcC after CPP training, and these changes were reversed by lncLingo2 overexpression. Collectively, lncLingo2 may function as a precursor of miR-876-5p in NAcC, hence modulating the development of opioid-associated behaviours in mice, which may serve as an underlying biomarker and therapeutic target of opioid addiction., (© 2024 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Centaurea paphlagonica (Bornm.) Wagenitz is an endemic plant in Turkey. Pyrocatechol, vanillic acid, 3,4-dihydroxy benzoic acid, 5-O-caffeoylshikimic acid, tamarixetin, chlorogenic acid methyl ester, quercetin, 1,3-dicaffeoylquinic acid, tamarixetin-7-O-β-D-glucopyranoside, quercimetrin, daucosterin, paphlagonicanin B, tamarixetin-7-O-β-rutinoside, rutin, chlorogenic acid, isoorientin, orientin, 3-O-feruloylquinic acid, quercetagetin-3-methyl ether 6-O-β-glucopyranoside, diosmetin 6-C-β-glucopyranoside, quercetagetin 4'-methyl ether 7-O-β-glucopyranoside, paphlagonicanin A, nepetin, cirsiliol, desacylcynaropicrin, and 8α-O-(2',3'-dihydroxyisobutyryl) desacylcynaropicrin were isolated from both flower and aerial parts of C. paphlagonica. These compounds were identified using 1D and 2D NMR methods and ESI-MS. The MTT assay assessed the antiproliferative activities of all isolated (known and new compounds) compounds on Caco-2, LNCaP, A549, HeLa, and HEK-293 cell lines. The 8α-O-(2',3'-dihydroxyisobutyryl) desacylcynaropicrin demonstrated the highest activity against CaCo-2 and HeLa cancer cell lines., (© 2023 John Wiley & Sons Ltd.)

Background Information: Coiled-coil domain-containing protein-124 (Ccdc124) is a conserved eukaryotic ribosome-associated RNA-binding protein which is involved in resuming ribosome activity after stress-related translational shutdown. Ccdc124 protein is also detected at cellular localizations devoid of ribosomes, such as the centrosome, or the cytokinetic midbody, but its translation-independent cellular function is currently unknown., Results: By using an unbiased LC-MS/MS-based proteomics approach in human embryonic kidney (HEK293) cells, we identified novel Ccdc124 partners and mapped the cellular organization of interacting proteins, a subset of which are known to be involved in nucleoli biogenesis and function. We then identified a novel interaction between the cancer-associated multifunctional nucleolar marker nucleophosmin (Npm1) and Ccdc124, and we characterized this interaction both in HEK293 (human embryonic kidney) and U2OS (osteosarcoma) cells. As expected, in both types of cells, Npm1 and Ccdc124 proteins colocalized within the nucleolus when assayed by immunocytochemical methods, or by monitoring the localization of green fluorescent protein-tagged Ccdc124., Conclusions: The nucleolar localization of Ccdc124 was impaired when Npm1 translocates from the nucleolus to the nucleoplasm in response to treatment with the DNA-intercalator and Topo2 inhibitor chemotherapeutic drug doxorubicin. Npm1 is critically involved in maintaining genomic stability by mediating various DNA-repair pathways, and over-expression of Npm1 or specific NPM1 mutations have been previously associated with proliferative diseases, such as acute myelogenous leukemia, anaplastic large-cell lymphoma, and solid cancers originating from different tissues., Significance: Identification of Ccdc124 as a novel interaction partner of Nmp1 within the frame of molecular mechanisms involving nucleolar stress-sensing and DNA-damage response is expected to provide novel insights into the biology of cancers associated with aberrations in NPM1., (© 2023 The Authors. Biology of the Cell published by Wiley-VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ‐carboxylation might be of interest. The expression and functional activity of vitamin K‐dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ‐carboxylation that is mediated by the enzymes γ‐carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX‐producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT‐PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme‐linked immunosorbent assay and coagulation test. In addition, to quantify γ‐carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX‐producing HEK cells, resulting in a 3.2‐fold higher expression of functional FIX, which displayed a 1.4‐fold enhanced specific activity. Moreover, a 3.9‐fold enhanced recovery of fully γ‐carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ‐carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs. [ABSTRACT FROM AUTHOR]

Despite the successful reduction in the malaria health burden in recent years, it continues to remain a significant global health problem mainly because of the emerging resistance to first-line treatments. Also because of the disruption in malaria prevention services during the COVID-19 pandemic, there was an increase in malaria cases in 2021 compared to 2020. Hence, the present study outlined the in silico study, synthesis, and antimalarial evaluation of 1,3,5-triazine hybrids conjugated with PABA-glutamic acid. Docking study revealed higher binding energy compared to the originally bound ligand WR99210, predominant hydrogen bond interaction, and involvement of key amino acid residues, like Arg122, Ser120, and Arg59. Fourteen compounds were synthesized using traditional and microwave synthesis. The in vitro antimalarial evaluation against chloroquine-sensitive 3D7 and resistant Dd2 strain of Plasmodium falciparum showed a high to moderate activity range. Compounds C1 and B4 showed high efficacy against both strains and a further study revealed that compound C1 is non-cytotoxic against the HEK293 cell line with no acute oral toxicity. In vivo, study was performed for the most potent antimalarial compound C1 to optimize the research work and found to be effectively suppressing parasitemia of Plasmodium berghei strain in the Swiss albino mice model., (© 2023 John Wiley & Sons Ltd.)

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs., (© 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS‐PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N‐terminal deletions, site‐directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N‐terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation. [ABSTRACT FROM AUTHOR]

Sensitive skin is a well- known skin condition showing sensory irritation to daily used products such as cosmetics or pharmaceuticals, possibly containing sensory irritants. Methylparaben (MP), widely used as a preservative, is a representative sensory irritant and hydrolyzed in the skin. We aimed to clarify the relationship between MP sensory irritation and MP hydrolysis. First, we investigated the percutaneous penetration and hydrolysis of MP by using an ex vivo pig skin system and confirmed that topically applied MP was immediately hydrolyzed to p-hydroxybenzoic acid (PHBA). We next evaluated whether MP or PHBA causes sensory irritation using a well-used stinging test in human skin and found that MP, but not PHBA, induced irritation. Additionally, MP, but not PHBA, increased intracellular calcium in cultured TRPA1-expressed HEK293 cells, supporting the stimulatory activity of MP. Five and 10 individuals with sensitive and non-sensitive skin, respectively, were selected by a questionnaire and stinging test. In their biopsied skin samples, MP hydrolytic activity was significantly lower in sensitive than non-sensitive skin. Finally, we examined the activity of carboxylesterase (CES), which promptly hydrolyzes MP to PHBA. By using specific inhibitors of CES and CES2, we found that CES1 was responsible for MP metabolism. Our study suggests that low skin metabolism of topical agents is one of the causes of skin sensory irritation and resultant sensitive skin., (© 2023 Japanese Dermatological Association.)

Lactate and ammonia accumulation is a major factor limiting the performance of fed‐batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by‐products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine‐free fed‐batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2‐overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed‐batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine‐free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 and 1 mM, respectively. In flask, this resulted in more than a 2.5‐fold increase in IFNα2b titer in comparison to the control glutamine‐supplied fed‐batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC‐MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high‐quality therapeutic glycoproteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:494–504, 2018 [ABSTRACT FROM AUTHOR]

The focus of this study is on a rapid and cost‐effective approach for the synthesis of silver nanoparticles (AgNPs) using Artemisia quttensis Podlech aerial parts extract and assessment of their antioxidant, antibacterial and anticancer activities. The prepared AgNPs were determined by ultraviolet–visible spectroscopy, X‐ray diffraction, Fourier transform infra‐red spectroscopy, transmission electron microscopy, scanning electron microscopy, energy‐dispersive spectroscopy, and dynamic light scattering and zeta‐potential analysis. The AgNPs and A. quttensis extract were evaluated for their antiradical scavenging activity by 2, 2‐diphenyl, 1‐picryl hydrazyl assay and anticancer activity against colon cancer (human colorectal adenocarcinoma cell line 29) compared with normal human embryonic kidney (HEK293) cells. Also, the prepared AgNPs were studied for its antibacterial activity. The AgNPs revealed a higher antioxidant activity compared with A. quttensis extract alone. The phyto‐synthesised AgNPs and A. quttensis extract showed a dose–response cytotoxicity effect against HT29 and HEK293 cells. As evidenced by Annexin V/propidium iodide staining, the number of apoptotic HT29 cells was significantly enhanced, following treatment with AgNPs as compared with untreated cells. Besides, the antibacterial property of the AgNPs indicated a significant effect against the selected pathogenic bacteria. These present obtained results show the potential applications of phyto‐synthesised AgNPs using A. quttensis aerial parts extract. [ABSTRACT FROM AUTHOR]

Aim: In our pilot study, we found an increase in tyrosine hydroxylase (Th) mRNA expression in the prefrontal cortex of 72-h REM sleep-deprived (SD) rats, a mania model. Additionally, the expression levels of miR-325-3p, miR-326-3p, and miR-330-5p, the predicted target miRNAs on TH, were significantly decreased. Based on these results, in this study, we investigated whether miRNA-325-3p, miR-326-3p, and miR-330-5p modulate TH and manic-like behaviors in SD rats., Methods: Manic-like behaviors were assessed using the open field test (OFT) and elevated plus-maze (EPM) test. The direct binding activity of miRNAs to the 3'-untranslated region (3'-UTR) of the Th gene was measured in HEK-293 cells using a luciferase reporter system. We also examined mRNA and protein expression of TH after intracerebroventricular (ICV) injection of miR-330-5p agomir to SD rats, along with manic-like behaviors., Results: We observed an upregulation in mRNA and protein expression of TH and downregulation in miRNA-325-3p, miR-326-3p, and miR-330-5p expressions in the prefrontal cortex of SD rats, together with increased manic-like behaviors. The luciferase reporter assay showed that miR-330-5p could repress TH expression through direct binding to its target site in the 3'-UTR of Th, whereas miR-326-3p and miR-330-5p could not. In addition, ICV injection of miR-330-5p agomir alleviated the increase in TH expression in the prefrontal cortex of SD rats and manic-like behaviors., Conclusions: TH expression regulation through miR-330-5p may be implicated in the pathophysiology of mania in SD rats., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with Ca 2+ leak predominantly caused by ryanodine receptor 2 (RyR2) mutations. We identified a R1760W-RyR2 mutation located between the N-terminal domain and the central domain of RyR2 in a CPVT patient by DNA sequencing. Recombinant mutant RyR2 -2801mcherry plasmid generated by the overlap extension polymerase chain reaction and seamless cloning was transfected in HEK293 cells for the cell model. Single-cell luminal and cytosolic Ca 2+ imaging was measured by endoplasmic reticulum (ER) luminal Ca 2+ -sensitive protein D1ER and Fura-2 AM on a confocal laser scanning microscope, respectively. We found that in RyR2 mutant cells, the propensity for store-overload-induced Ca 2+ release (SOICR) was enhanced representing increased Ca 2+ oscillations, reduced activation and termination thresholds of spontaneous Ca 2+ release; and the sensitivity to cytosolic Ca 2+ activation was increased manifesting reduced steady state ER Ca 2+ levels. Our results indicated that R1760W-RyR2 mutation induced calcium leak, representing a gain of function. Further, antiarrhythmic drugs propafenone and flecainide significantly suppressed SOICR caused by the R1760W-RyR2 mutation at a concentration of 20 μM, which was lower than the concentration at which carvedilol suppressed SOICR., (© 2022 John Wiley & Sons Australia, Ltd.)

Background: The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported., Methods: To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor., Results: In both subunit types, ethanol, at approximate whole-cell IC 50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (p o ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states., Conclusions: These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating., (© 2022 Research Society on Alcoholism.)

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Aims: To describe the in vitro characteristics and antidiabetic in vivo efficacy of the novel glucagon-like peptide-1 receptor agonist (GLP-1RA) GL0034., Materials and Methods: Glucagon-like peptide-1 receptor (GLP-1R) kinetic binding parameters, cyclic adenosine monophosphate (cAMP) signalling, endocytosis and recycling were measured using HEK293 and INS-1832/3 cells expressing human GLP-1R. Insulin secretion was measured in vitro using INS-1832/3 cells, mouse islets and human islets. Chronic administration studies to evaluate weight loss and glycaemic effects were performed in db/db and diet-induced obese mice., Results: Compared to the leading GLP-1RA semaglutide, GL0034 showed increased binding affinity and potency-driven bias in favour of cAMP over GLP-1R endocytosis and β-arrestin-2 recruitment. Insulin secretory responses were similar for both ligands. GL0034 (6 nmol/kg) led to at least as much weight loss and lowering of blood glucose as did semaglutide at a higher dose (14 nmol/kg)., Conclusions: GL0034 is a G protein-biased agonist that shows powerful antidiabetic effects in mice, and may serve as a promising new GLP-1RA for obese patients with type 2 diabetes., (© 2022 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

As a prerequisite for serotonin secretion, the P-STS ileal enterochromaffin cell line responds to acetylcholine (ACh) stimulation with an increase in intracellular calcium mediated by the muscarinic ACh receptor M3 (M3R). Histamine increases intracellular calcium via histamine H1 receptor (H1R) in P-STS cells and pre-incubation with histamine specifically augments the response to ACh but not to epinephrine or nicotine. We aimed to elucidate whether histamine receptors are involved in this synergism. Astonishingly, HEK-293 T cells-known to express M3R, but only a very low amount of histamine receptor messenger RNA-showed a similar enhancement of the calcium response to ACh by pre-incubation with histamine. Despite the much lower level of H1R protein detected in HEK-293 T cells as compared to P-STS cells, in both cell lines pre-treatment with H1R antagonists inhibited the synergism between histamine and ACh. No indication for an involvement of histamine H2 or H4 receptors in the synergism was found. Furthermore, pre-incubation with the cAMP-inducing compound forskolin had no influence on the intracellular calcium response to ACh. Serotonin secretion from P-STS cells was increased after challenge with ACh and histamine added simultaneously compared to ACh alone, suggesting that histamine increases ACh-induced serotonin secretion from enterochromaffin cells. In conclusion, our data suggest that histamine enhances the M3R-mediated intracellular calcium response to ACh via activation of H1R. This probably increases serotonin secretion from enterochromaffin cells and thereby affects intestinal motility in histamine intolerance, food allergies and irritable bowel syndrome., (© 2022 The Authors. Clinical and Experimental Pharmacology and Physiology published by John Wiley & Sons Australia, Ltd.)

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca 2+ -permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)-induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4-overexpressed HEK cells, we observed TCA-evoked direct inhibition of TRPC4. Compared with TRPC4-knockout mice, we identified that muscarinic cationic current (mI cat ) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Ventricular septal defect (VSD) is the most common congenital heart disease. Although the coding region of MEF2C is highly relevant to cardiac malformations, the role of MEF2C gene promoter variants in VSD patients has not been genetically investigated. We investigated the role of MEF2C gene promoter variants in 400 Han Chinese subjects (200 patients with isolated and sporadic VSD and 200 healthy controls). The promoter region of the MEF2C gene was sequenced that identified 10 variants. Expression vectors encompassing the variants and the firefly luciferase reporter gene plasmid (pGL3-basic) were constructed and subsequently transfected into HEK-293 cells. The luciferase activities were measured by Dual-luciferase reporter assay system. MEF2C gene promoter transcriptional activity was significantly reduced in 4 of the 10 variants in HEK-293 cells (P < 0.05). In addition, the JASPAR database was used to perform bioinformatics analysis, which showed that these variants disrupt the putative binding sites of transcription factors and affected the expression of MEF2C protein. This study for the first time identified the variants in the promoter of the MEF2C gene in Han Chinese population and revealed the role of these variants in the formation of VSD., (© 2022 Wiley Periodicals LLC.)

The issue of emerging resistance to antitubercular drugs has created a formidable barrier in the effective prevention and cure of tuberculosis globally. In an effort to search for new antimycobacterial agents, possibly comprising new pharmacophore, novel triazole-isatin derivatives were designed as Mycobacterium tuberculosis shikimate kinase inhibitors and synthesized by microwave-assisted method. The synthesized molecules were evaluated for their antimycobacterial activity by MABA assay against M. tuberculosis H37Rv. The molecule 5h demonstrated MIC of 0.8 μg/ml and good safety profile with higher selectivity index with HEK293 cell line. The antimycobacterial activity was further substantiated with molecular docking studies. The triazole-isatin derivatives showed significant binding interactions with amino acid residues in the active site of the enzyme. These studies revealed that molecule 5h could act as a potential lead molecule for further studies to find new target-directed molecules., (© 2022 John Wiley & Sons A/S.)

Aim: To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABA A ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury., Materials and Methods: Allosteric modulation of the GABA A receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABA A channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting., Results: Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABA A channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABA A receptor. In HepG2 cells, which expressed the main GABA A receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI., Conclusions: HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs., (© 2022 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Conotoxins are small cysteine-rich peptides secreted by the Conus venom glands, which act on ion channels or membrane receptors with high specificity and potency. Conotoxins are invaluable sources for neuroscience research and drug leads, but their application is hindered by the limited successes in quantitative engineering using either chemical or biotechnological approaches. Here, we explore the Pichia pastoris to express 23 selected conopeptides using a GFP-based fluorescence screen. We found that, in a protease-deficient strain PichiaPink™ Strain 4 (ade2 prb1 pep4), most of the recombinant conopeptides were expressed as two major folding variants including a compact form that was somehow resistant to reduction and high temperature. The GFP-αTxIA was the only one displaying a single band that showed a dose-dependent neurotoxicity on larvae of the insect Plutella xylostella, with a 48-h LD 50 lower than 1.12 pmol mg -1 body weight. Furthermore, the recombinant αTxIA after cleavage from the fusion was able to inhibit cell proliferation of the LYCT and HEK293T cell lines with an appearance IC 50 of 341 ± 8 and 235 ± 15 nM, respectively. This screening method is straightforward and easy to scale up, providing a versatile tool for further optimization of conotoxin production in the yeast cell., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

Pefilipin 2 (PLIN2) is the most abunda lipid droplet (LD)-associated protein in nonadipose tissue, and its expression correlates with intracellular lipid accumulation. Here we identified a missens polymorphism, Ser251Pro, that has major effect on protein structure and function, along with an influence on human plasma triglyceride concentration. The evolutionarily conserved Ser251Pro polymorphism was identified with the ClustalW program. Structure modeling using 3D-JigSaw and the Chimera package revealed that the Pro251 allele disrupts a predicted α-helix in PIXq2 Analyses of macrophages from individuals carrying Ser251Pro variants and human embryonic kidney 29 (HEK293) cells stably transfected with either of the alleles demonstrated that the Pro251 variant caus increased lipid accumulation and decreased fipolysis Analysis of LD size distribution in stably transfected cells showed that the minor Pro251 allele resulted in an increased number of small LDs per cell and increased perilipin 3 protein expression levels as compared with cells carrying the major Ser251 allele. Genotyping of 2113 individuals indicated that the Pro251 variant is associated with decreased plasma triglyceride an very low-density lipoprotein concentrations. Altogether, these data provide the first evidence of a polymorphis in PLIN2 that affects PLIN2 function and may influence the development of metabolic and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of th receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (K12= 10 nM-1 was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extraceUular region (aa 32-229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95-141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (K12 = 19-24 nM-l). The two LDLR-A domains (aa 54-89 and 132-167) with the negatively charged acidic residues involved in Ca2+ binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPL-GLL motif and the PDZ binding motifs (QERL/KESL appear to be involved in initiating and completing th process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency. [ABSTRACT FROM AUTHOR]

P2X7 receptors trigger Ca2+-dependent exocytotic glutamate release, but also function as a route for non-exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full-length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18-amino acid cysteine-rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non-competitive way by extracellular Mg2+, did not require the recruitment of pore-forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine-rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca2+ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca2+-dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non-exocytotic glutamate efflux. [ABSTRACT FROM AUTHOR]

Localized and sustained delivery of DNA using biomaterial scaffolds would increase the applicability of gene therapy in cancer treatment and tissue engineering. The most promising approach is the application of hydrogel scaffolds to encapsulate and deliver DNA in the form of nanocomplex to the target sites. In the present work, histidine conjugated trimethylated chitosan (HTMC) was synthesized and nanocomplexes fabricated with pDNA at different N/P ratios. The zeta potential and size of the HTMC/pDNA nanoparticles were determined using dynamic light scattering technique and the results were confirmed by scanning electron microscopy (SEM). The morphology of the nanoparticles was found spherical in shape having core-shell nanostructure. Based on gel retardation assay, polyplex dissociation following incubation with heparin, protection against DNase Ι, cytotoxicity and transfection experiments, HTMC/pDNA at N/P 15 was selected as an optimized formulation and loaded into CTS/PF127 and HA/PF127 hydrogel. The optimized HTMC/pDNA nanocomplex was homogenously distributed in the CTS/PF127 hydrogel. Transfection experiments were carried out on HEK293T cell lines and the results revealed that HTMC/pDNA complexes are stable and active inside the hydrogel, however, the transfection efficacy was decreased after incorporation into the hydrogel., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

Heterozygous de novo missense pathogenic variants in PTDSS1 that result in gain-of-function of phosphatidylserine synthase 1 are associated with Lenz-Majewski hyperostotic dwarfism (LMHD). We identified the novel heterozygous de novo variant p.(Leu137Phe) in PTDSS1 in a child with mild-to-moderate developmental delay. Skeletal survey revealed no evidence of LMHD in this patient. Functional assessment of the p.Leu137Phe variant was performed by overexpressing the mutant protein into HEK293 cells. Following C 14 -serine labeling and TLC analysis of lipids, we observed that the p.(Leu137Phe) variant displayed no catalytic activity compared to the wild-type enzyme. We conclude that p.(Leu137Phe) variant has decreased enzymatic activity and that is likely to be the etiology of the patient's symptoms given the gene's constraint in the population. This is the first report of the clinical phenotype seen in an individual with a heterozygous loss-of-function variant in PTDSS1. This phenotype is distinct from LMHD, which results from gain-of-function pathogenic variants in the same gene. Evaluation of the neurodevelopmental phenotype of additional individuals with loss-of-function variants in PTDSS1 is indicated to determine the spectrum of associated phenotypes., (© 2022 Wiley Periodicals LLC.)

We report the discovery of two new 2-aminothiophene based small molecule positive allosteric modulators (PAMs) of glucagon-like peptide 1 receptor (GLP-1R) for the treatment of type 2 diabetes. One of the chemotypes, (S-1), has a molecular weight of 239 g/mol, the smallest molecule among all reported GLP-1R PAMs. When combined with GLP-1 peptide, S-1 increased the GLP-1R activity in a dose-dependent manner in a cell-based assay. When combined with the peptide agonist of vasoactive intestinal polypeptide receptor 1 (VIPR1), S-1 showed no specific activity on VIPR1, another class B GPCR present in the same HEK293-CREB cell line. Insulin secretion studies found S-1 combined with GLP-1 increased insulin secretion by 1.5-fold at 5 μM. In a mechanistic study, evidence is provided that the synergistic effect of S-1 with GLP-1 may be partly due to the enhanced impact on CREB based phosphorylation. Given the favorable profile of these chemotypes, the work reported herein suggests that 2-aminothiophene derivatives are a new and promising class of GLP-1R PAMs., (© 2022 John Wiley & Sons Ltd.)